Molecular Investigation of Radiation Resistant Cyanobacterium Arthrospira Sp

Molecular investigation of radiation resistant cyanobacterium Arthrospira sp. PCC8005

Hanène Badri

A dissertation presented for the degree of Doctor (PhD) in Microbiology

23rd January 2014

Jury

Prof. Patrick Flammang, UMons - President

Prof. David Gillan, UMons - Secretary

Prof. Ruddy Wattiez, UMons - Promoter

Dr. Ir. Natalie Leys, SCK•CEN - Mentor

Dr. Annick Wilmotte, ULg, Belgium - External member

Dr. Daniela Billi, University of Rome, Italy - External member

Je dédie ce travail

A la mémoire de mon cher père, pour tant de sacrifices consentis et d’encouragement.

Je te dis cher père que ton rêve est réalisé, je t’envoie ce cadeau aujourd’hui tant attendu. J’ai voulu tant que tu sois présent aujourd’hui pour ma graduation, que tu sois fier de moi mais le seigneur en a décidé autrement.

Paix à ton âme.

A ma tendre mère qui m’a soutenu tout au long de mon chemin, avec ses encouragements

A mon cher époux Salem qui m’a donné tant d’amour, tendresse et d’encouragement pendant ces dures années, on a partagé tant de moments difficiles mais aussi de joies.

A vous mes frères Mohamed Taher et Mohamed Mehdi

Et une spéciale dédicace pour mon futur Bébé

Abstract

The cyanobacterium Arthrospira sp.PCC 8005 was selected by European Space Agency (ESA) for producing oxygen and food during future long-duration manned space missions, as part of the bioregenerative life support system 'MELiSSA'. For this task, it is essential that Arthrospira sp. PCC 8005 continues to produce oxygen and conserves high nutritive value while exposed to cosmic radiation in space. This led us to investigate in detail the tolerance and the response of Arthrospira sp. PCC 8005 to ionizing radiation. Our study showed that the Arthrospira cells are resistant to high doses of ionizing radiation, including both electromagnetic and particle irradiation. The live planktonic cells of Arthrospira sp. PCC 8005 are able to survive and fully recover their photosynthetic growth after exposure to doses of 6400 Gy of gamma irradiation, and 1000 and 2000 Gy of He and Fe particle radiation. This supports the classification of Arthrospira sp. PCC 8005 as a radiation resistant bacterium. Using a newly designed microarray chip and an optimized RNA extraction and protein analysis procedure, the molecular response of Arthrospira to high doses of gamma rays was investigated. In essence, the dynamic gene expression changes of Arthrospira sp. PCC 8005 in response to ionizing radiation over time, showed two main stages. During the early 'emergency' response, Arthrospira cells switched quickly from an active growth state to a growth arrest mode, during which the cells shut down photosynthesis and carbon fixation, and reroute their resources into cellular protection and repair. Arthrospira cells activated various antioxidant systems, such as glutathione, to detoxify the reactive oxygen species generated by the radiation, to protect essential lipids, proteins and DNA from oxidation. Arthrospira cells also activated ssDNA repair systems and systems to remove damaged amino acids and nucleic acids from the cells. During recovery, the cells induced a newly discovered cluster of genes, the arh genes, coding for proteins with unknown function but which are highly and specifically expressed in response to radiation, in a dose dependent manner. Finally, the cells restarted the vital energy and metabolic pathways, and full recover of photosynthetic proliferation could be obtained. These results, confirm that Arthrospira sp. PCC 8005 is valuable candidate for biotechnological applications in environments exposed to ionizing radiation, in space and on Earth.

Table of contents

Abstract ...... ii

Table of contents ...... iii

List of figures ...... x

List of tables ...... xiv

List of acronyms ...... xvi

Chapter I Arthrospira, a pioneer cyanobacterium for life support on Earth and in Space ...... 1

I.1 Abstract ...... 1

I.2 Ecology of the cyanobacterium Arthrospira ...... 1

I.3 Morphology of the cyanobacterium Arthrospira ...... 2

I.4 Taxonomy of the cyanobacterium Arthrospira ...... 4

I.4.1 The genus Arthrospira ...... 4

I.4.2 The species within the genus Arthrospira ...... 7

I.4.3 The origin and taxonomy of Arthrospira sp. strain PCC 8005 ...... 9

I.5 The genome of the edible cyanobacterium Arthrsopira sp. PCC8005 ...... 10

I.5.1 Genome sequencing ...... 10

I.5.2 Tiling-Array Design ...... 11

I.6 Oxygen production by Arthrospira: the photosynthesis process ...... 12

I.6.1 Photosynthesis measurement ...... 14

I.7 Arthrospira as food supplement ...... 18

I.7.1 Animal consumption of Arthrospira ...... 18

I.7.2 Human consumption of Arthrospira ...... 18

I.7.3 The therapeutic effects of Arthrospira food supplements ...... 20

I.7.4 Radiation protection effect ...... 22

I.8 Arthrospira to support life in space ...... 22

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I.9 References ...... 25

Chapter II Ionizing radiation ...... 29

II.1 Abstract ...... 29

II.2 Different types of ionising radiation ...... 29

II.2.1 Ionizing radiation ...... 29

II.2.2 Electromagnetic waves ...... 29

II.2.3 Particulates ...... 30

II.2.4 Measuring Ionising Radiation ...... 31

II.2.5 Linear Energy Transfer ...... 31

II.3 Natural ionising radiation on Earth and in Space ...... 33

II.4 Biological effects of ionizing radiation ...... 36

II.4.1 Radiation-induced production of reactive oxygen species (ROS) ...... 37

II.4.2 Lipid peroxidation ...... 38

II.4.3 Protein oxidation ...... 39

II.5 DNA damage ...... 39

II.6 References ...... 40

Chapter III Susceptibility of cyanobacteria to ionising radiation ...... 42

III.1 Abstract ...... 42

III.1.1 Introduction ...... 42

III.2 Cyanobacteria and electromagnetic waves ...... 42

III.3 Management of oxidative stress and radiation damage in cyanobacteria ...... 43

III.4 Tolerance and response of cyanobacteria to intense VIS and UV radiation ...... 44

III.4.1 Avoidance by migration and morphological changes ...... 46

III.4.2 Protection by UV-absorbing molecules ...... 48

III.4.3 Protection and repair of PSII ...... 51

III.4.4 Protection of lipids and enzymes via antioxidants for ROS scavenging ...... 53

III.4.5 Protection and repair of DNA ...... 59

III.4.6 Controlled Shut-down or self-destruction ...... 64

III.4.7 Radiation, ROS and damage sensing, signal transduction, and response regulation 64

III.5 Tolerance and response of cyanobacteria to X- and Gamma radiation ...... 69

III.5.1 Protection of enzymes by ROS-scavenging antioxidants, and degradation of damaged proteins ...... 71

III.5.2 Protection and repair of DNA ...... 73

III.6 Tolerance and response of cyanobacteria to particle radiation ...... 74

III.7 References ...... 76

Chapter IV Thesis aims ...... 87

Chapter V Molecular investigation of the radiation resistance of edible cyanobacterium Arthrospira sp. PCC 8005 ...... 90

Abstract ...... 91

V.1 Introduction ...... 92

V.2 Materials and Methods ...... 94

V.2.1 Strain and culture conditions ...... 94

V.2.2 Irradiation conditions ...... 94

V.2.3 Post-irradiation recovery and proliferation ...... 94

V.2.4 Photosynthetic potential measurement ...... 95

V.2.5 Pigments analysis ...... 95

V.2.6 RNA extraction ...... 96

V.2.7 Microarray design...... 97

V.2.8 RNA analysis via microarrays...... 97

V.2.9 Microarray data analysis ...... 98

V.2.10 Protein extraction and analysis ...... 98

V.2.11 Statistical analysis...... 100

V.3 Results ...... 101

V.3.1 Recovery and proliferation ...... 101

V.3.2 Functionality of PSII system: Quantum yield ...... 101

V.3.3 Pigment content...... 102

V.3.4 Photosynthesis and energy production ...... 103

V.3.5 Photosensing and cell motility ...... 108

V.3.6 Carbon fixation and secondary metabolite biosynthesis ...... 108

V.3.7 Stress response and antioxidants ...... 110

V.3.8 Protein damage and recycling ...... 111

V.3.9 DNA-repair and genetic modifications ...... 111

V.3.10 Differentially expressed conserved hypothetical proteins ...... 112

V.4 Discussion ...... 114

V.5 Conclusion ...... 123

V.6 Acknowledgements ...... 123

V.7 References ...... 124

V.8 Supplemental data ...... 129

Chapter VI Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays 159

Abstract ...... 160

VI.1 Introduction ...... 161

VI.2 Materials and methods ...... 162

VI.2.1 Strain and culture conditions ...... 162

VI.2.2 Irradiation conditions ...... 162

VI.2.3 Post-irradiation recovery and proliferation analysis ...... 163

VI.2.4 Photosystem II quantum yield ...... 163

VI.2.5 Pigments analysis ...... 164

VI.2.6 Glutathione measurement ...... 165

VI.2.7 RNA extraction ...... 165

VI.2.8 RNA analysis via microarrays ...... 166

VI.2.9 Microarray data analysis ...... 167

VI.2.10 Statistical analysis ...... 168

VI.3 Results ...... 168

VI.3.1 Global Gene expression Dynamics ...... 168

VI.3.2 Gene specific response patterns ...... 173

VI.3.3 Glutathione measurement ...... 177

VI.3.4 Photosynthesis efficiency and Pigments ...... 179

VI.3.5 Survival, Recovery and proliferation ...... 180

VI.4 Discussion ...... 181

VI.5 Conclusion ...... 188

VI.6 References ...... 191

VI.7 Supplemental data ...... 195

Chapter VII Response of the spaceflight-relevant cyanobacterium Arthrospira sp. PCC 8005 to high doses of charged-particle radiation ...... 208

Abstract ...... 209

VII.1 Introduction ...... 210

VII.2 Materials and methods ...... 212

VII.2.1 Strain and culture conditions ...... 212

VII.2.2 Irradiation procedure ...... 213

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VII.2.3 Post-irradiation analysis ...... 214

VII.3 Results ...... 215

VII.3.1 Recovery and Photosynthetic growth after irradiation ...... 215

VII.3.2 Photosynthetic activity ...... 217

VII.3.3 Pigments analysis ...... 218

VII.4 Discussion ...... 223

VII.5 Conclusion ...... 227

VII.6 Acknowledgements ...... 227

VII.7 References ...... 228

VII.8 Supplemental data ...... 231

Chapter VIII General Discussion, Conclusion and Perspectives ...... 234

VIII.1 The Development and Optimisation of Experimental set-ups and Analysis methods 235

VIII.2 The radiation tolerance of the cyanobacterium Arthrospira sp. PCC 8005 ...... 238

VIII.3 The impact of ionising radiation on Arthrospira sp. PCC 8005 ...... 240

VIII.4 The “emergency” response of Arthrospira sp. PCC 8005 immediately after irradiation 241

VIII.5 The mitigation of radiation damage in Arthrospira sp. PCC 8005: protection & detoxification ...... 245

VIII.6 The recovery of Arthrospira sp. PCC 8005 from radiation damage: repair & restart 249

VIII.7 Conclusion ...... 252

VIII.8 Perspectives ...... 254

VIII.9 References ...... 259

Acknowledgments ...... 265

Curiculum Vitae ...... 268

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List of figures

Figure I-1: Floating clumps of Arthrospira sp. PCC 8005 cultures ...... 2 Figure I-2: Optical microscopy images of Arthrospira sp. PCC 8005 (200X magnification), A: Helix form, B: Mixture of straight and flat spiral forms ...... 3 Figure I-3: Life cycle of Arthrospira [9] ...... 4 Figure I-4: A Maximum-likelihood phylogenetic cyanobacteria species tree, generated using phylogenomic methods by concatenating 31 conserved proteins from full genome sequence of cyanobacteria, as recently published by Shih et al. [10]...... 5 Figure I-5: The Arthrospira genus, belonging to the Bacteria-Cyanobacteria- Oscillatoriophycideae- Oscillatoriales lineage, currently groups 8 described species, and many strains not yet assigned to any species, such as strain PCC 8005[28]...... 9 Figure I-6: Dendrogram built by the neighbor-joining method, applied to a distance matrix calculated with the Jukes and Cantor corrections and based on the ITS sequences from 16 living strains, 3 dihés, and 1 commercial product (pill). [29] ...... 10 Figure I-7: (A) A 12x135K Array format of Nimblegen and (B) the Location and Numbering of Sample Tracking Control Probes on a 12x135K Array [34]...... 12 Figure I-8: Photosynthetic electron transport chain and1 ATP synthesis in cyanobacteria [40]. .. 14 Figure I-9: Energy dissipation of the excited chlorophyll ...... 15 Figure I-10 DUAL PAM1-100 device (Walz, Effertlich, Germany) ...... 16 Figure I-11: Fluorescence quenching analysis using saturation-pulse method [41] ...... 17 Figure I-12: The MELiSSA compartments for the recycling of organic waste into food, water, and oxygen. Adapted from [72]...... 24 Figure II-1 Types of ionizing radiation [5] ...... 30 Figure II-2: Induction of DNA damage and cell death by ionizing radiation...... 32 Figure II-3: The average radiation dose from natural sources for an adult in the United States is about 1.5-6 mSev/yr. Radon accounts for more than half of an adult’s total radiation exposure, whereas background radiation (terrestrial and cosmogenic) and exposure from medical sources account for about 15% each [12] ...... 34 Figure II-4: Sources of ionizing radiation in interplanetary space...... 35

Figure II-5:Mechanism of lipid peroxidation [24]...... 39 Figure III-1: Electromagnetic Spectrum [3] ...... 43 Figure III-2: Levels of ozone at various altitudes, blocking of different types of ultraviolet radiation. Essentially all UVC (100–280 nm) is blocked by di-oxygen (from 100–200 nm) or else by ozone (200–280 nm) in the atmosphere, while fraction of UVB (280–315 nm) and most of UVA (315–400 nm) will reach the surface of the Earth [7]...... 44 Figure III-3: Model showing the effects of UVR and mitigation strategies employed by cyanobacteria [21] ...... 46 Figure III-4: Aspects of scytonemin biology...... 49 Figure III-5: Structural and spectral properties of Mycosporines, UV absorbing pigments. [32] . 51 Figure III-6: A: Photo-inhibition of Photosystem II (PSII) leads to loss of PSII electron transfer activity. PSII is continuously repaired via degradation and synthesis of the D1 protein. Lincomycin can be used to block protein synthesis [63]. B: Scheme depicting the major regulatory steps of psbA gene (encoding D1 protein) regulation in cyanobacteria[58]...... 53 Figure III-7:Antioxidant enzymes involved in superoxide detoxification[67]...... 54 Figure III-8: Pathway depicting the interaction between the alpha-tocopherol (Vitamin E), ascorbate (Vitamin C) and gluthatione (thiol) oxidant cycles...... 55 Figure III-9: Conversion of ascorbic acid into different reduced forms at various pH indicating possible binding sites and free electrons responsible for their antioxidant and chelating property ...... 56 Figure III-10: Foyer- Halliwell- Asada cycle...... 57 Figure III-11: DNA's bases may be modified by deamination or alkylation. The position of the modified (damaged) base is called the "abasic site" or "AP site". In E.coli, the DNA glycosylase can recognize the AP site and remove its base. Then, the AP endonuclease removes the AP site and neighboring nucleotides. The gap is filled by DNA polymerase I and DNA ligase...... 61 Figure III-12: In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing the damaged nucleotides (e.g., the dimer induced by UV light). The gap is then filled by DNA polymerase I and DNA ligase. In yeast, the proteins similar to Uvr's are named RADxx ("RAD" stands for "radiation"), such as RAD3, RAD10...... 62 Figure III-13: Mismatch repair...... 63 Figure III-14: Adaptive response to superoxides...... 67

Figure III-15: Adaptive response to peroxides...... 69 Figure III-16: Model of IR-Driven Mn and Fe Redox Cycling [82] ...... 72 Figure III-17: The 2 main processes of genome reconstruction in Deinococcus radiodurans. .... 74 Figure V-1: Growth curves of Arthrospira sp. PCC 8005 following exposure to different doses of gamma rays...... 101 Figure V-2: PSII quantum yield of Arthrospira sp. PCC 8005 after gamma irradiation...... 102 Figure V-3: Significant reduction in light harvesting antenna pigments (allophycocyanine and phycocyanine) while stable chlorophyll A pigment content of Arthrospira sp. PCC 8005 after irradiation. A: allophycocyanin content, B: Phycocyanin content; C: Chlorophyll A content. ... 103 Figure V-4: A conceptual model describing the response of Arthrospira sp. PCC 8005 to gamma irradiation...... 122 Figure VI-1: Scatter plot showing the differentially expressed genes of Arthrospira sp. PCC 8005 in response to gamma irradiation plotted accordingly to their change in mRNA concentration (Log2 fold change values), for 3 radiation doses (800, 1600 and 3200 Gy) and 3 time points after radiation (0 hours, 2 hours, 5 hours)...... 169 Figure VI-2: Dynamic changes in gene expression using the Mfuzz clustering software, according to their gene expression profile during recovery time (0 hours, 2 hours, 5 hours), for 3 radiation doses (800, 1600 and 3200 Gy)...... 171 Figure VI-3: Principal Component Analysis (PCA) of the cluster centres...... 172 Figure VI-4: Gene Set Enrichment Analysis (GSEA) in the clusters of differentially expressed genes based on the Clusters of Orthologs Groups (COG) functional categories...... 175 Figure VI-5: Intracellular glutathione concentrations of Arthospira sp. PCC 8005 after irradiation...... 178 Figure VI-6: Photosynthetic capacity of Arthrospira sp. PCC 8005 after gamma irradiation. .... 179 Figure VI-7: Pigment content of Arthrospira sp. PCC 8005 after irradiation...... 180 Figure VI-8: General overview of the main Transcriptional response events of Arthrospira sp PCC 8005 after exposure to different doses of 60Co gamma rays...... 189 Figure VII-1: Percentage of Arthrospira sp. PCC 8005 cells, following exposure to different doses of (A) Helium and (B) Iron particle irradiation...... 216

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Figure VII-2: Growth curves of Arthrospira sp. PCC 8005 following exposure to different doses of (A) Helium and (B) Iron particle irradiation. Data represent mean of three independent biological replicates (n=3), and error bars present the standard error of the mean (SEM)...... 217 Figure VII-3: Photosynthetic potential, measured as PSII quantum yield, of Arthrospira sp. PCC 8005 after exposure to different doses of Helium and Iron particle irradiation...... 218 Figure VII-4: A: Photographs showing Arthrospira sp PCC 8005 cultures and pigmentation (A) and morphology (microscopy (200X Magnification) (B) after exposure to 1000 Gy of He particle irradiation...... 219 Figure VII-5: Absorption spectrum of Arthrospira sp. PCC 8005 cells between 400 and 700 nm. A: Control sample; B: Irradiated with 1000 Gy He particles. The arrow indicates phycocyanin absorption region ...... 220 Figure VII-6: Changes in pigmentation in Arthrospira sp. PCC 8005 cultures in function of the dose of He irradiation recieved...... 221 Figure VII-7: Changes in pigmentation in Arthrospira sp. PCC 8005 cultures in function of the dose of Fe irradiation received...... 222

Figure S 1: RITA Facilty of the Belgian reactor (BR2) at SCK•CEN...... 129 Figure S 2: Transcriptomic expression profile for the 5854 coding DNA sequences (CDS, or “genes”) of Arthrospira sp. PCC 8005 after exposure to 3200 Gy and 5000 Gy of gamma rays 131

Figure VI-S 1: Growth curves of Arthrospira sp. PCC 8005 following exposure to different doses of gamma rays...... 195

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List of tables

Table I-1: A typical biochemical composition of a whole dried biomass,'spirulina' product[48] . 19 Table V-1: Transcriptomic (microarray) results for genes known to be involved in photosynthesis...... 104 Table V-2: Transcriptomic (microarray) results for genes known to be involved in pigment biosynthesis and degradation...... 106 Table V-3: Transcriptomic (microarray) results for genes known to be involved in Carbon fixation...... 109 Table V-4: Transcriptomic and proteomic results for conserved hypothetical proteins, specifically expressed in response to ionising radiation...... 113 Table VII-1: Time required for irradiation to achieve the predefined total dose of He particles or Fe particles...... 213

Table S 1: Specific growth rate for the cultures grown after irradiation ...... 130 Table S 2: Transcriptomic (microarray) results for genes known to be involved in photosensing, signalling, and motility...... 132 Table S 3: Transcriptomic (microarray) results for genes known to be involved in secondary metabolite production...... 133 Table S 4: Transcriptomic (microarray) results for genes known to be involved in stress response and antioxidant defense...... 134 Table S 5: Transcriptomic (microarray) results for genes known to be involved in protein repair and recycling...... 135 Table S 6: Transcriptomic (microarray) results for genes known to be involved in DNA repair...... 136 Table S 7: Transcriptomic (microarray) results for genes known to be involved in genetic rearrangement, in specific genes from “Transposases”...... 137 Table S 8: Transcriptomic (microarray) results for genes known to be involved in genetic rearrangement, in specific genes from phage-like (Fax) Elements...... 138 Table S 9: Transcriptomic (microarray) results for genes known to be involved in genetic rearrangement, in specific genes from “CRISPRs” elements...... 140

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Table S 10: Transcriptomic (microarray) results for genes coding for conserved hypothetical proteins of “unknown function”...... 141 Table S 11: Proteomics results for 3200 Gy ...... 156 Table S 12: Proteomics results for 5000 Gy ...... 157

Table VI-S 1: Specific growth rate for the cultures grown after irradiation ...... 196 Table VI-S 2: Genes differentially expressed Arthrospira sp. PCC 8005 during the early emergency reponse after irradiation ...... 197 Table VI-S 3: Genes silenced Arthrospira sp. PCC 8005 during the early emergency response after irradations...... 201 Table VI-S 4: Genes expressed during recovery, belonging to clusters 1, 2, 7, 8 and 9 ...... 205 Table VI-S 5: Genes expressed during emergency response and throughout recovery belonging to cluster 8 and 9 ...... 206

Table VII-S 1: The specific growth rate for the cultures grown after irradiation...... 231 Table VII-S 2: The specific growth rate for the cultures grown after irradiation...... 232

List of acronyms

A FC: Fold Change M

AA: Amino acid FDR: False Discovery Rate MELiSSA: Micro Ecological Life-Support System Alternative ATP: Adenosine Triphosphate FNR: Ferredoxin–NADP+ oxidoreductase MGE's: mobile genetic elements ARTeMISS: Arthrospira sp. gene Expression and FADU: Fluorometric analysis of MMR: mismatch repair system mathematical Modelling in the DNA unwinding International Space Station MaGe: Microbial Genome G Annotation & Analysis Platform APC: Allophycocyanin GSH: glutathione MAAs: Mycosporine-like ANOVA: Analysis of Variance Amino Acids GS: glutamine synthetase C N GOGAT: glutamate synthase CEP: crude ethanol precipitate NER: Nucleotide excision repair GCR: Glactic Cosmic Rays Chlorophylla: Chla NPQ: non-photochemical GSEA: Gene set enrichment quenching CRISPR: regions of phage analysis immunity O H CDS: Coding sequences OD: optical density HZE: high-ionizing energy cDNA: Complementary DNA particles P

CPD : cis-syn cyclobutane HSP: Heat Shock Proteins PSI: Photosystem I pyrimidine dimers I PSII: Photosystem II 60Co: Cobalt 60 ITS: Internally Transcribed PBS: Phycobilisomes COG: Cluster of Orthologous Spacers Groups PQ: Plastoquinone ISS: International Space station CBB: Calvin–Benson–Bassham PQH2: Reduced palstoquinones ICP/MS D PC: Plastocyanin IR: Ionizing radiation DSBs: double-strand breaks PAR: photo-synthetically active L radiation E LEO: Low Earth orbit PUFA: polyunsaturated fatty ESA: European Space Agency acid LET: Linear Energy Transfer ESDSA: extended synthesis- R dependent strand annealing LCMSMS: Liquid process Chromatography/Mass RCS: Reaction Center spectrometry ESI: Electron spray ionization RBE: relative biological source LHCII: light harvesting complex effectiveness of PSII F ROS: Reactive Oxygen Species

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RM restriction modification system

SOD: Superoxide Dismutase

SSBs: single strand breaks T

TCA: tricarboxylic acid U

UV: Ultra-Violet W

WOC: Water-oxidizing complex

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Part I: Introduction

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space

Chapter I Arthrospira, a pioneer cyanobacterium for life support on Earth and in Space

I.1 Abstract

The cyanobacterium Arthrospira, naturally occuring in tropical saline alkaline lakes, is a gram- negative bacterium that carries out oxygenic photosynthesis. Arthrospira is a natural crucial source of food to many large aquatic organisms, such as fishes. Many Arthrospira species are also edible for man and are used as nutritious and health promoting food supplements. It is currently also investigated for oxygen production in artificial bioreactor systems, such as the MELiSSA system, to provide oxygen to man in confined environments, as would be needed in long-duration space exploration. Here we describe the ecology, morphology, taxonomy, and genetic characteristics of the cyanobacterium Arthrospira, and edible strain PCC 8005 in specific.

I.2 Ecology of the cyanobacterium Arthrospira Cyanobacteria flourish widespread on Earth: they populate fresh water and marine environments, hot springs and cold dry valleys, and present tolerance to salinity, light, UV radiation, pH, dryness (desiccation), temperature, and pressure. In addition, the biotopes of the members of the Arthrospira genus are versatile. Arthrospira has been reported to exist in environments varying in their temperature and salt concentrations, but most of them being of high alkalinity [1]. Arthrospira species have been isolated mainly from alkaline, and saline waters in tropical and semitropical regions [2]. Generally, the water populated by Arthrospira has a mean salinity of 37 g.L−1. However, Arthrospira has been found at salinity levels ranging from 8.5 to 200 g.L−1 and in at least one case up to 270 g.L−1 [3]. In the highly saline lakes, the species A. fusiformis is present as nearly monospecific populations, only a few other phototrophs being found. In contrast, in the mesohaline lakes, Arthrospira co-exists with a number of other phototrophs, including other cyanobacteria, diatoms and dino flagellates. A massive population of the species A. maxima was found in water −a 2− − + highly rich in HCO3 and CO3 , Cl and Na and the total salt concentration ranges from 11 to 39 g.L−1 [1].

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space I.3 Morphology of the cyanobacterium Arthrospira Most Arthrospira species, including strain PCC 8005, are planktonic1 (e.g A.maxima A. fusiformis), but some Arthrospira species are also benthic2 (e.g. A. jenneri, A. platensis). Most Arthrospira can be cultured as suspension cultures, which are blue-green of color. The presence of gas-filled vacuoles in the cells, together with the helical shape of the filaments may result in floating clumps and mats (Figure I-1) [4]. Arthrospira filaments can also display gliding motility on solid media or surfaces.

Figure I-1: Floating clumps of Arthrospira sp. PCC 8005 cultures Arthrospira is a blue-green pigmented filamentous cyanobacterium, with a gram-negative type cell membrane. Cells are arranged in multicellular trichomes, mostly spiral-shaped as an open left-hand helix along the entire length (Figure I-2). The trichomes are composed of cylindrical shorter than broad cells, and are enveloped by a thin sheath. The average width of the helicoildal Arthrospira trichomes, i.e. the cell diameter, varies between 6 to 12 µm (16 µm) [5]. Filaments are solitary and free floating. These main morphological features of the Arthrospira genus are easily visible under light microscopy.

1 Planktonic: Plankton is a diverse group of organisms that live in the water column and cannot swim against a current. 2 Bentic: The benthic zone is the ecological region at the lowest level of a body of water such as an ocean or a lake, including the sediment surface and some sub-surface layers. 2

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space

A B

Figure I-2: Optical microscopy images of Arthrospira sp. PCC 8005 (200X magnification), A: Helix form, B: Mixture of straight and flat spiral forms

The Arthrospira trichomes can, however, display a variety of coiled forms, from tightly coiled helix to flat spiral and almost linear forms, depending on the environmental conditions. The simultaneous presence of helical and straight forms of A. fusiforms [3], A. maxima [3] and Arthrospira sp. PCC 8005 (Figure I-2) in the same culture, has been observed frequently in lab conditions. But the triggering factor or the mechanisms for this spontaneous morphological conversion remains obscure. The helical shape change in Arthrospira was the topic of different studies. The rapid switch of Arthrospira from helix to spiral shape on solid media was described in the study of Van Liere [6] involving the effect of temperature in this modification. In the studies of Jeeji Bai [7,8], the authors reported the effect of physical and chemical conditions on the helix geometry of Arthrospira. They concluded that the change of helix geometry is an irreversible phenomenon: once a strain has converted from the helix to the straight form either naturally due to a physical or chemical treatment, it does not usually revert to the helical form. Jeeji Bai [8] suggested that this phenomenon may be due to a mutation affecting some trichomes under certain growth conditions. The blue-green filaments are composed of non-heterocystous vegetative cells, showing easily visible transverse cross walls. Cell division occurs by binary fission on one plane at right angles to the long axis of the trichomes. Trichome elongation occurs through multiple intercalary cells division all along the filaments. Trichome multiplication occurs only by fragmentation: the trichome breakage is trans-cellular by destruction of an intercalary cell, also called lysing cell or sacrificial cell (necridium). Necridia act as unique specialized cells allowing the breakup of a

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space trichome, with the formation of shorter segments or, occasionally of hormogonia. Hormogonia cells undergo an enlargement and maturation process following a development cycle described and redrawn by Cifferi (Figure I-3) [9].

Figure I-3: Life cycle of Arthrospira [9]

I.4 Taxonomy of the cyanobacterium Arthrospira

I.4.1 The genus Arthrospira The cyanobacteria are a relatively large and morphologically complex group of bacteria, grouping many different orders, families, genera, species and strains. The taxonomy and nomenclature of the cyanobacteria has been subject of a long debate. Historically the different cyanobacteria were classified based on their morphology and phenotypic traits. More recently additional genetic characteristics, such as 16S and 23S rRNA gene sequences, and even full genome sequences, have been taken into account to taxonomically classify cyanobacteria and to draw-up phylogenetic trees, representing evolutionary changes, as for example illustrated in (Figure I-4).

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space

Figure I-4: A Maximum-likelihood phylogenetic cyanobacteria species tree, generated using phylogenomic methods by concatenating 31 conserved proteins from full genome sequence of cyanobacteria, as recently published by Shih et al. [10].

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space The names in red represent the 51 new cyanobacterial genomes, which were sequenced in the study of Shih et al. [10]. Branches are colour coded according to morphological subsection (I to V). Nodes supported with a bootstrap of ≥ 70% are indicated by a black dot. The phylogetic tree confirmed 8 independent morphological transitions during evolution, i.e. acquisition of the filamentous morphology (subsection III), or of the ability to form baeocytes (subsection II), which are denoted by blue triangles, annotated by events 1–8. [10].

The Arthrospira genus belongs to the Oscillatoriophycideae order and Oscillatoriales family. There has been a long lasting confusion between the genus names Arthrospira and Spirulina. Early on, Stizenberger (1854) and Gomont (1892-1893) separated the cyanobacterial forms with regularly coiled filaments and visible septa within the genus Arthrospira (Stinzenberger 1852) and those with invisible septa within the genus Spirulina (Turpin 1892). However, later Geitler (1925, 1932) [11,12]unified within the genus Spirulina (Turpin 1829), all those Oscillatoriacean organisms having the property of helically coiled trichomes along the entire length of the multicellular filaments, independent of the presence of more or less visible cross-walls under the light microscope [13]. This led not only to the merging of Arthrospira into the genus Spirulina [14] but also the genus of Oscillatoria (Vacher 1904), within the Oscillatoriales family, along with Spirulina [15,16]. Later, Castenholz (1989) [17] suggested to distinguish the Spirulina and Arthrospira genera in Bergey’s Manual of Systematic Bacteriology, based on multiple phenotypic and genotypic characteristics such as trichome helicilty, trichome size, cell wall structure and pore patterns [18], thylakoid pattern [19], trichome motility and fragmentation [19], and GC content [20]. In 2001, with the Second Edition of Bergey’s Manual of Systematic Bacteriology, Castenholz updated the criteria to separate Arthrospira from Spirulina. On the phenotypic level, the two genera were distinguished according to the trichome helix, cross-walls whether invisible or visible, and cell diameter. Arthrospira can be mainly be differentiated from Spirulina based on the degree of inclination of the pitch of the trichome helix, which forms an angle >45° from the transverse axis, the presence of easy visible septa and the distribution of junctional pores in one circular row around the cross-walls, and a cell diameter of typically 6–12 µm in Arthrospira versus 2–4 µm in Spirulina. The validity of the traditional morphological approach (Geitler’s conception) for taxonomic classification was well recognized by Wilmotte and Golubic (1991) [21] for differentiating complex cyanobacterial species such as the Arthrospira species, but they also suggest to include genetic characteristics. Among these genetic taxonomy methods, one can find DNA base

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space composition (GC content), DNA/DNA or DNA/RNA hybridisation, DNA restriction fingerprint, and base sequence catalogues of certain marker genes. In 2002, Manen and Falquet [22] investigated the genetic diversity of 23 natural, cultivated and commercial strains of Arthrospira by comparing the genus with 20 other non-Arthrospira cyanobacterial strains, utilizing the cpcB and cpcA genes coding for the beta and alpha subunits of the enzyme involved phycocyanine synthesis. These studies confirmed once again, that Arthrospira is not related to Spirulina and for the first time showed that the former genus constitutes a strongly sustained monophyletic group. The incorrect use of the name Spirulina for the genus Arthrospira, is however unfortunately still commonly used, although it dates from Geitler’s work, is outdated and should not be used [11,12]. Many species and strains currently listed as Spirulina should therefore be re-included in the Arthrospira genus. These include also all those Arthrospira strains commercially grown and sold as 'spirulina'. These 'spirulina' products are so widely known under this name that it seems inevitable that the name will persist. In this thesis, these products are referred as 'spirulina', i.e. written without italics and in between quotes.

I.4.2 The species within the genus Arthrospira In addition, the taxonomy and nomenclature of the different species withing the Arthrospira genus, has been not whitout debate (Figure I-4). The most common Arthrospira species: A. platensis, A. maxima and A. fusiformis, were defined by Komarek and Lund (1990) [23]. They maintained the inclusion of the non-planktonic (bentic) forms in the species A. platensis (Gomont), but differentiate the planktonic forms based on the morphological characters and distribution into two different taxa: A. maxima (Setchell et Gardner) and A. fusiformis (Voronich.) comb. nova [23]. This classification was confirmed and further detailed by Komárek and Anagnostidis in 2005 [2] when they concluded that freshwater benthic Arthrospira stains (i.e.without gas vacuoles) united in a fine, mostly slimy thallus, had to be placed in A. platensis or A. jenneri, while Arthrospira stains with solitary and free floating trichomes in the planktonic forms (i.e. with gas vacuoles) of subtropical and tropical saline lakes had to be placed in A. maxima and A. fusiformis. However, the extensive studies carried out on A. platensis have never shown that this cyanobacterium, which forms massive water blooms in several tropical lakes, could be, considered non-planktonic. In addition, it remains uncertain whether the planktonic species A. maxima and A. fusiformis can be considered as different species. In 1992 and 1999, Desikachary and Jeeji Bai [24] and Jeeji Bai [25] proposed that all the calyptrate forms of A. fusiformis should be included in the new species

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space A. indica (Desikachary). This implied that the thickening of the apical cell wall (calyptra) should be, considered a significant taxonomic feature.

Komarek and Lund (1990) [23] also suggested that these species, have different geographical distributions. For the first time they considered A. platensis, which is similar to A. jenneri, to be a benthic species (i.e. without gas vacuoles) that is essentially restricted to South America. Moreover, they considered A. maxima (Setchell et Gardner) to be pan-tropical, while A. fusiformis (Komárek and Lund) was limited to Africa and tropical and central Asia. In the study of Scheldeman (1999), 51 Arthrospira strains from 4 different continents ( North and South America, Afrika and Asia) were clustered based on DNA restriction analysis of amplified sequences of the Internally Transcribed Spacers (ITS) between the 16S rRNA and 23S rRNA genes [26]. This study highlighted that the strains could be resolved in only two clusters (i.e. cluster I and II), each of them subdivided into two sub-clusters [26]. But it was concluded that no clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology. In 2010, 32 new strains of Arthrospira isolated from plankton sampled in Mexico, East Africa and India were investigated and compared with 53 strains or samples of earlier studies. The study included morphological features and molecular phylogenetic analyses on the basis of nucleotide sequences of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes and the cpcB and cpcA genes [27]. Both genetic regions of the strains in this study indeed showed a significant sequence divergence among Arthrospira strains from South America, East Africa, and India, indicating possible distinct evolutionary lineages. It was concluded that there are deep genotypic divergences between Mexican and African/Indian strains of Arthrospira, which represent two distinct genotypes, which were also distinguishable based on trichome morphology, and referable to the two main tropical planktonic species A. fusiformis and A. maxima. However, because of the high morphological plasticity observed particularly with wild and clonal forms of A. fusiformis, it is sometimes difficult to distinguish these two edible species of Arthrospira, i.e. A. fusiformis and A. maxima, by using only a morphological approach.

Today the genus Arthrospira, groups 8 descriped species (Figure I-5), but also still many strains which are not yet assigned to any species, such as strain PCC 8005. Perhaps in the future additional

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space Arthrospira genome sequencing, and full genome sequence analysis and comparison, can help further classify these Arthrospira strains into species.

Genus Arthrospira : . Arthrospira erdosensis . Arthrospira fusiformis . Arthrospira indica . Arthrospira innermongoliensis . Arthrospira jenneri . Arthrospira massartii . Arthrospira maxima . Arthrospira platensis

Figure I-5: The Arthrospira genus, belonging to the Bacteria-Cyanobacteria- Oscillatoriophycideae- Oscillatoriales lineage, currently groups 8 described species, and many strains not yet assigned to any species, such as strain PCC 8005[28].

I.4.3 The origin and taxonomy of Arthrospira sp. strain PCC 8005 The geographic origin and taxonomic classification of Arthrospira sp. PCC 8005 is still enigmatic. Originally, Dr. Jeeji-Bay isolated and deposited four different strains (including the PCC8005) isolated from four different locations, i.e. India, lake Sonachi in Kenya, Mexico and Peru [29], in the Pasteur Culture Collection (PCC) of Cyanobacteria in Paris, France [30]. Unfortunately, the records describing which of the four strains originated from which country have been lost over the years, and thus the exact origin of PCC 8005 is still unclear until today. In the study of Scheldeman (1999), clustering of 51 Arthrospira strains based on sequence differences of the Internally Transcribed Spacers (ITS) between the 16S rRNA and 23S rRNA genes highlighted that the differents strains could be resolved in only two clusters (i.e. cluster I and II), each of them subdivided into two sub-clusters [26]. Among the strains associated to the cluster I, Arthrospira sp. PCC 8005 and Arthrospira indica MCRC (a strain with straight filaments from India that was sent by Dr Jeeji-Bay to the authors) presented two unique nucleotide bases in their ITS sequences, and were associated within the sub-cluster Ib. Based on these results, the hypothesis was made that Arthrospira sp. PCC 8005 could have been originally isolated from India (Figure I-6). Based on some sequences taken from the genome sequence of PCC 8005, and its comparison to the other 3 Arthrospira genomes available (i.e. of A. maxima CS-328, A. platensis NIES-39, A. platensis Paraca) it has been grouped together with A. platensis NIES-39 (based on a concatenation of 29

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space conserved proteins) or with A. maxima CS-328 (Figure I-4), based on a concatenation of 32 conserved proteins). Analysing the genome as a whole, the genome of Arthrospira sp. PCC 8005 displays the highest overall synteny comparing to the genome of Arthrospira maxima CS-328 [31]. Hence, further research and additional detailed genome analysis and comparison (e.g. based on ANI scores [32]) has to be done to get more insight into the taxonomy of Arthrospira sp PCC 8005, and to determine whether it indeed belongs to the African/Indian species A. maxima or another species.

Figure I-6: Dendrogram built by the neighbor-joining method, applied to a distance matrix calculated with the Jukes and Cantor corrections and based on the ITS sequences from 16 living strains, 3 dihés, and 1 commercial product (pill). [29]

I.5 The genome of the edible cyanobacterium Arthrsopira sp. PCC8005

I.5.1 Genome sequencing Whole-genome sequencing of Arthrospira sp. strain PCC 8005 was performed by the Microbiology group at SCK•CEN (Belgium), in collaboration with the sequencing group of Dr. Valerie Barbe, at Genoscope (France), using Sanger sequencing (up to 96,000 longer reads) and 454 pyrosequencing technology (amounting to 400,000 reads). This led to an improved version 5 of the

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space genome, currently publically available at EMBL database (accession number: CAFN01000000) and imported in the Microbial Genome Annotation and Analysis Platform (MaGe) [33] allowing private expert annotation and analysis of the genes. The final assembly includes 6 contigs into 1 scaffolds representing 6 228 153 bases (6.2 Mbp) with an average GC content of 44.73%. This final scaffold was processed by the MaGe annotation platform [33] and predicted 6 345 protein- coding sequences (CDSs) and 173 genes encoding RNA. The MaGe annotation system assigned 58,87% of the CDSs to one or more functional COGs (clusters of orthologous groups), and reported 1,704 conserved hypothetical and 884 hypothetical proteins. Manual functional annotation of the genes by a team of experts (ARTANN consortium) via the MaGe platform showed that many genes belonging to the same pathway are dispersed over the genome [31]. The PCC 8005 genome is highly repetitive in nature, with more than 300 kb present as tandem sequences, and contains four clustered, regularly interspaced short palindromic repeats (CRISPRs), which may provide a cellular defense against phages and plasmids. The genome also contains at least 140 complete insertion elements (ISs) belonging to various families and seven copies of a putative genomic island [31].

I.5.2 Tiling-Array Design During the course of this Phd, Dr. Pieter Monsieurs from the Microbiology group at SCK•CEN (Belgium), designed a micro array specific for Arthrospira sp. PCC 8005 based on the Version 3 (692 contigs, ~6.8 Mbp) of sequenced genome [31], called 'Arthrospira HX12'. This microarray allowed full genome gene expression (RNA) analysis in response to various environmental conditions and stresses, including ionising radiation. The design and production of Arthrospira HX12 microarray chips was done in collaboration with the Roche NimbleGen company, and the 12x135k array format was used (Figure I-7), meaning that each slide contained 12 identical arrays, and each array contained maximum 135 000 probes, covering the full genome of Arthrospira sp. PCC 8005. The array design is a single channel array with probes length ranging from 50 to 72 nucleotides and an average length of 53 nucleotides, and an average spacing of 34 nucleotides between 2 different probes. In total, 135 367 probes were designed to cover to full genome of Arthrospira sp. PCC 8005 (version 3), excluding random and control probes. These probes were later mapped back to the improved version 5 of the genome, when this became available in January 2014, and could be grouped to 5854 CDS and 3141 intergenic regions. A unique set of Sample Tracking Controls (STC) probes (~ 1000 nt) were placed as repeating sets of 20 along the perimeter of each array and as two 4 x 5 blocks in the upper left corner and in the centre of the array

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space (Figure I-7) Roche NimbleGen recommends to add STC to each experimental and control (input) sample pair prior to loading onto 12x135K arrays to perform a sample tracking analysis and to visually check the STC features along the perimeter to confirm that the correct sample has been added to each array.

Figure I-7: (A) A 12x135K Array format of Nimblegen and (B) the Location and Numbering of Sample Tracking Control Probes on a 12x135K Array [34]. I.6 Oxygen production by Arthrospira: the photosynthesis process Cyanobacteria are bacteria that carry out oxygenic photosynthesis and are thought to be ancestors of plastids in algae and of chloroplasts in plants [35]. Oxygenic photosynthesis started about 3.5 billion years ago when ancient cyanobacteria-like organisms evolved an apparatus capable of capturing and utilizing visible light (400-700 nm) to extract electrons from H2O with concomitant release of O2. These electrons then could be used for the reduction of CO2 to energy-rich carbohydrates [36]. It is estimated that today, half of global photosynthesis is carried out by phytoplankton [37]. Indeed more than 25 % of current photosynthesis on Earth can be accounted by two genera of marine cyanobacteria Synechococcus and Prolochococcus [38].

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space Similar to all photosynthetic eukaryotes (algae, plants), cyanobacteria use special reaction centres

(RCs) in photosystem PSII and PSI, to use light energy to induced electron transfer from H2O to NADP+, to form NADPH that is used to power up the synthesis of carbohydrates [39]. One distinctive feature of cyanobacteria is the presence of the giant multidimensional extraneous light- harvesting antenna system on the cytoplasmic side of their thylakoid membranes, the phycobilisomes (PBSs), which give the specific blue-green color to cyanobacteria. However, cyanobacteria are not the sole photosynthetic organisms that have PBSs; red algae also employ the PBSs for harvesting sunlight.

The initial event of the photosynthetic light reaction starts with the absorption of light energy (photons) by the phycobilin pigments in the large antenna system (PBS) attached to the cytoplasmic surface of the photosynthetic membrane (Phycocyanin (PC) and allophycocyanin (APC). Then the phycobilin deliver the energy of absorbed light (excitation energy) to the large pigment protein complexes of PSII and PSI integrated in the thylakoids membrane. The PSII system uses the excitation energy to specifically energize the chlorophyll a molecule in the reaction centre (RC).

This allows to ultimately derived electrons from H2O by its oxidation at the water-oxidizing complex (WOC1) of PSII. These electrons are passed along the photosynthetic electron-transport chain, through several intermediates (coenzymes and cofactors), to plastoquinone (PQ). The reduced plastoquinone (PQH2) transfers then its electrons, via the cytochrome1 B6f complex and plastocyanin (PC) (or cytochrome c6), to PSI, and finally to ferredoxin (Fd). Then, ferredoxin– NADP+ oxidoreductase (FNR) transfers the electrons to NADP+ with the final production of NADPH. Protons (H+ ions) are released into the thylakoid lumen by the WOC as water is oxidized, as well as when PQH2 delivers electrons to Cyt b6/f complex. The generated proton gradient across the thylakoid membrane is used by ATP synthase to produce ATP from ADP and inorganic phosphate (Pi). The resulted ATP and NADPH are used to fuel the Calvin Benson cyc1e in the carboxysome/cytosol whereby atmospheric CO2 is reduced to organic compounds by1 ribulose biphosphate carboxylase/oxygenase (Figure I-8).

Their main photosynthetic pigment in the Arthrospira cells is phycocyanin, which is blue in colour, but the cells also contain chlorophyll contributing to their green colour. In addition to Chl a, phycocyanin, and allophycocyanin, most cyanobacteria contain carotenoids. Some cyanobacteria

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space contain also the pigment phycoerythrin in their PBSs, giving the cells a red or pink colour, but the pigment is absent most Arthrospira strains, including Arthrospira sp. strain PCC 8005.

Figure I-8: Photosynthetic electron transport chain and1 ATP synthesis in cyanobacteria [40].

I.6.1 Photosynthesis measurement Chlorophyll a (Chl a) fluorescence has become one of the most common, simple, fast and useful techniques to monitor a culture’s photosynthetic performance. Its non-invasiveness and sensitivity, and reliable measurement instruments are wide availability. In cyanobacterial cultures, Chla fluorescence reflects the performance of photochemical processes in the photosynthetic apparatus, especially PSII, and subsequently its biomass productivity (Figure I-9). When a molecule of chlorophyll a absorbs light it is promoted from its ground state to its first singlet excited state. The excited state then has three main fates. Either the energy is passed to another chlorophyll molecule by resonance energy transfer (in this way excitation is gradually passed to the photochemical reaction centers (photosystem I and photosystem II) where energy is used in photosynthesis (called photochemical quenching or qP) (2) ; or the excited state can return to the ground state by emitting

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space the energy as heat (called non-photochemical quenching or NPQ) (3), or the excited state can return to the ground state by emitting a photon (fluorescence) (1). The contribution of the PSI emission in the total signal is mostly small and for practical purposes is often neglected [41].

Figure I-9: Energy dissipation of the excited chlorophyll The fluorescence measurement was carried out in this thesis with the DUAL PAM-100 device (Walz, Effertlich, Germany) (Figure I-10). It is based on the Pulse-Amplitude-Modulation (PAM) technique, which means that by modulating the measuring light beam (microsecond-range pulses) and parallel detection of the excited fluorescence, the relative chlorophyll fluorescence yield can be determined not only in the dark, but also in the presence of ambient light. The excitation of the photosystem II (PS II) Chlorophyll a is done with a modulated beam of red light of 635 nm and the fluorescence emitted is detected at 700 nm with the help of a photosensor (photomultiplier or photodiode). In the cyanobacterial cells, the red light of 635 nm (photons) is first absorbed by the large antenna systems (PBSs) and the phycobilins deliver the energy of absorbed light (excitation energy) to the large chlorophyll-protein complexes, i.e. the reaction centers RC, in PSII and PSI.

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space

Figure I-10 DUAL PAM1-100 device (Walz, Effertlich, Germany) The measurement process occurs after 15 min of dark adaptation during which the PSII reaction centers are "open", meaning that all PSII reaction centres are fully oxidized and all photochemical processes are off. After this 15 min of dark adaptation, the cells are exposed to a measuring light (ML), to determine the minimum fluorescence “F0” (expressed in arbitrary units) (Figure I-11). This ML is a weak light source (red light of 635 nm, <0.3 mmol photons m−2 s−1), to weak to induce photosynthesis. The exposure to the ML thus will not activate the PSII system but allows a baseline measurement of fluorescence. Next the cells will be exposed to a saturating pulse (SP) of intense light (red light of 635 nm, SP= 8 000 mmol photons m−2 s−1, 0.8 s duration) (Figure I-11). This SP is a very short intense light, largly exceeding the capacity for light utilization in the PSII reaction centers, which will quickly fully reduce or "close" transiently all the available PSII reaction centers. This prevents energy of PSII being passed to downstream electron carriers, and thus will stop photochemical processes (i.e. photochemical quenching qP) to negligible levels. Because the flash is short it will not 'activate' the photosynthesis apparatus, and the non-photochemical quenching is not affected. As a result, a steep rise in fluorescence will be measured, which is recorded as the fluorescence reached in the absence of any photochemical quenching, known as the maximum fluorescence “Fm” (expressed in arbitrary units) (Figure I-11). From those measurements, the ratio Fv/Fm can then be calculated where the variable fluorescence Fv is equal to Fm – F0. The ratio Fv/Fm represents the difference between maximum fluorescence from fully reduced PSII reaction centre (Fm) and the intrinsic fluorescence (F0) from the fully oxidized PSII. Fv/Fm is thus a measure of the maximum efficiency of PSII (the efficiency if all PSII centres were open), and can be used to estimate the maximal "potential" efficiency of PSII.

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space Healthy Arthrospira cells normally have a yield Fv/Fm of ca. 0.6 [41], meaning that about 60% of the light energy potentially could use for photosynthesis, while photosynthetically non-functional (dead) cells have Fv/Fm of 0. As such, this Chlorophyll fluorescence can be used as a proxy of stress because environmental stresses, e.g. extremes of temperature, light and water availability, can reduce the ability of a cell to metabolize normally. This can mean an imbalance between the absorption of light energy by chlorophyll and the use of energy in photosynthesis.

Figure I-11: Fluorescence quenching analysis using saturation-pulse method [41] To determine the actual (real) efficiency of PSII of a sample, under a given light regime, one needs to assess the loss of light energy due to non-photochemical quenching. For this, one needs to determine the minimal and maximum fluorescence (F0' and Fm') of the illuminated sample (arbitrary units). The fluorescence level of the illuminated sample (F0') is lowered with respect to fluorescence level of the dark adapted sample (F0) by non-photochemical quenching. From those measurements, the ratio Fm' – F0'/Fm' can then be calculated which represents the actual proportion of light absorbed by PSII that is used in photochemistry. As such, it can give a measure of the rate of linear electron transport and so the overall Photosystem II efficiency. Chlorophyll a fluorescence thus appears to measure of photosynthesis and photosynthetic growth, but this is an over-simplification. Fluorescence can measure the efficiency of PSII photochemistry, which can be used to estimate the rate of linear electron transport but it cannot measure the exact amount of electrons (energy) used for carbon fixation. Electron transport and CO2 fixation can correlate well, but may also not correlate due to processes such as photorespiration and nitrogen metabolism. A more correct way to obtain a full picture of the photosynthetic activity of the cells is to simultaneously measure chlorophyll fluorescence and gas exchange, i.e. O2 production and

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space

CO2 fixation, at different light intensities, in non-photorespiratory inducing conditions. A plot of the CO2 fixation and PSII photochemistry can indicate the electron requirement per molecule CO2 fixed, and the extent of photorespiration.

I.7 Arthrospira as food supplement

I.7.1 Animal consumption of Arthrospira Arthrospira is a crucial natural source of food to many large aquatic organisms, such as fishes, mainly in tropical and saline (mineral) lakes and water reservoirs (A. maxima, A. fusiformis). Dry Arthrospira biomass is also used as animal feed, for fishes, chicken, quail, turkey and pigs [42].

I.7.2 Human consumption of Arthrospira In the sixteenth century, when the Spanish invaders conquered Mexico, they discovered that Aztecs living in the capital Tenochtitlan were collecting a “new food” from the lake Texcoco. Travellers to Mexico during this time described how the Aztecs used a soft blue green material, harvested with fine nets from the lack to make a kind of bread called “Tecuitlatl” [9]. Later, it was characterised as Arthrospira biomass. Also in Africa, naturally growing Arthrospira has been collected from Lake Chad, by the Kanembu population, which they still do today. The wet biomass is harvested from the water, collected, and then spread out in the sandy shore of the lake for sun drying. The semi-dried biomass, in the form of dark green cake, is called “dihé”. The cake is then cut into small pieces and used by the local population as part of their daily diet [43]. Also in the industrialised countries, there has been a growing interest in the use of Arthrospira as food supplement. Researchers pioneering the work on cyanobacterial blooms at Lake Texcoco, establish the first large scale production plant for the growth of Arthrospira at the “Institut Français du Pétrole” [44]. This work was afterwards enlarged by groups localized in Italy, France and Israël [3]. Today, there are several small scale Arthrospira producers and large industrial Arthrospira farms, distributed over the world, and the dried biomass or extracts thereof are sold as a wide variety of these products are sold under the name 'spirulina'. Mainly whole dried biomass, are used as health promoting dietary supplements. The PCC 8005 is commercially grown and sold on the European market by the AlgoSource Group, recognized as one of the major microalgae world expert [45]. The high nutritional value of 'spirulina' is mainly due to its rich content of proteins, essential amino acids, essential fatty acids, vitamins, and minerals. Spirulina is 60-70% protein by dry weight and 18

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space is a rich source of vitamins (Table I-1) [46]. Spirulina is one of the richest natural sources of phycocyanin and β-carotene. The amount of phycocyanine and carotenoids in spirulina, can go up to 15% and 0.5% of the dry weight, and the carotenoids content is almost 10 times more than that in carrots. Chemical screening of spirulina showed also the presence of large amounts of phenolics compounds and flavonoids. Quantification showed the presence of 0.71% (m/m) phenolics (calculated as gallic acid) and 0.24% (m/m) flavonoids calculated as catechin equivalents per 150 g of fresh mass [47]. The high alpha-tocopherol (vitamine E), ascrobic acid (vitamine C), gluthatione, carotenoid and flavonoid content all contribute to its potent antioxidant properties.

Table I-1: A typical biochemical composition of a whole dried biomass,'spirulina' product[48]

Spirulina（dried） Nutritional value per 100 g Energy 1,213 kJ (290 kcal) Carbohydrates 23.9 g Sugars 3.1 g Dietary fiber 3.6 g Fat 7.72 g Saturated 2.65 g Monounsaturated 0.675 g Polyunsaturated 2.08 g Protein 57.47 g Glutamic acid 8.386 g Aspartic acid 5.793 g Leucine 4.947 g Alanine 4.515 g Arginine 4.147 g Valine 3.512 g Isoleucine 3.209 g Glycine 3.099 g Lysine 3.025 g Serine 2.998 g Threonine 2.970 g Phenylalanine 2.777 g Tyrosine 2.584 g Proline 2.382 g Methionine 1.149 g Histidine 1.085 g Tryptophan 0.929 g Cystine 0.662 g Vitamins

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space Niacin (Vitamin B3) 12.82 mg (85%) Choline 66.00 mg (13%) Ascorbic acid (Vitamin C) 10.10 mg (12%) Tocopherol (Vitamin E) 5.00 mg (33%) Riboflavin (Vitamin B2) 3.67 mg (306%) Pantothenic acid (Vitamin 3.48 mg (70%) B5) Thiamine (Vitamin B1) 2.38 mg (207%) Vitamin B6 364 µg (28%) Folate (Vitamin B9) 94 μg (24%) Vitamin A equiv. 29 μg (4%) beta-carotene 342 μg (3%) lutein zeaxanthin 0 μg Vitamin K 25.5 μg (24%) Vitamin B12 0 μg (0%) Vitamin D 0 IU (0%) Trace metals Potassium 1363 mg (29%) Sodium 1048 mg (70%) Magnesium 195 mg (55%) Calcium 120 mg (12%) Phosphorus 118 mg (17%) Iron 28.5 mg (219%) Zinc 2 mg (21%) Manganese 1.9 mg (90%) Other constituents Water 4.68 g

I.7.3 The therapeutic effects of Arthrospira food supplements In addition to its high the nutritional value, there are also studies reporting an important therapeutic value of orally consumed Arthrospira. Spirulina is gaining a tremendous attention as potent edible antioxidant, which could have an important therapeutic value. Experimental and epidemiological evidences suggest that oxidative stress characterized by excessive generation of ROS is a critical player in many pathological states including the inflammatory diseases, neurodegenerative diseases, atherosclerosis, cardiovascular diseases, diabetes mellitus, cancer, and reperfusion injury [49]. I.7.3.1 Anti-oxidant effects Early biochemical laboratory studies reported that the alcohol extract of spirulina inhibited lipid peroxidation more significantly (65% inhibition) than chemical antioxidant like α-tocopherol

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space (35%), Butylated hydroxyanisole (BHA) (45%), and beta-carotene (48%) [50]. Also, the water extract of spirulina had more antioxidant effect (76%) than gallic acid (54%) and chlorogenic acid (56%). In another study, realised by Zhi-gang [51], the antioxidant effects of two fractions of a hot water extract of spirulina were studied using three systems that generate superoxide, lipid, and hydroxyl radicals. Both fractions showed significant capacity to scavenge hydroxyl radicals (the most highly reactive oxygen radical), but no effect on superoxide radicals. Later, in vivo test on humans, confirmed the anti-oxidant effects of Arthrospira food supplements. Geriatric patients administrated spirulina for 16 weeks showed a remarkable improvement in the antioxidant potential as measured by the increased levels of antioxidant status in blood plasma of these individuals [52]. A double-blind placebo controlled study performed on individuals after exercise showed decrease amount of creatine kinase in the blood (as indicator of muscular breakdown) when they were supplemented with spirulina. Moreover their exhaustion time in the treadmill exercise increased by 52 seconds. This could be explained by the antioxidant potential of spirulina [53]. I.7.3.2 Anti-cancer effects Spirulina is one of the richest natural sources of phycocyanin and β-carotene. It is assumed that because of the powerful antioxidant activity of both pigments, the DNA damage that is caused by free radicals is reduced and cancer is prevented. Spirulina has indeed been shown to be a potential cancer-fighting nutriceutical [54]. Numerous laboratory experiments using animal models have demonstrated the anticancer properties of spirulina [55-57]. The administration of phycocyanin to mice with liver cancer markedly increased their survival rate. I.7.3.3 Anti-inflammatory effect Spirulina has also specific anti-inflammatory properties. The high phycocyanin and carotenoids content are probably at the origin of this anti-inflamatoir effect, as it is known that antioxidants have the intrinsic anti-inflammatory characteristics because they quench ROS. In their studies, Bhat and Madyastha [58,59] examined C-phycocyanin and found that among its potent scavenging potential of free radicals, the pigment exhibited significant hepato-protective effects [60]. In addition, in the studies of Romay et al. [61], the potential effect of phycocyanin to inhibit inflammation in mouse ears was reported.

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space I.7.4 Radiation protection effect The radioprotective effect of a crude ethanol precipitate (CEP) of Arthrospira platensis was first studied in vitro with polychromatic erythrocytes (PCE) of mouse bone marrow using the micronucleus test. In this set-up, the Arthrospira extract caused a significant reduction of micronucleus frequencies induced by gamma rays. Gamma rays followed by treatment with CEP led to about the same radioprotective effect as CEP treatment followed by gamma-radiation. From this, the authors concluded that the protective compound probably acts as a DNA-stabilizing factor, and they excluded the possibility of a radical scavenging mechanism [62]. These in vitro results were confirmed by in vivo results from animal tests. Rats exposed to X rays (5 Gy) and fed with phycocyanin extract from spirulina, had a decreased dehydrogenase activity and energy rich phosphate level in the blood [63]. In addition, it was reported that spirulina promotes hematopoietic stem cells and progenitor cells to differentiate after lethal 60Co radiation and thus increased the rate of survival in mice [64]. Some studies involving human treatment with Arthrospira after the Chernobyl nuclear accident were reported in the communication of Amha Belay [65]. In the paper of Loseva [66] it was reported that feeding children with 5g of spirulina daily resulted in the reduction of Cesium-137.

I.8 Arthrospira to support life in space Realization of long-term manned Space mission such as the establishment of planetary bases on the Moon or Mars requires a number of critical technologies to be developed in order to supply the most important needs for the astronauts. Evidently, the most required need is the life support for crew. A life support comprises four main functions which are atmosphere generation, water recycling, food generation and waste treatment [67]. One astronaut demands daily 1 kg of O2, 2.8 kg of drinking water and 13 kg of hygiene water production, and 2.7 kg of food (freeze-dried + canned food), and produces daily 1.2 kg CO2 which needs to be removed [68]. This leads of about 22.4 kg of total requirements per person per day of different compounds to be produced or eliminated [69] which is for future long-distance and long-duration flights of larger crews not be manageable without recycling, for both economical and safety reasons. In attempted to find a solution to this problem, a bioregenerative life support system, called MELiSSA, was designed about 25 years ago, by the three fathers, Max Mergeay, Willy Verstraeten and Claude Chipaux [70]. The MELiSSA system, which was later adopted by the European Space Agency (ESA), aims the complete recycling of gas liquid and solid wastes during long-distance

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space space exploration missions. The MELiSSA loop mimics the natural ecosystem of a lake. The system consists of a closed loop of four interconnected bioreactors and a higher plant chamber where each one has a specific task to fulfil [68]. . First compartment (I): This compartment collects the waste produced by the crew (i.e faeces, urine …) and the non-edible output of the higher plant compartment (straw, roots...) and the non-edible microbial biomass from the system. The main task of compartement I is

to anaerobically transform this waste to volatile fatty acids, CO2, H2, ammonium, and minerals. For biosafety reasons, as well as degradation efficiency, the compartment operates in thermophilic conditions (i.e. 55°C). . Second compartment (II): This compartment is responsible for the elimination of the terminal products of the liquefying compartment I. In the initial MELiSSA concept, it is divided into two separate and independent compartments (i.e a photoautotrophic and a photoheterotrophic compartment). This separation was mainly due to an expectation of high

H2 production from the first compartment. Although the specific treatment of hydrogen is still being kept in mind, the very wide results for substrate degradation obtained with Rhodospirillum rubrum lead to simplification of the loop to only one second compartment. . Third compartment (III): The nitrifying compartment's main function is to convert the + NH4 released from the organic waste to nitrates, which is the most favourable source of nitrogen for the photosynthetic organisms in compartement IV. The compartment III is + – composed of a mixed Nitrosomas and Nitrobacter culture which oxidise NH4 to NO2 and – – NO2 to NO3 respectively. . Fourth compartement (IV): : The photo-autotophic compartment, is split into two parts: o The Cyanobacteria compartment (IVa) o The Higher Plant (HP) compartment (IVb) Both these compartments are essential for the regeneration of oxygen and water, the removal of carbon dioxide, and the production of food. The cyanobacteria compartment (IVa) has been extensively studied in the MELiSSA project. Stochiometries have been validated in optimal and several limitation conditions.

The organisms in compartment IVa should fulfil efficient O2 production and photosynthetic

CO2 fixation combined with limited volume ratio. Given these criteria, cyanobacteria are

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space the ideal candidate organisms. In addition, the most important criteria for the produced biomass to be useful as food supplement are a high biomass production rate and a high nutritious quality (carbohydrates, fat, and proteins as well as number of vitamins and minerals). The edible cyanobacterium is very digestive in contrast to many other microorganisms [71], and there is no need for cooking or any special treatment before human consumption allowing preservation of the integrity of components such vitamins and fatty acids. Hence the easily digestible cyanobacterium Arthrospira sp. PCC 8005 was selected to colonise the compartment IVa of the MELiSSA loop (Figure I-12) [67]. The higher plant compartment (HPC) (IVb): The activities on this compartment have been initiated with eight crops: wheat, tomato, potato, soybean, rice, spinach, onion and lettuce.

Figure I-12: The MELiSSA compartments for the recycling of organic waste into food, water, and oxygen. Adapted from [72].

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space The European Space Agency (ESA) selected thus Arthrospira sp. PCC 8005 to be used continuously in the MELiSSA loop for recycling oxygen, water, and food during future long-haul space missions. During such extended space missions, Arthrospira sp. PCC 8005 will be chronically exposed to artificial illumination in the photobioreactor, and reduced gravity and harmful cosmic radiation. The effects of such environmental stresses on the oxygen and biomass production and carbon dioxide fixation kinetics, as well as the nutritional value of the biomass produced, thus need to be investigated in detail. In the frame work of the project ARTeMISS standing for Arthrospira sp. gene Expression and Mathematical Modelling in the International Space Station, targeting the first flight experiment of the cyanobacterium Arthrospira sp. PCC8005 onboard ISS, the response of Arthrospira to spaceflight conditions at the culture level (cell density, cell interactions), cellular level (size, shape, colour, membrane structure), and molecular level (metabolomic, lipidomic, proteomic, transcriptomic and genetic level) will be investigated. Furthermore, the kinetic parameters for subsequent mathematical modelling of Arthrospira sp. strain PCC8005 reproduction and metabolism under spaceflight conditions will be determined.

I.9 References 1. Busson FF (1970) Spirulina platensis (Gom.) Geitler et Spirulina geitleri J. de Toni: cyanophycées alimentaires: Service de santé. 2. Komárek J, Anagnostidis K (2005) Süßwasserflora von Mitteleuropa, Bd. 19/2: Cyanoprokaryota: Bd. 2 / Part 2: Oscillatoriales: Spektrum Akademischer Verlag. 3. Sili C, Torzillo G, Vonshak A (2012) Arthrospira (Spirulina). In: Whitton BA, editor. Ecology of Cyanobacteria II: Springer Netherlands. pp. 677-705. 4. Food, Agriculture Organization of the United N, Habib MAB (2008) A review on culture, production and use of Spirulina as food for humans and feeds for domestic animals and fish. Rome, Italy: Food and Agriculture Organization of the United Nations. 5. Venkataraman LV (1997) Spirulina platensis (Arthrospira): Physiology, Cell Biology and Biotechnologym, edited by Avigad Vonshak. Journal of Applied Phycology 9: 295-296. 6. Van Liere L, Mur LR (1979) Growth Kinetics of Oscillatoria agardhii Gomont in Continuous Culture, Limited in its Growth by the Light Energy Supply. Journal of General Microbiology 115: 153-160. 7. Jeeji Bai N, Seshadri CV (1980) On coiling and uncoiling of trichomes in the genus Spirulina. Algological Studies/Archiv für Hydrobiologie, Supplement Volumes 26: 32-47. 8. Jeeji Bai N (1985) Competitive exclusion or morphological transformation? A case study with Spirulina fusiformis. Algological Studies/Archiv für Hydrobiologie, Supplement Volumes 38-39: 191-199. 9. Ciferri O (1983) Spirulina, the edible microorganism. Microbiological reviews 47: 551-578. 10. Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, et al. (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proceedings of the National Academy of Sciences of the United States of America 110: 1053-1058. 11. Geitler L (1925) Cyanophyceae. In: Die Süsswasser-Flora Deutschlands, Österreichs und der Schweiz. (Pascher, A. Eds). Jena: Gustav Fischer 12: 1-450. 12. Geitler L (1932) Cyanophyceae. In: Kryptogamen-Flora von Deutschland, Österreich und der Schweiz. Ed. 2. In: Rabenhorst LE, editor. Leipzig: Akademische Verlagsgesellschaft. pp. 673-1196.

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space 13. Hoffmann L (1985) Quelques remarques sur la classification des Oscillatoriaceae. Cryptogamie, Algologie pp. 71-79. 14. Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Journal of General Microbiology 111: 1-61. 15. Iltis A (1970) Phytoplancton des eaux natronées du Kanem (Tchad) : 4. Note sur les espèces du genre Oscillatoria, sous-genre Spirulina (Cyanophyta). Cahiers ORSTOM Série Hydrobiologie 4: 129- 134. 16. Caspers H (1974) Pierre Bourrelly: Ls Algues d'eau douce. Initiation à la systématique. Tome I: Les Algues vertes. Tome II: Chrysophyées, Xanthophycées et Diatomées. Tome III: Eugléniens, Péridiniens, Algues rouges et Algues bleues. – Avec 118 + 115 + 137 planches, 13+ 22+ 14 figures, 512 + 440 +544pp. Paris: Editions N. Boubée & Cie. 1966/1970. 120.–, 107.04, 150.– F. Internationale Revue der gesamten Hydrobiologie und Hydrographie 59: 286-287. 17. Castenholz RW (1989) Subsection III. Order Oscillatoriales. In: Bergey's Manual of Systematic Bacteriology. Staley, JT, Bryant, MP, Pfennig, N and JG Holt, Eds Williams and Wilkins, Baltimore 3: 1771-1780. 18. Guglielmi GC-B, G (1982) Structure et distribution des pores et des perforations de l'enveloppe de peptidoglycane chez quelques cyanobactéries Protistologica 18: 151. 19. Anagnostidis K, Komárek Jí (1988) Modern approach to the classification system of cyanophytes. 3 - Oscillatoriales. Algological Studies/Archiv für Hydrobiologie, Supplement Volumes 50-53: 327- 472. 20. Herdman M, Janvier M, Waterbury JB, Rippka R, Stanier RY, et al. (1979) Deoxyribonucleic Acid Base Composition of Cyanobacteria. Journal of General Microbiology 111: 63-71. 21. Wilmotte A, Golubic S (1991) Morphological and genetic criteria inthe taxonomy of Cyanophyta/Cyanobacteria. Algological Studies (Stuttgart). pp. 1-24. 22. Manen JF (2002) The cpcB--cpcA locus as a tool for the genetic characterization of the genus Arthrospira (Cyanobacteria): evidence for horizontal transfer. International Journal of Systematic and Evolutionary Microbiology 52: 861-867. 23. J K, JWG L (1990) What is “ Spirulina platensis ” in fact? Arch Hydrobiol Suppl 85, Algol Stud 58: 1- 6. 24. Desikachary TV JBN (1992) Taxonomic studies in Spirulina In: N JB, editor. Spirulina ETTA national symposium MCRC. Madras: Seshradi CV. pp. 12-21. 25. N JB (1999) A taxonomic appraisal of the genera Spirulina and Arthrospira. Mahato AK, Vidyavati (eds) New Delhi: Recent trends in algal taxonomy pp. 253-272. 26. Scheldeman P, Baurain D, Bouhy R, Scott M, Mühling M, et al. (1999) Arthrospira (‘Spirulina’) strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer. FEMS Microbiology Letters 172: 213-222. 27. Dadheech PK, Ballot A, Casper P, Kotut K, Novelo E, et al. (2010) Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences. Phycologia 49: 361-372. 28. NCBI TaxonomyBrowser http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=35823&lvl=3&lin =f&keep=1&srchmode=1&unlock. 29. Baurain D, Renquin L, Grubisic S, Scheldeman P, Belay A, et al. (2002) Remarkable Conservation of Internally Transcribed Spacer Sequences of Arthrospira ("Spirulina" ) (Cyanophyceae, Cyanobacteria) Strains from Four Continents and of Recent and 30-Year-Old Dried Samples from Africa1. Journal of Phycology 38: 384-393. 30. Pasteur I (2014) http://cyanobacteria.web.pasteur.fr/. 31. Janssen PJ, Morin N, Mergeay M, Leroy B, Wattiez R, et al. (2010) Genome sequence of the edible cyanobacterium Arthrospira sp. PCC 8005. Journal of bacteriology 192: 2465-2466.

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space 32. Rodriguez RL, Grajales A, Arrieta-Ortiz ML, Salazar C, Restrepo S, et al. (2012) Genomes-based phylogeny of the genus Xanthomonas. BMC microbiology 12: 43. 33. Vallenet D, Labarre L, Rouy Z, Barbe V, Bocs S, et al. (2006) MaGe: a microbial genome annotation system supported by synteny results. Nucleic acids research 34: 53-65. 34. NimbleGen (2011) NimbleGen Arrays User’s Guide Gene Expression Arrays In: NimbleGen R, editor. Version 6.0, Nov 2011 ed. 35. Lars Olof Björn G (2009 ) The evolution of photosynthesis and chloroplasts. CURRENT SCIENCE 96: 1466-1474. 36. Blankenship RE (2010) Early evolution of photosynthesis. Plant physiology 154: 434-438. 37. Whitton BA, Potts, M. (Eds.) (2000) The Ecology of Cyanobacteria: Their Diversity in Time and Space. 38. Partensky F, Hess WR, Vaulot D (1999) Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiology and molecular biology reviews : MMBR 63: 106-127. 39. Govindjee JFK, Lawrence Berkeley, Johannes Messinger, Umea (2010) Photosystem II. ENCYCLOPEDIA OF LIFE SCIENCES 40. M.S JDJ (2006) http://www.chm.bris.ac.uk/motm/oec/motm.htm. 41. Masojídek J, Vonshak A, Torzillo G (2010) Chlorophyll Fluorescence Applications in Microalgal Mass Cultures. In: Suggett DJ, Prášil O, Borowitzka MA, editors. Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications: Springer Netherlands. pp. 277-292. 42. Belay A, Kato T, Ota Y (1996) Spirulina (Arthrospira): potential application as an animal feed supplement. Journal of Applied Phycology 8: 303-311. 43. Abdulqader G, Barsanti L, Tredici MR (2000) Harvest of Arthrospira platensis from Lake Kossorom (Chad) and its household usage among the Kanembu. Journal of Applied Phycology 12: 493-498. 44. Lloyd RG, Buckman C, Benson FE (1987) Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme. J Gen Microbiol 133: 2531-2538. 45. Algosource (2015) http://www.algosource.com/. 46. Belay A (2002) The Potential Application of Spirulina (Arthrospira) as a Nutritional and Therapeutic Supplement in Health Management. The Journal of the American Nutraceutical Association 5. 47. T.Ramanathan RSa (2012) Antioxidant and Radical Scavenging Effect of Blue-Green Alga Spirulina platensis. International Journal of Pharmaceutical & Biological Archives 3: 4. 48. Wikipedia (2015) http://en.wikipedia.org/wiki/Spirulina_(dietary_supplement). 49. Soccio M, Toniato E, Evangelista V, Carluccio M, De Caterina R (2005) Oxidative stress and cardiovascular risk: the role of vascular NAD(P)H oxidase and its genetic variants. European Journal of Clinical Investigation 35: 305-314. 50. Colla LM, Furlong EB, Costa JAV (2007) Antioxidant properties of Spirulina (Arthospira) platensis cultivated under different temperatures and nitrogen regimes. Brazilian Archives of Biology and Technology 50: 161-167. 51. Zhou Z, Liu Z, Liu X (1997) Study on the isolation, purification and antioxidation properties of polysaccharides from Spirulina maxima. Acta Botanica Sinica 39: 77-81. 52. Mittal A, Kumar PV, Banerjee S, Rao AR, Kumar A (1999) Modulatory potential of Spirulina fusiformis on carcinogen metabolizing enzymes in Swiss albino mice. Phytotherapy research : PTR 13: 111- 114. 53. Trushina EN GO, Gadzhieva ZM, Mustafina OK, Pozdniakov AL (2007) The influence of Spirulina and Selen-Spirulina on some indexes of rat's immune status. Article in Russian Vopr Pitan 76: 21-25. 54. Peto R, Doll R, Buckley JD, Sporn MB (1981) Can dietary beta-carotene materially reduce human cancer rates? Nature 290: 201-208. 55. Schwartz J, Shklar G (1987) Regression of experimental hamster cancer by beta carotene and algae extracts. Journal of Oral and Maxillofacial Surgery 45: 510-515. 56. Zhang HQ, Lin AP, Sun Y, Deng YM (2001) Chemo- and radio-protective effects of polysaccharide of Spirulina platensis on hemopoietic system of mice and dogs. Acta pharmacologica Sinica 22: 1121- 1124.

Introduction Chapter I: Arthrospira, a pioneer cyanobacterium for life support on Earth and space 57. Mishima T, Murata J, Toyoshima M, Fujii H, Nakajima M, et al. (1998) Inhibition of tumor invasion and metastasis by calciumspirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue- green alga, Spirulina platensis. Clin Exp Metastasis 16: 541-550. 58. Bhat VB, Madyastha KM (2001) Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: protection against oxidative damage to DNA. Biochem Biophys Res Commun 285: 262-266. 59. Bhat VB, Madyastha KM (2000) C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochem Biophys Res Commun 275: 20-25. 60. Vadiraja BB, Gaikwad NW, Madyastha KM (1998) Hepatoprotective Effect of C-Phycocyanin: Protection for Carbon Tetrachloride andR-(+)-Pulegone-Mediated Hepatotoxicty in Rats. Biochemical and Biophysical Research Communications 249: 428-431. 61. Romay C, Ledón N, González R (1998) Further studies on anti-inflammatory activity of phycocyanin in some animal models of inflammation. Inflamm res 47: 334-338. 62. Qishen P, Baojiang G, Kolman A (1989) Radioprotective effect of extract from Spirulina platensis in mouse bone marrow cells studied by using the micronucleus test. Toxicology Letters 48: 165-169. 63. Karpov LM, Brown, II, Poltavtseva NV, Ershova ON, Karakis SG, et al. (2000) [The postradiation use of vitamin-containing complexes and a phycocyanin extract in a radiation lesion in rats]. Radiatsionnaia biologiia, radioecologiia / Rossiiskaia akademiia nauk 40: 310-314. 64. Subhashini J, Mahipal SV, Reddy MC, Mallikarjuna Reddy M, Rachamallu A, et al. (2004) Molecular mechanisms in C-Phycocyanin induced apoptosis in human chronic myeloid leukemia cell line- K562. Biochemical pharmacology 68: 453-462. 65. Amha Belay PD (2011) http://www.iherb.com/i/info/pic/Spirulina_RadiationA.pdf. 66. Loseva LPD (1993) Spirulina natural sorbent of radionucleides. Research Institute of Radiation Medicine, Minsk, Belarus. : 6th International Congress of Applied Algology, Czech Republic. 67. Hendrickx L, De Wever H, Hermans V, Mastroleo F, Morin N, et al. (2006) Microbial ecology of the closed artificial ecosystem MELiSSA (Micro-Ecological Life Support System Alternative): Reinventing and compartmentalizing the Earth's food and oxygen regeneration system for long-haul space exploration missions. Research in Microbiology 157: 77-86. 68. Farges B, Poughon L, Creuly C, Cornet JF, Dussap CG, et al. (2008) Dynamic aspects and controllability of the MELiSSA project: a bioregenerative system to provide life support in space. Applied biochemistry and biotechnology 151: 686-699. 69. Horneck G, Facius R, Reichert M, Rettberg P, Seboldt W, et al. (2003) Humex, a study on the survivability and adaptation of humans to long-duration exploratory missions, part I: Lunar missions. Advances in Space Research 31: 2389-2401. 70. MERGEAY M. VW, DUBERTRET G., LEFORT-TRAN M., CHIPAUX C. BINOT R. (1988) MELISSA - a microorganisms based model for CELSS development. Proceedings of the 3rd symposium on space thermal control & life support system. Noordwijk, The Netherlands. 71. van Eykelenburg C (1977) On the morphology and ultrastructure of the cell wall of Spirulina platensis. Antonie van Leeuwenhoek 43: 89-99. 72. Janssen PJ, Lambreva MD, Plumere N, Bartolucci C, Antonacci A, et al. (2014) Photosynthesis at the forefront of a sustainable life. Frontiers in chemistry 2: 36.

Introduction Chapter II: Ionizing radiation

Chapter II Ionizing radiation

II.1 Abstract Biological effect begins with the ionization of atoms. The mechanism by which radiation causes damage to human tissue, or any other material, is by ionization of atoms in the material. In this chapter, the different types of radiation and their properties are described. The potential biological effects of ionizing radiation including ROS production, lipid peroxidation, protein oxidation and DNA damage, are discussed.

II.2 Different types of ionising radiation

II.2.1 Ionizing radiation Ionizing radiation is defined, as any type of electromagnetic wave or particle that carries enough energy to ionize or remove electrons from an atom. There are two types of ionizing radiation, both produced by the decay of radioactive elements: electromagnetic radiation (X rays and gamma radiation) and particulate radiation (α particles, β particles (electrons), protons, neutrons, and ions) (Figure II-1).

II.2.2 Electromagnetic waves Gamma or X rays are energetic photons that form part of the electromagnetic spectrum that includes UV, visible light and radio waves and that can penetrate deeply into a cell or tissue. Photons radiation is called gamma rays if produced by a nuclear reaction, subatomic particle, or radioactive decay within the nucleus. The photons are called X rays if they are produced outside the nucleus. Nevertheless, the generic term photon is therefore, used for both descriptions. Electromagnetic waves (X or gamma rays) are radiations that have mainly indirect effects, hence they do not or less produce directly chemical or biological damage themselves but they produce charged particles and secondary electrons after energy absorption in the material [1]. They can generate ions by several types of energy-absorption events (most commonly by the Compton Effect) and ion production is accompanied by the release of energetic electrons. Multiple ions and electrons can be generated in one event. The generated ions can react with other molecules to produce free radicals [1].

Introduction Chapter II: Ionizing radiation

II.2.3 Particulates Particulate radiation includes α particles, β particles (electrons), protons, neutrons, and ions. Nuclei having high atomic number and energy are also called heavy ions or HZE particles [2]. HZE particles are particles with high (H) atomic number (Z) (Z >2, with Z the total number of protons found in a nucleus) and energy (E), and consist of a nucleus with no orbiting electrons, meaning that the charge of the ion is the same as the atomic number of the nucleus [3]. The amount of kinetic energy per nucleon (MeV/nucleon) of the charged-particle, is a function of its mass (atomic number) and velocity [4]. Depending on their velocity, charged particles of different atomic number can thus have similar kinetic energy per nucleon, or the same nucleus can have different kinetic energy per nucleon. The kinetic energy of the particle will determine its penetration depth, its linear energy transfer (LET, in eV/µm), and its biological effects.

Most particulate types of radiation are directly ionizing individual compounds. These particles can undergo strong electrostatic interactions with the electrons of atoms of the medium and can disrupt directly the atomic structure of the target medium generating then chemical and biological effects through their passage. The passage of the charged particles, causes random ionization and excitation processes and intense damage to molecules along their passage in the medium [1].

Figure II-1 Types of ionizing radiation [5]

Introduction Chapter II: Ionizing radiation

II.2.4 Measuring Ionising Radiation The human senses cannot detect radiation or discern whether a material is radioactive. However, a variety of instruments can detect and measure radiation reliably and accurately. The amount of ionising radiation, or 'dose', received by a person is measured in terms of the energy absorbed in the body tissue, and is expressed in gray. One gray (Gy) is one joule deposited per kilogram of mass. Equal exposure to different types of radiation expressed as gray, do not however necessarily produce equal biological effects. One gray of alpha radiation, for example, will have a greater effect than one gray of beta radiation. When we talk about radiation effects, we therefore express the radiation as effective dose, in a unit called the sievert (Sv). The use of the sievert implies that only stochastic effects (such as radiation-induced cancer) are being considered, and to avoid confusion deterministic effects are conventionally compared to values of absorbed dose expressed by the SI unit gray (Gy).

II.2.5 Linear Energy Transfer Normally when ionizing radiation goes through a material, it will lose gradually its energy with various interactions processes along the length of its path. Hence, for a particular absorber, the rate energy loss depends on the energy and the type of radiation as well as the density of the material. The terms “Linear Energy Transfer” refer to the density of energy deposition in a material. This parameter was defined as the average energy deposited per unit length of track of radiation and uses the unit keV/µm. LET depends on the nature of radiation as on material traversed. LET indicates essentially the quality of different types of radiation and their damaging power. The energy deposition along the track of the electromagnetic waves, such as Gamma and X rays, is sparse, which means that some photons can pass through a molecule without depositing any energy or causing any damage. These are called low LET radiations. Charged particles are classified 'densely ionizing radiation' or high LET radiation, because they deposit greater energy along their track compared to X or gamma rays. The energy loss events for high LET radiation are closely condensate and significantly deposited along all parts of the track. The LET varies along the length of track of a charged particle. The charged particles slow down along their deposition of energy. There rate of transferring energy increases as they slow down such that there is a peak of energy deposition at the end of track, called the “Bragg peak”. LET is important feature for the biological effect of a radiation. The biological effect of a radiation depends on its average LET. High LET radiation will be more damaging than low LET (Figure II-2,

Introduction Chapter II: Ionizing radiation

A). Low LET radiation will cause less cell damage due to its “sparsely ionization” relatively to the dimension of biomolecules such the DNA. High LET radiation produces clusters of ions and radicals along their passage through the cell and causes more locally condensated damage [245. Thus ionising particles have a higher biological effectiveness than gamma or X-rays [6]. The relative biological effectiveness (RBE) of a given particle radiation (r) can be defined as: RBE

(r) = DX / Dr, where Dr and DX are the doses of particle radiation and X-radiation (typically of 250 keV), respectively, required for the same biological effect [7]. It is assumed in general, that RBE of a radiation increases with its average LET, up to 100 keV/µm (Figure II-2, B). As such, HZE and neutrons represent the radiation components with the highest relative biological effectiveness [8]. Above 100 keV/µm the RBE value, it starts to decline due energy deposition in excess of what is needed to cause the lethal damage to the cells. This is called the “overkill effect” [9]. In addition to the LET, also the total amount and the rate at which the radiation dose is received, i.e. the total absorbed dose (Gy) and the dose rate (Gy/hour), will determine the biological impact of the radiation.

A B

Figure II-2: Induction of DNA damage and cell death by ionizing radiation. A: High LET radiation with a high density of ionization and excitation along its track, induces a clustered DNA damage site which is defined as multiple lesions within a few nm in a DNA molecule (a). Low LET radiation with sparsely ionisation events along its track creates randomly isolated damage (b). These cases have the same number of lesion but differently distributed over the DNA molecule [10]. (B) The relative biological effectiveness (RBE) of a radiation increases

Introduction Chapter II: Ionizing radiation with its average LET, up to 100 keV/µm. Above 100 keV/µm the RBE value, it starts to decline due energy deposition in excess of what is needed to cause the lethal damage to the cells. This is called the “overkill effect” Kalpana M. Kanal [11].

II.3 Natural ionising radiation on Earth and in Space We are continuously exposed to measurable background radiation from a variety of natural sources, which, on average, is equal to about 1.5–6 mSev/yr (Figure II-3). One component of background radiation is cosmic rays, high-energy particles and γ rays emitted by the sun and other stars, which bombard Earth continuously. Because cosmic rays are partially absorbed by the atmosphere before they reach Earth’s surface, the exposure of people living at sea level (about 0.30 mSev/yr) is significantly less than the exposure of people living at higher altitudes (about 0.5 mSev/yr in Denver, Colorado). Every 4 hours spent in an airplane at greater than 30,000 ft (Feets), adds about 0.01 mSev to a person’s annual radiation exposure.

A second component of background radiation is cosmogenic radiation, produced by the interaction of cosmic rays with gases in the upper atmosphere. When high-energy cosmic rays collide with oxygen and nitrogen atoms, neutrons and protons are released. These, in turn, react with other atoms to produce radioactive isotopes, such as 14C.

The carbon atoms react with oxygen atoms to form CO2, which is eventually washed to Earth’s surface in rain and taken up by plants. Tritium (3H) is also produced in the upper atmosphere and falls to Earth in precipitation. The total radiation dose attributable to 14C is estimated to be 0.01 mSev/yr, while that due to 3H is about 1000 times less.

The third major component of background radiation is terrestrial radiation, which is due to the remnants of radioactive elements that were present on primordial Earth and their decay products. For example, many rocks and minerals in the soil contain small amounts of radioactive isotopes, such as 232Th and 238U, as well as radioactive daughter isotopes, such as 226Ra. The amount of background radiation from these sources is about the same as that from cosmic rays (approximately 0.30 mSev/yr). These isotopes are also found in small amounts in building materials derived from rocks and minerals, which significantly increases the radiation exposure for people who live in brick or concrete-block houses (0.60–1.60 mSev/yr) instead of houses made of wood (0.10–0.20 mSev/yr). Because radon is a dense gas, it tends to accumulate in enclosed spaces such as

Introduction Chapter II: Ionizing radiation basements, especially in locations where the soil contains greater-than-average amounts of naturally occurring uranium minerals. Under most conditions, radioactive decay of radon poses no problems because of the very short range of the emitted α particle [12].

Figure II-3: The average radiation dose from natural sources for an adult in the United States is about 1.5-6 mSev/yr. Radon accounts for more than half of an adult’s total radiation exposure, whereas background radiation (terrestrial and cosmogenic) and exposure from medical sources account for about 15% each [12] Earth is also continuously irradiated from all directions by galactic cosmic rays (GCR) and radiation from solar particle events (SPE).

Introduction Chapter II: Ionizing radiation

Figure II-4: Sources of ionizing radiation in interplanetary space. The galactic cosmic rays are high-energy charged particles that originate from sources in the cosmos, beyond our solar system. They are composed of 98% ions and 2% electrons and positrons. The ions are composed of 87 % protons, 12% Helium ions and the remaining 1% represents heavier ions (Baryons). 2He2+ nuclei, or alpha particles, make up 14% of the cosmic radiation [13]. HZE are only a minor fraction (<1%) of GCRs, but Iron nuclei are relatively plentiful compared to the other high Z ions [14]. The energies of GCR particles range from from 1 MeV/nucleon to more than 103 GeV/nucleon.

GCR particles lose kinetic energy principally by ejecting orbital electrons from the atoms with which they interact, and thus cause severe damage to the material they pass.

Solar protons events correspond to fluxes of high-energy protons from the sun, which vary greatly in time, and can reach up to energetic radiation of 1 GeV.

Most of the charged particles from the cosmos and from the sun, however, never reach the Earth surface, but remain trapped in the geomagnetic field of the Earth and make the Earth radiation belts, the Van Allen belts. The population of charged particles stably trapped by the Earth's magnetic field consists mainly of electrons with energies between 50 keV and 5 MeV and protons with energies between 100 keV and several hundred MeV, and some low energy heavy ions [15]. The Earth's trapped particle radiation belts were discovered at the beginning of the space age and were immediately recognised as a considerable hazard to space missions.

Introduction Chapter II: Ionizing radiation

Astronauts and cosmonauts aboard a spacecraft in low Earth orbit (LEO), such the NASA Space shuttle, the Russian soyuz module and ISS, or aboard spacecraft travelling outside the Earth orbit on missions to and from Moon or Mars, are continuously exposed to higher levels of ionizing radiation; i.e. fluxes of complex radiations of variable energies and intensities. There are three major sources of primary ionizing radiation in space: (1) energetic electrons and protons which are trapped in the Earth geomagnetic field, (2) galactic cosmic rays, and (3) solar particle events which generate high fluxes of charged particles. It has been suggested that half the dose on-board ISS in LEO is expected to come from trapped protons and half from galactic cosmic rays [14].

Thus, the type of radiation the living organisms are exposed to in space during space travel is very different from the natural radiation experienced on Earth. Although the HZE particles contribute in number only as a small proportion to cosmic rays and thus the radiation exposure in space, their high charge and high energies contribute significantly to the overall biological impact of cosmic rays in space [16]. 56Fe26+ nuclei possess high LET and are highly penetrating, and therefore considered as the most dangerous heavy ionized nuclei components of cosmic radiation [17] [14]. HZE particles are able to traverse the human body and several meters of dense solid shielding, depending on their energy.

II.4 Biological effects of ionizing radiation The biological effects of ionising radiation are induced though either direct energy absorption by the biological molecules (such lipids, proteins and nucleic acids) or indirectly via interactions of those molecules with radiation induced radicals produced by water radiolysis.

In general, the biological impact of radiation can be summarized as follow:

 The radiation will cause ionization and excitation of electrons in atoms and molecules s leading to the production of free radicals in the cell.  The free radicals will cause breakage of chemical bonds and production of new chemical bonds and cross-linkage between macromolecules.  The molecules that regulate vital cell processes (e.g. lipids, proteins, RNA and DNA) will be damaged.  The cells will try to repair the damage, but if damage is beyond repair the cell will dye.

Introduction Chapter II: Ionizing radiation

II.4.1 Radiation-induced production of reactive oxygen species (ROS) VIS radiation and the photosynthesis process in phototrophic bacteria produces dioxygen gas from water, which will be dissolved in the water. But the photosynthesis process generates also singlet 1 oxygen ( O2) (also written as O2*). Singlet oxygen is generated by the photosensitizer pigments of the PSII and PSI reaction centres, i.e. the light-harvesting chlorophyll molecules. Singlet oxygen is an electronically excited state of molecular oxygen (O2), which is not as stable as the normal triplet oxygen, but highly reactive and a strong oxidant [18].

In active respiratory cells, photrophic or heterotrophic, dissolved oxygen (O2) is an important natural source of ROS in the cells. Dissolved O2, either derived from the atmosphere or generated –aq – endogenously (e.g. by photosynthesis), reacts with e to form superoxide anion (O2• ) (Equation

1) [18], which can reform to hydroperoxyl radicals (HO2•) and finally hydrogen peroxide (H2O2) + – in the presence of protons (H ) (Equations 2 and 3). The superoxide anions (O2• ), hydroperoxyl radicals (HO2•) and hydrogen peroxide (H2O2) produced from oxygen are relatively inert and can be neutralised enzymatically by the cell. Ionising radiation, however, strongly induces additional ROS production. Radiation interacts with matter primarily through ionization and excitation of electrons in atoms and molecules, which may convert intro free radicals in pico to femto seconds after physical interaction with atoms (10-13 to -15 10 s). Water (H2O) is the most abundant chemical found in living cells. The primary ROS which arise during the radiolysis of H2O, is the hydroxyl radical (•OH) (Equation 4) [19]. These hydroxyl radicals (•OH) produced from water radiolysis are extremely oxidizing and damaging, and cannot be easily neutralised by the cell. Hydroxyl radicals (•OH) will indiscriminately react with any organic molecule and oxidize lipids, proteins, RNA and DNA [18,19]. However, the short lifetime of the •OH precludes damage to molecules beyond a few Angstroms from where •OH is formed.

The hydroxyl radicals can react with each other to generate hydrogen peroxide (H2O2) (Equation 5) [19], which can diffuse throughout the cell [18,19]. The hydroxyl radicals (•OH) can also react – with hydrogen peroxide (H2O2), and produce superoxide anions (O2• ) (Equation 6). A secondary source of hydroxyl radicals (•OH) in cells during irradiation, are the Haber Weiss and Fenton chain reactions, which are the most powerful oxidizing reactions known. It involves the – catalytic decomposition of H2O2, to a hydroxyl radical (•OH) and a hydroxide ion (OH ), by ferrous ions (Equation 7) [20]. This is referred to as the Fenton’s reaction. The Fe ions can be recycled, via - 3+ a reduction with superoxide anion (O2• ) which can donate an electron to iron (Fe ) to yield again

Introduction Chapter II: Ionizing radiation

2+ a reduced state of iron (Fe ) (Equation 8), which then can reduce again H2O2 and start the reaction again. It is thus a cyclic chain reaction, which is difficult to stop. The total net reaction through - – which H2O2, O2• and iron rapidly generate hydroxyl radicals (•OH) and hydroxide ion (OH ) is called the Haber Weiss reaction (Equation 9). The cells will need a large pool of strong antioxidants to neutralise the produced hydroxyl radicals (•OH) and hydroxide ions (OH–) (Equations 10 and 11). –aq – 1) O2+ e → O2• (superoxide anion) – + 2) O2• + H → HO2• (hydroperoxyl radical) + 3) HO2• + H → H2O2 (hydrogen peroxide) + –aq 4) H2O → •OH (hydroxyl radical) + H (proton) + e (hydrated electron)

5) 2 •OH → H2O2 (hydrogen peroxide) – + 6) •OH + H2O2 → O2• (superoxide anion) + H2O + H 2+ – 3+ 7) H2O2 + Fe → •OH (hydroxyl radical) + OH (hydroxide ion) + Fe − 3+ 2+ 8) O2• + Fe → O2 + Fe − − 9) H2O2 + O2• → •OH + OH + O2

10) •OH + HO2•→ H2O + O2 – + 11) OH + H → H2O

II.4.2 Lipid peroxidation The cell membrane, which is composed of polyunsaturated fatty acids, is a primary target for reactive oxygen attack [21]. Reactive oxygen species (ROS) in the cell can cause lipid peroxidation. Lipid peroxidation can be defined as the oxidative deterioration of lipids that contain carbon– carbon double bonds. This will lead to the formation of lipid hydro-peroxides, via enzymatic or non-enzymatic reactions that are mediated by ROS, which are responsible for the destruction and damage of cell membranes [22]. Lipid hydro-peroxides are non-radical oxidised lipids that are derived from unsaturated fatty acids, phospholipids, glycolipids, and cholesterol esters. The non- enzymatic formation of lipid hydro-peroxides is initiated by the presence of molecular oxygen and is facilitated by Fe2+ ions [23] The enzymatic formation of lipid hydro-peroxides occur in the presence of lipo-oxygenases, a family of lipid peroxidation enzymes that oxygenate free and esterified polyunsaturated fatty acid (PUFA) generating as consequence peroxy-radicals.

Introduction Chapter II: Ionizing radiation

Figure II-5:Mechanism of lipid peroxidation [24]. II.4.3 Protein oxidation In addition, cellular proteins are important targets of damaging radiation. Exposure of proteins to reactive oxygen may cause fragmentation, denaturation, crosslinking, aggregation, and loss of function. Ionizing radiation can generate a wide range of protein damages due to oxidative stress, such as amino acid modifications, carbonyl group formation, formation of protein-protein crosslinks, and formation of S–S bridges [25].

Carbonylation is an irreversible oxidative process and one of major causes of radiation-induced protein damage. Carbonylation is the direct oxidation of the side chains of lysine, arginine, proline, and threonine amino acids residues, and can also be formed by secondary reactions with reactive carbonyl compounds on carbohydrates (glycoxidation products), lipids, and advanced glycation/lipoxidation end product [26]. Protein carbonylation is indeed widely used as marker of protein oxidation [27] .

II.5 DNA damage IR-induced ROS will also have a negative impact on DNA. In addition to the widespread ‘indirect DNA damage’ caused in cells by IR-induced ROS, IR also can inflict ‘direct DNA damage’ when the macromolecules absorb the energy from X-ray and γ-ray photons, or ionising particles [19]. Ionizing radiation generates multiple types of DNA damage: (i) base damage, (ii) DNA single strand breaks (SSBs), (iii) DNA double strand breaks (DSBs), and (iiii) interstrand cross-links. The

Introduction Chapter II: Ionizing radiation

DNA bases are the most affected with more than 80 different types of structural modifications induced by ionizing radiations [28]. The DNA single strand breaks (SSBs) are usually quickly repaired and most often safely repairable. But as the dose of ionizing radiation (IR) increases, the linear density of base damages and single strand breaks (SSBs) increases on both strands, which gives rise to double-strand breaks (DSBs). On average for every 20 SSBs induced by gamma rays in DNA there is 1 DSB [29]. Other sources list that a dose of gamma rays IR typically causes 40 times more SSBs than DSBs [19,30]. If not repaired, the DSBs prevent the replication of genomes and lead to cell death.

II.6 References 1. IAEA (2010) Radiation Biology: A Handbook for Teachers and Students. 2. Hassler DM, Zeitlin C, Wimmer-Schweingruber RF, Ehresmann B, Rafkin S, et al. (2014) Mars' surface radiation environment measured with the Mars Science Laboratory's Curiosity rover. Science 343: 1244797. 3. Schwank JR, Shaneyfelt MR, Dodd PE (2013) Radiation Hardness Assurance Testing of Microelectronic Devices and Integrated Circuits: Test Guideline for Proton and Heavy Ion Single-Event Effects. Ieee T Nucl Sci 60: 2101-2118. 4. Horneck G, Klaus DM, Mancinelli RL (2010) Space microbiology. Microbiology and molecular biology reviews : MMBR 74: 121-156. 5. MIRION (2015) https://www.mirion.com/introduction-to-radiation-safety/types-of-ionizing-radiation/. 6. Urushibara A, Shikazono N, Watanabe R, Fujii K, O'Neill P, et al. (2006) DNA damage induced by the direct effect of He ion particles. Radiation protection dosimetry 122: 163-165. 7. Protection ICoR (2003) Relative Biological Effectiveness, Radiation Weighting and Quality Factor. 8. Hastings D, Garrett H (2004) Spacecraft-Environment Interactions Series CAaSS, editor. 9. Hall EJ, Hei TK (2003) Genomic instability and bystander effects induced by high-LET radiation. Oncogene 22: 7034-7042. 10. JAEA (2007) Dependence of Yield of DNA Damage Refractory to Enzymatic Repair on Ionization & Excitation Density of Radiation. 2188-1456. 11. Kalpana M. Kanal P, DABR (2010) Biological Effects of Ionizing Radiation How are LET and RBE Related ? Radiation Biology pp. 15. 12. Bruce A. Averill PE (2012) Principles of General Chemistry. 13. Hellweg CE, Baumstark-Khan C (2007) Getting ready for the manned mission to Mars: the astronauts' risk from space radiation. Die Naturwissenschaften 94: 517-526. 14. Benton ER, Benton EV (2001) Space radiation dosimetry in low-Earth orbit and beyond. Nuclear instruments & methods in physics research Section B, Beam interactions with materials and atoms 184: 255-294. 15. Abel B, Thorne RM, Vampola AL (1994) Solar cyclic behavior of trapped energetic electrons in Earth's inner radiation belt. Journal of Geophysical Research 99: 19427. 16. Dartnell LR (2011) Ionizing radiation and life. Astrobiology 11: 551-582. 17. Saenger EL (2000) The National Council on Radiation Protection and Measurements: problems and prospects. AJR American journal of roentgenology 175: 1509-1511. 18. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. (2007) Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS Biol 5: e92. 19. Armstrong WD (1958) The chemical basis of radiation damage. Postgraduate medicine 23: 499-507.

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20. Imlay JA (2008) Cellular defenses against superoxide and hydrogen peroxide. Annual review of biochemistry 77: 755-776. 21. Chance B, Sies H, Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiological reviews 59: 527-605. 22. Marnett LJ (1999) Lipid peroxidation-DNA damage by malondialdehyde. Mutation research 424: 83- 95. 23. Repetto MG, Ferrarotti NF, Boveris A (2010) The involvement of transition metal ions on iron- dependent lipid peroxidation. Archives of toxicology 84: 255-262. 24. Young IS, McEneny J (2001) Lipoprotein oxidation and atherosclerosis. Biochemical Society transactions 29: 358-362. 25. Matallana-Surget S, Wattiez R (2013) Impact of Solar Radiation on Gene Expression in Bacteria. Proteome Sci. 26. Requena JR, Levine RL, Stadtman ER (2003) Recent advances in the analysis of oxidized proteins. Amino acids 25: 221-226. 27. Moller IM, Jensen PE, Hansson A (2007) Oxidative modifications to cellular components in plants. Annual review of plant biology 58: 459-481. 28. Bjelland S, Seeberg E (2003) Mutagenicity, toxicity and repair of DNA base damage induced by oxidation. Mutation research 531: 37-80. 29. Krisch RE, Flick MB, Trumbore CN (1991) Radiation chemical mechanisms of single- and double- strand break formation in irradiated SV40 DNA. Radiation research 126: 251-259. 30. Daly MJ (2009) A new perspective on radiation resistance based on Deinococcus radiodurans. Nature reviews Microbiology 7: 237-245.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Chapter III Susceptibility of cyanobacteria to ionising radiation

III.1 Abstract The Susceptibility of bacteria in general to ionizing radiation and cyanobacteria in specific has gained a great interest last decades. The response of cyanobacteria to UV radiation has been well documented. Here in this chapter we tried to report the main response mechanisms of cyanobacteria to UV, X and Gamma radiation, and the impact of particle radiation on cyanobacteria.

III.1.1 Introduction Cyanobacteria are a primitive group of photoautotrophic bacteria that appeared on Earth at least 2.5 billion years ago and that have and still contribute markedly to the release of oxygen (O2), and global carbon dioxide (CO2) and nitrogen (N2) fixation on Earth [1]. Cyanobacteria have withstood the challenges of large evolutionary environmental changes. They are one of the largest and most versatile groups of bacteria. They can be found almost in any environment, including extreme ones such as cold and hot deserts, Antarctic dry valleys, and tropical rain forests [2]. Obviously, cyanobacteria have developed different strategies to protect themselves, allowing them to survive and proliferate under challenging conditions, including habitats exposed to high light intensities and ionising radiation.

III.2 Cyanobacteria and electromagnetic waves Oxygenic photosynthetic organisms such cyanobacteria need sufficient light of a certain spectrum and certain intensity to drive photosynthesis.

In general, they use visible (VIS) light, i.e. photons with a wavelength between 400-700 nm, for photosynthesis (Figure III-1). The light or radiation within this region of the electromagnetic spectrum provides enough energy (ca 1.8 eV) to split water and is referred to as Photosynthetically Active Radiation (PAR). UV photons (3.5 eV energy), X rays (200 KeV energy) and Gamma rays (e.g: from 60Co) (1.17 MeV & 1.33 MeV energy) are highly energetic photons, containing much greater amount of energy than visible light (VIS) (Figure III-1), and cannot be used efficiently for photosynthesis.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Figure III-1: Electromagnetic Spectrum [3]

III.3 Management of oxidative stress and radiation damage in cyanobacteria The normal photosynthesis process, that occurs with normal fluxes of visible light, generates 1 inevitably reactive oxygen species (ROS) including the singlet oxygen ( O2), the superoxide anion - (O2• ), hydrogen peroxide (H2O2) and the hydroxyl radical (OH•), which are causing oxidative stress and damage in the cell [4].

High photon fluxes or high energetic photons cause excessive heating and ROS generation and damage to the cells. High fluxes of VIS light or UV radiation cause little damage, which can be repaired. X- and Gamma rays are penetrating radiation and cause much more severe damage [5].

Thus, the natural phototrophic nature of cyanobacteria requires not only the management of light energy harvesting but also the management of oxidative stress continuously produced by normal photosynthetic electron transport. And the cells require additional mechanisms to prevent and mitigation of the damage caused by excessive radiation, e.g. by UV, X and gamma radiation.

Cyanobacteria have developed numerous strategies in order to manage the oxidative stress and radiation damage within the cells, i.e. by (i) Avoidance of cell exposure to radiation by migration and morphological changes,

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation (ii) Protecting the cells by radiation-absorbing molecules, (iii) Protecting and repairing the PSII by energy dissipation and photo-inhibition, (iv) Protecting the membranes and enzymes by ROS-scavenging antioxidants, and (v) Protecting and repairing the DNA from radiation-induced damage

The cells can activate some or all of these systems depending on the type and severity of oxidative stress and radiation damage experienced. Different types of radiation may induce different responses.

III.4 Tolerance and response of cyanobacteria to intense VIS and UV radiation During the early Proterozoic era, when there was no oxygen in the atmosphere, the aqueous environment of the ancient Earth was exposed to high doses of UV-radiation. Today, however, solar UVR is only a very small proportion of the total irradiance on the Earth’s surface, as most of it is absorbed by the Earth’s stratospheric ozone layer (Figure III-2). UVA (315–400 nm) comprises the largest portion of the total solar UV radiation (<7%) reaching the Earth. Most of the extra- terrestrial UVB (280–315 nm) (<1%) and all of the UVC (~180–280 nm) is practically absent (0%) [6].

Figure III-2: Levels of ozone at various altitudes, blocking of different types of ultraviolet radiation. Essentially all UVC (100–280 nm) is blocked by di-oxygen (from 100–200 nm) or else

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation by ozone (200–280 nm) in the atmosphere, while fraction of UVB (280–315 nm) and most of UVA (315–400 nm) will reach the surface of the Earth [7].

Although UVB radiation accounts for less than 1% of the total energy reaching the Earth’s surface, it is a highly active component of the solar spectrum and can have significant effects on the biota [8]. It has been well documented that UVB radiation has a great negative effect on various physiological and biochemical processes in cyanobacteria. It can cause for example impairment of photosynthesis [9], loss of pigmentation [10], delay in growth [11], and imbalance of nitrogen metabolism [12], and loss of motility [13]. The exposure of Arthrospira platensis to UVB and UVA resulted in significant inhibition of the effective quantum yield and growth, although high levels of PAR caused most of the inhibition [14]. Arthrospira sp. strains 439 and D-0083 exposed to UVB radiation showed a gradual loss of the oxygen-evolving activity to about 56% after 180 min exposure [15]. A significant decrease in the number of motile filaments of Anabeana variabilis, Oscillatoria tenuis and Phomidium uncinatum, was observed within 10-30 min of UVR exposure [13]. UVB radiation can also change the motility and alter the morphology of filamentous cyanobacterium as Arthrospira platensis [16]. For UVA, which is more abundant but less damaging, mainly indirect effects via the energy transfer from UVA-stimulated chromophores to the DNA target, or via the production of ROS by the photosensitized chlorophyll and phycobilins, were reported [9]. In general, however, cyanobacteria have been reported to be quite resistant to UVR. The cyanobacterium Chrococcidiopsis for example survived 548 days of exposure to UVR in low Earth orbit [17] and can even withstand a few minutes of unattenuated Martian UV Flux [18]. Moreover, it has been reported that the cyanobacterium Chrococcidiopsis can endure UVC doses as high as 13 W/m2 [19]. Cyanobacteria were probably already challenged by ultraviolet radiation (UVR) on the Early Earth without an ozone layer [20], and they thus had to evolve mechanisms to cope and live with this stress. It is clear that cyanobacteria have indeed developed multiple mechanisms to counteract UV damage. These strategies include multiple defence lines (Figure III-3), such as:

(vi) Avoidance of cell exposure to radiation by migration and morphological changes, (vii) Protecting the cells by radiation-absorbing molecules, (viii) Protecting and repairing the PSII by energy dissipation and photo-inhibition,

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation (ix) Protecting the membranes and enzymes by ROS-scavenging antioxidants, and (x) Protecting and repairing the DNA from radiation-induced damage.

Figure III-3: Model showing the effects of UVR and mitigation strategies employed by cyanobacteria [21]

III.4.1 Avoidance by migration and morphological changes The avoidance mechanism is the first line of defence of cyanobacteria against UVR, which involves

(i) migration form high to low UVR levels, (ii) changing their morphology called self-shading, (iii) synthesis of extracellular polysaccharides, and (iv) formation of mats [22].

It has been reported, that Spirulina subsalsa and Ocillatoria laetevirens have the ability to move up or downwards in the hyper-saline mats of Guerrero Negro to protect their photosynthetic machinery from high UVB and UVA exposure [23]. Cyanobacteria actually possess different systems for motility. The presence of gas vacuoles, grants a buoancy property for many

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation cyanobacteria, including Arthrospira, allowing vertical movement. This property allows the cyanobacteria to migrate up and down in the water or on wetted surfaces, in attempt to find the optimal light exposure areas. Many of the filamentous cyanbacterial species are also motile via a gliding mechanism, allowing gliding along their long axis. Arthrospira for example shows vigorous gliding motility of filamentous cells (trichomes) with rotation along their long axis under the microscope. The gliding aspect appears to be powered by a “slime jet” mechanism, in which the cells extrude a gel that expands quickly as it hydrates providing a propulsion force [24]. Many of such gliding cyanobacteria display also photomovement, by which a trichome modulates its gliding according to the incident light. The latter has been found to play an important role in guiding the trichomes to optimal lighting conditions, which can either inhibit the cells if the incident light is too weak, or damage the cells if too strong. The cyanobacteria Arthospira platensis has the capability to change its cellular and filament morphology in response to the environmental conditions [16]. It has been reported that the shape of Arthospira platensis was modified from a loose helix shape to a tight compacted helix in response to UVR as a way for self-shading [16]. And it was shown that self-shading by compression of the spirals contributes significantly in protection of the Arthrospira platensis filaments against deleterious UVR [16].

One of the most common protective mechanisms within the cyanobacteria is their ability to synthesize external polysaccharides (EPS) layers, a sheath, which protects the cells from adverse environmental conditions. Many studies have shown that a coating of extracellular polysaccharides material can protect bacteria against dehydration, phagocytosis, antibody recognition, and even lysis by viruses [25,26]. It has been proposed that these polysaccharides can form hydrogen bonds with proteins, lipids and DNA, thus replacing the water shell that usually surrounds these macromolecules [27]. The exopolysaccharides, owing hydrophilic/hydrophobic characteristics, are able to trap and accumulate water and to create a gelatinous layer around the cells, thus regulating water uptake and loss and stabilizing the cell membrane during periods of desiccation [28]. A number of cyanobacteria are capable of surviving nearly without water, producing both internal and external polysaccharides, which help them to stabilize the macromolecular constituents of the cell, as well as the cellular structure. Upon rehydration, cyanobacteria can rapidly recover metabolic activities and repair cellular components [29]. But, such EPS capsules and slime protect

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation the cyanobacterial cells also from the harmful effects of UV radiations. It was reported that high levels of UVB radiation (1.0 W/m2) increase the synthesis of exopolysaccharides and induce the formation of a sheath surrounding the filaments of Nostoc commune [10]. In addition, some cyanobacteria use their motility and EPS production capacity to from aggregated and mats to protect from UV radiation. The endurance of Chrococcidiopsis to UVC doses as high as 13 Wm-2, for example, is partially ascribed to their multicellular aggregates [19]. Benthic cyanobacteria tend to make mat formations attached to surfaces, whereas planktonic cyanobacteria sometimes produce floating aggregates or mats.

III.4.2 Protection by UV-absorbing molecules The synthesis of UV-absorbing compounds, that serve as passive protective mechanism, is widespread in microorganisms, plants and animals [30]. The accumulation of UV-absorbing compounds also plays a vital role in the protection of cyanobacteria against the deleterious effects of UV irradiation [31]. Cyanobacteria can produce naphthalene-based melanins, sytonemin, and mycosporine as UV absorbing/screening compounds to provide photo-protection against UVA and/or UVB radiation [32]. Scytonemin is a yellow–brown, lipid-soluble dimeric pigment with a molecular mass of 544 Da. Its structure is based on indolic and phenolic subunits, with an in vivo absorption maximum at 370 nm [33]. A purified scytonemin showed a maximum absorption for UVA at 386 nm, nevertheless, this compound could also absorb significantly UVB at 252, 278, and 300 nm. It is deposited in extracellular cyanobacterial sheaths, and absorbs the UVA irradiation before it reaches the cell. The accumulation of scytonemin in the extracellular sheath minimizes 90% of UVA penetration into the cell and decreases the damage to biological targets [34,35]. Scytonemin can protect also cyanobacteria when other ultraviolet-protective mechanisms such as active repair of damaged cellular components are ineffective. Scytonemin was proposed to be synthesized from secondary metabolites of aromatic amino acid biosynthesis by condensation of tryptophan and phenylpropanoid-derived subunits [33]. The synthesis of scytonemin is induced by UVA radiation whereas blue, green, or red VIS light, even at the same fluency rates, does not have significant influence [34]. The observation that scytonemin synthesis was induced by UVA irradiation, but not with UVB, is in agreement with its photoprotective role against UVA [36]. Accumulation of scytonemin occurred for example in the terrestrial cyanobacterium Chlorogleopsis sp. to protect

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation the photosynthetic machinery from UVA damage [36]. In the model cyanobacterium Nostoc punctiforme ATCC 29133, which was used to study UVA response by analysing global gene expression patterns, 27 of the up-regulated genes encodes proteins associated with secondary metabolism, including those involved in the biosynthesis of the UVA sunscreen scytonemin. It has been reported that also repeated desiccation activates scytonemin synthesis under UVA exposure [37]. There is no evidence for the biosynthesis of scytonemin compounds by members of the genus Arthrospira.

Figure III-4: Aspects of scytonemin biology. a | Chemical structure of scytonemin. b | Comparison of absorption spectra for pure scytonemin in ethyl acetate (blue), wild-type, whole-cell, scytonemin-containing Nostoc punctiforme (red) and scytonemin-null mutant N. punctiforme (green).C | Image of two filaments of Stigonema spp. The left bacteria filaments do not contain scytonemin in their extracellular sheaths, whereas the bacteria on the right do. [32]

Mycosporines are small (<400 Da) colourless water-soluble molecules, having absorption maxima for UVA between 310 and 365 nm, and composed of either a cyclohexenone or cyclohexenimine chromophore carrying nitrogen or imino-alcohol substituents. When substituted with amino acids residues, they are called Mycosporine-like Amino Acids (MAAs), which can have altered absorption properties, allowing also absorption of UVB [38]. Anabaena strains have been characterized to produce several MAAs in response to UVR [39,40]. The MAA shinorine is synthesized in Anabaena variabilis PCC 7937 under various abiotic stressors with and without UVR [41]. The biosynthesis of three MAAs, i.e. mycosporine-glycine, porphyra-334, and shinorine, was reported in Anabaena doliolum [42]. Induction of the MAA shinorine in response to UVB has also been shown for three heterocystous N2-fixing cyanobacteria

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation [43], and in the cyanobacterium Chlorogleopsis PCC 6912 [44]. It has been reported also that Trichodesmium and some other marine and freshwater planktonic cyanobacteria are known to accumulate large quantities of MAAs (mycosporine-like amino acids) that are intracellular UVR absorbers. It was long assumed that the planktonic cyanobacterium Arthrospira did not accumulate UV sunscreen compounds to counteract the deleterious UV radiation, in contrast to many other cyanobacteria. However, the ability of Arthrospira sp. CU2556 to synthetize MAA mycosporine- glycine (M-Gly) under UVB exposure was reported recently [45]. In all but one of the cyanobacteria examined, Nostoc, these MAA compounds are located in the cytoplasm and could serve as alternative targets for about 10–30% of UVB photons penetrating a cell [46]. In terrestrial Nostoc commune, however, MAAs are bound to oligosaccharides in the inner external glycan sheath (EPS) [10]. There is clear evidence that the presence of MAA protects vital functions in phytoplankton from deleterious short wavelength irradiation. MAAs protects the cells by absorbing highly energetic UVR and then dissipating excess energy in the form of heat to their surrounding [47]. The synthesis of MAAs in cyanobacteria is dependent on the availability of nitrogen and growth media [40]. Various factors stimulate MAA synthesis such as seasonal effect, stage of development and exposure to UV rays [48]. MAA synthesis in cyanobacteria is enhanced by photo-synthetically active radiation (PAR, 400-700nm), UVA and UVB radiation. It has been reported that the enhancement with UVB light was the highest of the three light regimes [22]. MAA biosynthesis can be activated in an indirect manner via ROS accumulation as a primary event caused by UVB irradiation.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Figure III-5: Structural and spectral properties of Mycosporines, UV absorbing pigments. [32]

Some species of cyanobacteria, however, do not synthesize and accumulate UV-absorbing compounds like scytonemin or MAAs on or in their cells [49]. But in cyanobacteria, the apoproteins of the light-harvesting complex of photosystem II (PSII), which are complexed with chlorophyll and carotenoids like xanthophylls, can also serve as an UV-antenna complex, absorbing more than 99% of UVB [50]. Cyanobacteria possess a wide variety of carotenoids including for instance myxoxantophyll, β-carotene and its derivate zeaxanthine, and echinenone [51]. Carotenoids absorption maxima occur mainly in the visible spectrum (400-700 nm) but often extend into the UVA region (<400 nm), while some have even a small absorption peak in the UVB region [10,52]. The cyanobacterium Nostoc commune changes its carotenoids content in response to UVB irradiation in favour of myxoxantophyll and echinenone, which act as outer membrane bound UVB photoreceptors [10]. Zeaxanthin has been shown to be particularly important for photo-acclimation during UVB stress in Synechococcus sp. PCC 7942 [53].

III.4.3 Protection and repair of PSII One mechanism to protect PSII from radiation damage is energy dissipation in the form of heat, which is considered as preventive or photo-protective mechanism against intense light [54]. In plants, it is done by non-photochemical quenching (NPQ) of the excitation energy via the light- harvesting complex of PSII (LHCII), containing carotenoid pigments. This process is triggered by

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation the acidification of the thylakoid lumen resulting in the modification of the carotenoid violaxantin to zeaxantine, leading to better energy dissipation. Since cyanobacteria lack the plant-like LHCII and the violaxantin pigment, it has been assumed that cyanobacteria lack this photo-protective mechanism. However, recently it has been reported that cyanobacteria use 3 different systems for energy dissipation and photo-protection : the carotenoids [55], the high light inducible protein (Hli) [56], and the chlorophyll-binding (CP43’) protein [57]. If the photon radiation is too intense, it will damage the light harvesting complexes of PSII, causing photo-inhibition. The photo-inhibition process is a light dependent process, and is typically induced by strong blue VIS and UV light. Photo-inhibition causes inactivation of the photosystem (Figure III-6-A). The inactivation of the photosystem occurs in two steps: firstly, the damage of the oxygen-evolving complex, and secondly the degradation and the turnover of D1 protein (Figure III-6-A). The D1 protein is the primary target of the damage, and it is sacrificed in order to avoid complete inactivation and disassembly of PSII [58]. Intense VIS and UV radiation has direct negative effects on the key reaction centre proteins, D1 and D2, which are degraded by irradiation [59]. In addition to the direct damage caused, D1/D2 proteins may also be destroyed indirectly by the reactive oxygen species produced by high intensity blue/violet light [60]. In contrast to photodamage by visible light, where mainly the D1 subunit is damaged and repaired, UVB radiation damages the D1 and D2 proteins to almost the same extent [60]. To avoid permanent inhibition of PSII function after the loss of the D1/D2 heterodimers, a tightly regulated repair process occurs in the thylakoid membranes of cyanobacteria to replace the damaged proteins with new, fully functional copies [61](Figure III-6-B). The FtsH protease plays a key role in the cleavage of damaged D1 protein during the reparation of PSII [62]. The repair process after UV-damage includes de novo synthesis of both subunits, D1and D2 [60].

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Figure III-6: A: Photo-inhibition of Photosystem II (PSII) leads to loss of PSII electron transfer activity. PSII is continuously repaired via degradation and synthesis of the D1 protein. Lincomycin can be used to block protein synthesis [63]. B: Scheme depicting the major regulatory steps of psbA gene (encoding D1 protein) regulation in cyanobacteria[58].

The rapid de novo synthesis of D1/D2 proteins is an essential part of the PSII repair process. An increased turnover of D1 and D2 proteins of PSII reaction center was found to be responsible for UVB resistance in the cyanobacteria Synechosystis [60]. The high turnover of the D1 protein is also an important defense mechanism to counteract the UVB induced damage of PSII in A. platensis [15]. Using the Synechococcus PCC 7942, it has been demonstrated that also an ATP- dependent Clp protease is essential for the acclimation to UVB [64]. The activity of this Clp protease results in the rise of a modified D1 PSII protein more resistant to UVB stress [64].

III.4.4 Protection of lipids and enzymes via antioxidants for ROS scavenging Once produced, the accumulation of ROS can be prevented via antioxidants which actively scavenge and neutralize the ROS. Cyanobacteria use a wide variety of enzymatic and non- enzymatic antioxidant systems. − Cyanobacteria possess diverse cellular enzymes to neutralise (i) superoxide radicals (O2• ) and hydrogen peroxide (H2O2) formed in the photosynthetic electron chain in the presence of dissolved oxygen, and (ii) hydroperoxyl radicals (HO2•) produced by the photo- and radiolysis of water. These enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), peroxiredoxins (PRX) (i.e. alkylhydroperoxidase (AHP), and other peroxidases), glutaredoxins (GRX), thioredoxins (TRX), and enzymes involved in alpha-tocopherol cycle, the ascorbate cycle

53 Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), or the glutathione cycle such as the glutathione peroxidase (GPX), and glutathione reductase (GR) [51]. − Superoxide dismutase (SOD) scavenges superoxide radicals (O2• ) and converts them to hydrogen peroxide (H2O2) which is further converted to water and O2 via a combined catalase-peroxide system [65]. SODs are ubiquitous metallo-enzymes that exist in four forms depending in their catalytic metals: FeSOD, MnSOD, Cu/ZnSOD and NiSOD [66]. Comparative genomic analysis revealed that NiSOD is the only SOD found in primitive cyanobacteria. FeSOD and MnSOD are found in the higher orders of cyanobacteria, while Cu/ZnSOD is rare in cyanobacteria. Arthrospira sp. PCC 8005 possesses only 1 FeSOD (Sod). Although most cyanobacteria contain catalase genes, they are absent in members of the Arthrospira genus (Figure III-7).

Figure III-7:Antioxidant enzymes involved in superoxide detoxification[67].

Peroxiredoxins (PRX), glutaredoxins (GRX) and thioredoxins (TRX) are thiol-specific antioxidant enzymes that possess an active centre disulfide bond that function as electron carrier. They exists in either a reduced form with 2-SH groups or an oxidized form where the two cysteine residues are linked in an intramolecular disulfide bond -SS-. Peroxiredoxins (PRX) catalyse the reduction of hydrogen peroxide, alkyl hydroperoxides and peroxynitrite, using other thiol-containing reducing agents (such as gluthation, glutaredoxin or thioredoxin) as electron donors [68]. Glutaredoxins (GRX) are small enzymes (ca. 100 AA) that can reduce peroxiredoxins, dehydroascorbate, and methionine sulfoxide reductase, using glutathione as a cofactor. Glutaredoxins are oxidized by ROS, and reduced non-enzymatically by glutathione. No oxidoreductase exists that enzymatically specifically reduces glutaredoxins. Glutaredoxins also function as electron carriers in the glutathione-dependent synthesis of deoxyribonucleotides by the

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation enzyme ribonucleotide reductase, and they were shown to bind iron-sulfur clusters and to deliver the cluster to enzymes on demand. Thioredoxins (TRX) are related to and share many of the functions of glutaredoxins, but they have a characteristic tertiary structure termed the thioredoxin fold. The thioredoxins are kept in the reduced state by the flavoenzyme thioredoxin reductase (TR) (the TrxR protein), which also contains a redox-active disulfide bond. Thioredoxins act as antioxidants by facilitating the reduction of other enzymes, including peroxidase and ribonucleotide reductase. It has been reported that they can also regulate the activity of several transcription factors involved in redox homeostasis and replication, which in turn control cell proliferation. Beside enzymatic antioxidants, most bacteria also contain non-enzymatic antioxidants. Non- enzymatic antioxidants can neutralise ROS compounds for which no specific enzymatic antioxidants systems exist, such as the hydroxyl radical (•OH), and can serve as an antioxidant stock to protect a variety of cellular components from oxidation. Alpha-tocopherol (Vitamine E), ascorbate (Vitamine C) and glutathione are well-known antioxidant systems (Figure III-8).

Figure III-8: Pathway depicting the interaction between the alpha-tocopherol (Vitamin E), ascorbate (Vitamin C) and gluthatione (thiol) oxidant cycles. Alpha-tocopherol (Vitamin E) has a methylated phenol structure, is fat soluble and a potential antioxidant. Because of its property of being fat soluble this vitamin can easily get bound to lipid membrane and plays role against lipid peroxidation of the membrane. During the redox reaction α- tocopherol is converted to α-tocopherol radical and with the aid of ascorbic acid (vitamin C), α- 55

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation tocopherol is regenerated. Thus vitamin C and E act in great accordance in water and fat environment, respectively[69].

Ascorbic acid (Vitamin C) is a very powerful and effective antioxidant which is functional in - aqueous environment. It is a diacid (AscH2) and majorly present as AscH under normal 2- - physiological conditions and in lesser amount it is present as AscH2 and Asc . The AscH form combines with free radical to form a tricarbonyl ascorbate free radical AscH•, which is resonance stabilized and is relatively inert. Thus the production of AscH• marks the end of reaction and protects the organism from the oxidative stress. Vitamin C also acts a defense against membrane oxidation (Figure III-9)

Figure III-9: Conversion of ascorbic acid into different reduced forms at various pH indicating possible binding sites and free electrons responsible for their antioxidant and chelating property

Glutathione (GSH) is the most abundantly found intracellular thiol antioxidant. Glutathione (GSH) is the reduced form and glutathione disulphide (GSSH) is the oxidized form. GSH plays role in the redox signaling and maintains a reducing environment in the cell, allowing normal DNA expression and DNA repair by proteins containing sulfhydryl. GSH acts as a cofactor to many detoxifying enzymes. Glutathione helps to neutralise ROS via the glutathione peroxidases (GPX) enzyme. This enzyme exists in two forms: one is selenium-independent glutathione-S-transferase and other is selenium-dependent glutathione peroxidase. The enzymes reduce peroxides to form selenoles (Se-

OH).The important substrates of GPX are H2O2 and organic peroxide (ROOH). Glutathione also

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation facilitates the regeneration of alpha-tocopherol and ascorbate vitamin C and E back to their active forms [70] (Figure III-10).

Figure III-10: Foyer- Halliwell- Asada cycle. Enzymes and intermediates of the cycle (also known as ASC-GSH cycle) are reported. In white boxes the enzymes active in both animal and plant cells; in gray box the enzyme exclusively presents in plant cells. APX, ascorbate peroxidase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase[71]. Besides glutathione, there are also other non-enzymatic thiol-based antioxidants such as lipoic acid. Lipoic acid, also called α-lipoic acid (ALA) or thioctic acid, is a disulphide derivative of octanoic acid. It is readily soluble in both aqueous and lipid media due to which it also referred to as “universal antioxidant”. It is usually stored in cells as dihydro lipoic acid (DHLA). Both ALA and DHLA are powerful antioxidant and protect the cell by neutralisation of ROS, regeneration of other antioxidants like Vitamin C and E and also chelation of oxidative metal cations (Cu2+ and Fe2+) preventing the promotion of the oxidative chain reaction. In cyanobacteria, and phototrophs in general, also carotenoids are very strong non-enzymatic antioxidants with an important role in oxidative stress mitigation. Carotenoids are tetraterpenoid pigments. There are over 600 types of carotenoids, which can be divided in 2 types: xanthophylls, which contain oxygen, and carotenes, which are made up of only hydrocarbon and do not have oxygen. Carotenoids have the ability to delocalize the unpaired electron through conjugated double bond structure. One of the roles of carotenoids in photosynthetic systems is to prevent damage caused by produced singlet oxygen by either absorbing excess light energy (triplet state energy) from chlorophyll molecules or quenching the singlet oxygen molecules directly [72]. The

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation carotenoid β-carotene, for example, can efficiently scavenge ROS and protect lipids from peroxidative damage. Cyanobacteria can contain large amounts of phenolic compounds and flavonoids[73]. Flavonoids are secondary metabolites and important polyphenolic antioxidants. The phenolic antioxidants (Ph- OH) can react with radicals (ROO∙), producing ROOH and PhO•. Since the PhO• radical so formed is a stable molecule, and the oxidation reaction is terminated. Some of these enzymatic and non-enzymatic antioxidants contribute significantly to the UV resistance of cyanobacteria. It has been shown that filamentous cyanobacterium Nostoc punctiforme ATCC 29133 induces mainly sod and cat genes to cope with oxidative stress generated by UVA exposure [74]. The unicellular Synechosystis sp. PCC 6803 increased the transcription level of sod and gpx upon UVB exposure [75]. Filamentous Anabeana sp. PCC7120 was able to cope with oxidative stress induced by salt and UVB by increasing the transcription levels of peroxiredoxin [76]. The accumulation of peroxiredoxine proteins was induced in response to UVB stress in Anabaena PCC 7120 [76]. In Anabeana sp. PCC 7120, the Ahp alkylhydroperoxide reductase protein played an important role in combating a multitude of stresses, including heat, copper, or salt stress and ionizing radiations such as UVB [77]. Synechocystis sp PCC 6803, enhanced in addition to superoxide dismutase and glutathione peroxidase, also the production of carotenoids upon UVB exposure [78]. Accumulation of carotenoids enhanced also UVC resistance in cyanobacteria [19]. The role of small antioxidants molecules in radiation resistance has been widely discussed for several bacteria. It has been reported that, the amount of protein damage in irradiated cells is strongly influenced by their antioxidant status, where yields of radiation-induced protein oxidation can be 100 times greater in sensitive bacteria than in resistant bacteria [79]. It has been suggested, that naturally sensitive bacteria are killed by ionizing radiation mainly owing to the susceptibility of their repair proteins to oxidative inactivation, which could render even minor DNA damage lethal. In contrast, manganese complexes in extremely resistant bacteria may prevent oxidative protein damage, which could protect the activity of enzymes, and thereby greatly increase the efficiency of DNA repair [80]. DNA repair-proficient bacteria which are unable to protect their proteins from ionizing radiation- induced ROS succumb to relatively minor DNA damage [81,82]. While impaired DNA DSB repair provides the best available correlation with radiation-induced cell-killing, protection of proteins in

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation radiation resistant bacteria by Mn-dependent chemical antioxidants generally provides an explanation for extreme resistance without invoking the need for novel repair pathways or unusual forms of DNA packaging[80] Furthermore the trehalose, a compatible solute of similar size as Mannosylglycerate (MG) and Di- myo-inositol phosphate (DIP) but carrying no charges, was highly protective of protein activity against IR, either alone or in combination with Mn2+. Trehalose is present in a wide variety of organisms including bacteria, yeast, fungi, plants, and invertebrates and was found to protect proteins from heat, osmotic stress, desiccation, and oxidation[83]. Additionally, strains of Chroococcidiopsis, a desiccation and IR-resistant cyanobacterium, were shown to accumulate trehalose [84]. The antioxidant properties of trehalose has been widely discussed in combination with Mn and phosphate, this small organic molecule forms the basis for the high radiation resistance found in R. xylanophilus and R. radiotolerans. The current model of Mn-based antioxidants scavenging IR-generated ROS that was established for aerobic bacteria and archaea [85,86]. In addition to its antioxidant activity, Mn may also act by functionally substituting for Fe in the Fe–S cluster of enzymes and thereby mitigating the deleterious effects of Fenton chemistry during oxidative stress.

III.4.5 Protection and repair of DNA In all living organisms, including cyanobacteria, DNA is the most precious target to protect from solar UVR. UVR can cause a myriad of types of DNA-damage leading to cell death. The UV- induced lesions consist of dimeric photoproducts such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs) [87]. The formation of such dimeric photoproducts inhibit the progression of DNA polymerase, and thus will adversely affects DNA synthesis and RNA transcription, ultimately leading to mutation or death of the organism [88,89]. Cyanobacteria have developed different repair mechanisms to protect and repair DNA from radiation induced damage, including (i) DNA-binding antioxidant proteins (ii) Photo-reactivation by photolyase, which converts UV-induced dimers into monomers, (iii) Dark or excision repair, including base excision repair (BER) and nucleotide excision repair (NER) (iv) Recombinational repair. (v) Mismatch and repair

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Dps, the DNA-binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. The protective ability of Dps stems, in part, from its ability to effectively sequester iron atoms that would alternatively be used in the production of highly toxic free radicals via the Fenton reaction. The capacity to sequester and bind DNA in an extremely organized and rapid manner during times of nutritional deprivation and ⁄ or environmental stress is an additional approach utilized by Dps to provide protection. These protective strategies have proven to be essential for stress resistance in E. coli[90]. During the process of photo-reactivation, UVA/Bleu light is used by the DNA photolyase enzyme to split pyrimidine dimers caused by exposure to UVB/C radiation. Photo-reactivation by photolyase was demonstrated in all cyanobacteria studied so far. This mechanism has been well investigated in Anabaena strains [91]. Homologs of the photolyase gene (pha), have been identified and functionally characterized for their role in photo-reactivation in Synechocystis sp. PCC 6803 [92,93]. Moreover, a study realized on a wild type and a photolysase deficient mutant of Synechocystis sp PCC 6803, showed that a significant amount of damaged DNA was accumulated during UVB exposure in the photolysase deficient mutant, accompanied with decreased amount of D1 protein and PSII activity [94]. In comparison to photoreactivation, excision repair (dark repair) is a more complex pathway where damaged DNA is replaced by new nucleotides by means of a complementary DNA strand. Excision repair can be distinguished into base excision repair (BER) and nucleotide excision repair (NER). The BER pathway has probably been evolved to protect cells specifically from DNA damage induced by hydrolytic deamination, strong alkylating agents, or ionizing radiation (IR), and proceeds through a series of repair complexes that act at the site of DNA-damage [95]. The key enzymes involved in BER are DNA glycosylases, which remove different types of modified bases by cleavage of the N-glycosidic bond between the base and the 2-deoxyribose moiety of the

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation nucleotides residues. The enzyme Fpg (mamidopyrimidine-DNA glycosylase) was reported to be involved in DNA repair and UV resistance in the cyanobacterium Synechococcus elongatus [96].

Figure III-11: DNA's bases may be modified by deamination or alkylation. The position of the modified (damaged) base is called the "abasic site" or "AP site". In E.coli, the DNA glycosylase can recognize the AP site and remove its base. Then, the AP endonuclease removes the AP site and neighboring nucleotides. The gap is filled by DNA polymerase I and DNA ligase.

In contrast to BER, NER is one of the most versatile and flexible repair systems that recover a wide range of DNA lesions, including CPDs and 6-4PPs caused by UVR, bulky chemical adducts, DNA-intrastrand crosslinks, and some forms of oxidative damage. This mechanism is present in most organisms and highly conserved in eukaryotes. Discovery of NER was first described in E. coli [97] where about six proteins are recruited to complete the repair, including UvrA, UvrB, and UvrC (known as ABC-complex, which shows excinuclease activity), UvrD (helicase II), DNA polymerase I (pol I), and DNA ligase.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Figure III-12: In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing the damaged nucleotides (e.g., the dimer induced by UV light). The gap is then filled by DNA polymerase I and DNA ligase. In yeast, the proteins similar to Uvr's are named RADxx ("RAD" stands for "radiation"), such as RAD3, RAD10. Recombination repair is one of the most prevalent mechanisms that can efficiently repair single- strand breaks/gaps (SSBs) as well as double-strand breaks (DSBs) in damaged DNA. Recombination repair proceeds by a series of biochemical reactions and fills the daughter strand gaps by moving a complementary strand from a homologous region of DNA to the site opposite the damage. The lesion is left unrepaired and after the cell cycles through another replication, the damaged base is available as a substrate for excision repair [98]. The high importance of RecA protein in DNA-repair and UV-radiation resistance has been reported for many phototosynthetic bacteria [99]. An increase in recA transcript was reported in Anabaena variabilis PCC 7937 after UV exposure [100]. Synechosystis sp. PCC 6803 recA mutant, showed high sensitivity towards UV light when growing under subsequent dark then low light condition. However, the mutant displayed similar growth rate as the wild type strain when growing under only low light condition. Moreover, the mutant recA460 of Synechocystis sp. PCC 6803 provided

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation evidence that additional genetic mechanism exist, independent of RecA, that cause enhanced UVC resistance on light to dark transition [101].

To repair mismatched bases, the system has to know which base is the correct one. This is achieved by a special methylase called the "Dam methylase", which can methylate all adenines that occur within (5')GATC sequences. Immediately after DNA replication, the template strand has been methylated, but the newly synthesized strand is not methylated yet. Thus, the template strand and the new strand can be distinguished

Figure III-13: Mismatch repair. The repairing process begins with the protein MutS which binds to mismatched base pairs. Then, MutL is recruited to the complex and activates MutH which binds to GATC sequences. Activation of MutH cleaves the unmethylated strand at the GATC site. Subsequently, the segment from the cleavage site to the mismatch is removed by exonuclease (with assistance from helicase II and SSB proteins). If the cleavage occurs on the 3' side of the mismatch, this step is carried out by exonuclease I (which degrades a single strand only in the 3' to 5' direction). If the cleavage occurs on the 5' side of the mismatch, exonuclease VII or RecJ is used to degrade the single stranded DNA. The gap is filled by DNA polymerase III and DNA ligase.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation III.4.6 Controlled Shut-down or self-destruction If the radiation damage is very severe, the cyanobacterial cells may temporarily shut-down their vegetative cellular activities, including photosynthesis and carbon fixation, leading to a growth arrest, and the conversion to a more stress-tolerant, metabolic inactive, 'static' or 'dormant' state This state resembles the state of persisted cells or spores, described for other bacteria. This will allow the cells to temporarily direct all energy and resources to cellular detoxification and repair. If, the damaged cyanobacterial cells are beyond repair, they may also undergo apoptosis or programmed cell death (PCD). The nitrogen fixing cyanobacteria Trichodesmium sp. induces a process of autocatlytic PCD following high irradiance, iron starvation or oxidative stress [102].

Similarly, recent study showed that H2O2 treatment induced PCD programme in the Microcystis aeruginosa [103]. This cyanobacterium expressed several genes encoding the caspase enzyme, which is involved in PCD in eukaryotes [104]. Thus, PCD plays an important role under oxidative stress in cyanobacteria, but the exact role of this mechanism under UV stress still needs to be confirmed.

III.4.7 Radiation, ROS and damage sensing, signal transduction, and response regulation Thus several mitigation strategies (described above) were developed by cyanobacteria to deal with the detrimental effects of UVR. Nevertheless, before responding, the cells need to (i) sense the environmental signals and (ii) transduce them to the corresponding regulatory apparatus to mediate the response. Cyanobacteria have several classes of photo-receptors to sense the light environment and prevent the damage induced by UVR exposure [105]. The model Synechocystis sp. PCC 6803 utilizes more than three distinct classes of photo-sensors to control UVA induced photo-taxis. These include the UV intensity sensor UirS [106], phytochrome-related cyanobacteriochromes (CBRCRs) such as PixJ1 or TaxD1 [107] and Cph2 [108], and cryptochrome DASH (Cry-DASH) [109]. UirS is a membrane-associated a UV-A photosensor protein, part of a 2-component RS regulatory system UirS-UirR, which controls gene expression, via the release of the bound UirR transcriptional regulator protein, which targets the lsiR promoter [106]. Cyanobacteriochromes are phytochrome-related photoreceptor proteins found only in the cyanobacteria. The cyanobacteriochrome protein covalently binds a linear tetrapyrrole molecule in the GAF domain. The GAF domain of cyanobacteriochrome protein is related to but different from the phytochrome GAF domain. The spectral properties of cyanobacteriochromes also significantly

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation differ from those of phytochromes. Cyanobacteriochromes perform reversible photoconversion between blue (~430 nm) and green (~530 nm) absorbing forms or between green (~530 nm) and red (~660 nm) absorbing forms, while phytochromes photoconvert between red (~660 nm) and far- red (~700 nm) absorbing forms. Cryptochromes (Cry proteins) are a class of flavoproteins that are are derived from and closely related to photolyases and that are sensitive to blue light. They have an N-terminal photolyase homology (PHR) domain, that binds two light-harvesting chromophores : a pterin (in the form of 5,10-methenyltetrahydrofolic acid (MTHF)) and a flavin (in the form of adenine dinucleotide FAD), which both can absorb a photon. Studies have suggested a phototransduction model by which energy captured by pterin is transferred to flavin FAD, which then would be reduced to FADH and could mediate the phosphorylation of a certain domain in cryptochrome, triggering a signal transduction chain, possibly affecting gene regulation. Cryptochrome DASH is a specific class cryptochromes, present in bacteria and plants, of which it shown that it can directly function as a transcriptional repressor in Synechosystis. The Cry-DASH coding gene (Syn-Cry), from the Synechosystis sp. PCC 6803, is required for efficient restoration of photosystem activity following PAR and UVB induced photo-damage [110]. It has been suggested that a reduced pterin may also serve as the chromophore in the photosensory induction of MAA biosynthesis under UVB radiation in cyanobacterium Chlorogloeopsis sp. PCC 6912 [111]. Additional support came from findings demonstrating that light-dependent induction of MAA is impaired by inhibitors of the pterin biosynthetic pathway [111]. Later on, a study investigating photoreceptors of Nostoc commune using inhibitors of the shikimate pathway, photosynthesis, proteins and pterin synthesis, had difficulties to confirm the unique involvement of pterin molecules as photoreceptors for MAA production in Nostoc commune [112]. In cyanobacteria the underlying mechanism of UVB signal transduction is not well documented. In higher plants the transduction of UVB signals relies on second messengers including calcium, kinases and the catalytic formation of ROS. In the plant Arabidopsis, ROS increases the concentration of calcium which, together with calmodium, regulates the catalase activity [113]. Similar observations were seen in the cyanobacteria Anabaena sp. and Nostoc commune, following exposure to UVB [98,114]. Other signals such cAMP and cGMP are also found in cyanobacteria. Change in cellular homeostasis of cGMP was found to be involved in the signal transduction under

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation UVB radiation in Synechosystis sp. PCC 6803 [115]. But further research is required to characterize more in detail UV perception and transduction for cyanobacteria.

III.4.7.1 SOS response The SOS response is a coordinated cellular response well described in E.coli and aids in the survival of the organism by affecting the expression of proteins that are involved in cellular division, replication, recombination, and excision repair. The basic mechanism of regulation is relatively simple. Under normal conditions, the LexA repressor binds to the SOS box, with the sequence to maintain repression of the SOS regulon. Directly or indirectly, DNA damage or other replication blocks generate single-stranded regions that lead to the formation of single-stranded DNA, on which the RecA protein polymerizes. The RecA-single-stranded DNA filament binds to LexA and stimulates its auto-proteolysis activity. Cleavage of LexA inactivates its repressor activity, allowing transcription of the SOS genes, including lexA and recA themselves. Cell division stops, excision and recombination activities increase, and the capacity of replicase to bypass the damage increases. All of these enable the cell either to eliminate the DNA damage or to survive with damaged DNA. Upon recovery, the inducing signal disappears, and LexA accumulates and represses the cognate genes. The excision repair genes uvrA, uvrB, and uvrD are under the control of the LexA protein; consequently, the cellular excision repair activity increases transiently as part of the SOS response reaction and then drops down to pre-induction levels after repair [116].

III.4.7.2 Adaptive Response to Oxidative Damage The activation of various regulators in response to an increase in ROS concentrations can often modulate the transcription of a subset of genes, allowing an appropriate response to the stress sensed. The way bacteria respond to elevated concentrations of either superoxide or H2O2 is completely different, illustrating the ability of these simple organisms to sense and to discriminate between these two signals. The SoxRS regulon is a two-component regulatory system that responds to superoxide stress. SoxR is an iron-sulfur protein that is sensitive to superoxides, and when oxidized it becomes a transcriptional activator for SoxS. SoxS is a positive regulator of genes encoding superoxide- detoxifying and DNA-repair enzymes. The ferredoxin-like protein SoxR is a 34-kDa homodimer containing one essential [2Fe-2 S] cluster per polypeptide chain. Both the reduced and oxidized

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation forms of SoxR bind to the SoxS promoter, with comparable affinities, although binding of the reduced form does not induce transcription [4].

Figure III-14: Adaptive response to superoxides. The SoxR protein has a [2Fe-2 S] active center. The protein binds to the soxS promoter in reduced form, but does not turn it on. Upon oxidation by superoxides, it overwinds the promoter of soxS, making it a high-affinity site for RNA polymerase. Increased transcription of soxS leads to increased SoxS protein, which is a transcriptional activator that turns on genes involved in inactivating superoxides (zwf, sodA) or repairing DNA (nfo).

The OxyR system belongs to the LysR family of single-component bacterial regulators, in which the same protein functions both as a sensor and as a signal transducer-regulator. The OxyR protein acts as both a sensor and transducer of the oxidative stress signal. OxyR activation by peroxide is based on cysteine chemistry is regulated by the oxidation status of two cysteine residues, Cys199 and Cys208. Peroxides lead to formation of a disulfide bridge between the two cysteine residues.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation Both disulfide and dithiol forms of OxyR bind to the oxyR promoter, but only the disulfide form binds to the katG (hydroperoxidase) and ahpC (hydroperoxide reductase) promoters. Binding of reduced OxyR to its own promoter represses its gene, whereas the disulfide form turns on this gene, as it does to the katG, ahpC, gorA, and grx1 genes. The members of this regulon are involved solely in antioxidant defense and not DNA repair. Not all bacteria have an OxyR homologue. For example, the Fur-like peroxide-sensing repressor PerR, first identified in Bacillus subtilis, was shown to be conserved in other Gram-positive and Gram-negative bacteria lacking OxyR. Up to now, no SoxR/SoxS homologues have been reported in cyanobacteria and the regulator sustaining the superoxide response is still unclear in these phototrophs. However, a PerR orthologue has been identified in Synechocystis PCC 6803 using a microarray approach [117] . Transcription of the perR gene in Synechocystis PCC 6803 (slr1738) was induced by peroxide, and the inactivation of perR derepressed, even in the absence of peroxide, the expression of a set of genes including the dps-homologue mrgA and ahpC alkylhydroperoxidase.

While in the study of [118] Houot et al, highlights the possibility that PerR may function as a peroxide-sensing repressor in Synechocystis sp. PCC 6803. In contrast to its clear role in B. subtilis, PerR does not act as the master regulator of the peroxide response in cyanobacteria.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Figure III-15: Adaptive response to peroxides. Peroxides convert two cysteine thiol groups on OxyR to a disulfide bond. This activates OxyR as a transcription factor, which turns on genes whose products combat peroxides. In the absence of peroxides, glutaredoxin reduces OxyR and converts it to the inactive form

III.5 Tolerance and response of cyanobacteria to X- and Gamma radiation A wide variation in X-ray and gamma-ray tolerance exists, and several organisms can also survive high doses. A whole‑body exposure to 10 Gy is lethal to most vertebrate animals, including humans [119]. But the freshwater invertebrate animal Philodina roseola [120], the water bear Milnesium tardigradum [121] and the roundworm Caenorhabditis elegans [122] can tolerate 3 000 – 5 000 Gy, although they become sterile. Most bacteria cannot survive 200 Gy [119]. But the archaeal species Halobacterium sp. NRC‑1 can resist to 5 000 Gy [123], whereas some bacteria from the Deinococcus–Thermus group can survive doses of more than 12 000 Gy [124]. Deinococcus radiodurans is 30-fold and 1000-fold more resistant to ionizing radiation than E. coli and humans respectively. Also cyanobacteria showed a wide range of radiation tolerance for X- and gamma ray, and some of them are higly radiation resistant [125]. They were grouped into three categories. The first group showed a low resistance with a D90 less than 4 000 Gy. This group include Microcystis aeruginosa,

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation Anacystis nidulans and some species of Schizothrix calcicola. The second group showed a moderate resistance with a D90 between 4 000 Gy and 12 000 Gy. These criteria were valid for Oscillatoria brevis, Anabaena variabilis and Scytonema hoffmannii. The third group showed the highest resistance, with a D90 greater than 12 000 Gy. Species of this group include Plectonema boryanum, Lyngbya estuarii, Nostoc muscorum and Microcoleus vaginatus. Most of the cyanobacteria with high radiotolerance, i.e. from the second and third group, are filamenteous. There are a significant number of studies on the effect of gamma or X rays as low LET ionizing radiation on cyanobacteria, but most report only on the physiological impact [126-130]. Only few reports, investigated the molecular mechanisms behind. It seems that the damage introduced by X- or gamma irradiation shares features with the damage that results from other stresses to which bacteria have adapted. For example, desiccation introduces many DNA double-strand breaks into the genomes of D. radiodurans [131] and members of the cyanobacterial genus Chroococcidiopsis [126]. Both organisms are tolerant to desiccation and are resistant to the potentially lethal effects of X- or gamma radiation, which might indicate that the radioresistance of these species is a fortuitous consequence of their ability to tolerate desiccation-induced DNA double-strand breaks. Not much is reported on the molecular mechanism responsible for the radiation resistance of cyanobacteria to X or gamma rays. But in principle the cells could rely again on the same systems as induced by intense VIS or UV radiation: (i) Avoidance of cell exposure to radiation by migration and morphological changes, (ii) Protecting the cells by radiation-absorbing molecules, (iii) Protecting and repairing the PSII by energy dissipation and photo-inhibition, (iv) Protecting the membranes and enzymes by ROS-scavenging antioxidants, and (v) Protecting and repairing the DNA from radiation-induced damage

However, probably not all of these systems can provide for X-ray and gamma-ray resistance.

Therefore, the following section will deal mainly with the mechanism of radiation resistance which have been described to essential for Deinococcus radiodurans. The key radiation resistance mechanisms of Deinococcus radiodurans to X or gamma rays have been linked to: (iv) protecting the enzymes by ROS-scavenging antioxidants, including superoxide dismutase, catalase, peroxiredoxins, glutaredoxins, thioredoxins bacithiol, carotenoids and manganese, and degradation of damaged proteins

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation (v) protecting and repairing the DNA from radiation-induced damage, via extended synthesis-dependent strand annealing and homologous recombination (ESDSA and HR) and degradation of oxidized nucleotides by hydrolases.

III.5.1 Protection of enzymes by ROS-scavenging antioxidants, and degradation of damaged proteins It has been reported that the robustness of D. radiodurans to high doses of gamma rays is due to its strong oxidative stress resistance mechanism that protects vital proteins from oxidative damage [82]. Proteins especially involved in DNA repair and replication must be protected from damage by radiation before DNA repair can begin. D. radiodurans possesses an efficient enzymatic superoxide ROS scavenging system mediated by four superoxide dismutase, three catalases, and two peroxidases [86]. D. radiodurans encodes also other thiol-based antioxidant enzymes such as alkyl hydroperoxide reductase, peroxiredoxins, glutaredoxins, thioredoxins and thioredoxin reductase [86]. The classical glutathione cycle, with gluthatione, glutathione peroxidase and gluthation reductase, are absent in Deinococcus [132], but is compensated by bacithiol, also a thiol-based molecule which is considered as a substitute for glutathione. It has been reported recently that bacithiol is largly responsible for its extreme resistance to gamma rays [133]. The DNA is protected against oxidative damage by two DNA- binding antioxidant DPS proteins [86]. The non-enzymatic scavenger mechanisms of D. radiodurans include also carotenoids which are efficient scavengers of ROS and especially of singlet oxygen and peroxyl radicals [86,134]. Recently, it has been demonstrated that Mn-dependent antioxidant mechanisms can protect proteins from ROS-induced damage in many species, including D. radiodurans [82,135]. Leibowitz et al. demonstrated that D. radiodurans contains approximately 100 times more Mn2+ than Escherichia coli when grown in a defined minimal medium [136]. The high intracellular concentrations of manganese ions are known to alleviate oxidative stress in several bacterial species, as these ions can interact with different ROS compounds, depending on their oxidation state and their binding with different molecules. It has been reported there is a relationship between intracellular Mn/Fe concentration ratios and bacterial survival following exposure to IR Radiation resistant cells contained about 300 times more Mn and about three times less Fe than sensitive cells [137]. The benefits of manganese accumulation compared to iron accumulation in irradiated cells are related to the limitation of Fenton chemistry [138]. Mn2+ ions that accumulate in resistant bacteria form

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation complexes that shield Fe–S cluster containing proteins from ROS generated during irradiation. By preventing the release of Fe2+ from Fe–S‑cluster containing proteins, the global damaging effects of the Fenton reaction during irradiation will be minimized, allowing enzyme systems involved in recovery to survive and function efficiently.

Figure III-16: Model of IR-Driven Mn and Fe Redox Cycling [82] If radiation-induced ROS cannot be neutralised by antioxidants, lipids and proteins will be oxidised and potentially toxic and mutagenic oxidized derivates will be generate. These damaged amino acids need to be degraded as quickly as possible to sanitize the cells. This process is highly pronounced in radiation-resistant Deinococcus radiodurans. The dysfunctional oxidized proteins, damaged beyond reparation, are removed by proteolysis and rapidly resynthesized [139]. Deinococcus encodes Lon proteases, Clp protease and ATPase subunits such ClpA, ClpB , ClpC and ClpX [140]. The level of intracellular proteolytic activity is increased following radiation exposure [135]. The degradation process activates also GroEL and DnaK [141]. Similar ROS-scavenging antioxidants, and protein degradation processes also exist in cyanobacteria, and thus could also play a role in the X- and gamma ray resistance of cyanobacteria. Cyanobacteria contain also superoxide dismutase, catalase, peroxiredoxins, glutaredoxins, thioredoxins bacithiol, and carotenoids (described above).

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation III.5.2 Protection and repair of DNA Exposure to 5000 Gy of 60Co radiation reduces the genome of any bacterium to hundreds of fragments. However, Deinococcus seems to take this catastrophe in stride. Deinococcus can repair approximately 200 DSBs or 190 cross links per genome copy without a loss of viability [142]. An extremely efficient DNA repair process rapidly reassembles all the fragments into functional chromosomes during the post irradiation recovery [143,144]. Over a period of 3–4 hours, overlapping fragments are stiched together into complete chromosomes, afterwards cells resume normal growth [145]. Such genome reconstruction can be monitored by analysis of the DNA by the Pulsed Field Gel Electrophoreses method (PFGE). This genome reconstruction process comprises essentially three distinct phases: (i) The DNA degradation period with little repair , (ii) The re-joining of DNA fragments in parallel with extensive DNA synthesis, and (iii) The resumption of growth upon the completion of DNA repair. The second step, the reconstruction and the reassembly of the genome includes 2 processes : a process named extended synthesis-dependent single strand DNA annealing (ESDSA), and double strand breaks repair via homologous recombination (HR) [146,147] (Figure III-17). The first process, extended synthesis-dependent single-strand annealing (ESDSA) is dominated by nuclease and DNA polymerase activity. The second process is a more conventional RecA-mediated double- strand break repair process focused on the final splicing of large chromosomal segments. Damaged nucleotides and potentially toxic nucleoside diphosphate derivatives are detoxified and recycled by Nudix (nucleoside di-phosphate) hydrolases [132]. In addition, it has been reported that Uvr2, closely related to the ABC transport, may be in charge of oligonucleotide export [132].

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation

Figure III-17: The 2 main processes of genome reconstruction in Deinococcus radiodurans. Extended synthesis-dependent single strand DNA annealing (ESDSA), and double strand breaks repair via homologous recombination (HR) [145]. Similar genome reconstruction processes also exist in cyanobacteria, and thus could also play a role in the X- and gamma ray resistance of cyanobacteria. Moreover, the efficient DNA repair system in cyanobacteria includes also other systems such as the excision and strand break repair systems and the photo-reactivation system (described above) [148]. It has been shown that Chrococcidiopsis was able to reconstruct and repair its genomic DNA after exposure to 5000 Gy of X-rays exposure, after 24 h of recovery [126]. A diazotrophic culture of Anabaena 7120, having a 20–24 h generation time, showed an efficient recovery following 6000 Gy of gamma radiation exposure, by the extensive reparation of DNA damage within 2–3 days [130]. Similarly, the nitrogen fixing Anabaena L-31 exposed to 6000 Gy of 60Co gamma rays could repair its DNA completely after 4 days post irradiation recovery [128]. Currently it is not possible, to comment whether such DNA repair in Anabaena is error-free (like in D. radiodurans) or generates mutants. Nevertheless, at least no serious morphological or metabolic impairments were showed in cultures that recovered from such stress.

III.6 Tolerance and response of cyanobacteria to particle radiation The tolerance and response of cyanobacteria to particle radiation has been very little studied.

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation The resistance of bacteria to particle radiation, has been mainly investigated by using spores of Bacillus [149]. The impact HZE particles, on Bacillus spores were investigated within Biostack device during Apollo 16 and 17 NASA missions [150,151]. Spores of Bacillus subtilis were significantly more sensitive to high LET (Iron and Helium particles) irradiation than low LET (X rays). [152]. But a substantial fraction of dormant spores of B. subtilis 168 were capable of surviving the exposure of galactic cosmic rays (GCR), i.e. X rays and high energy charged particles, up to 500 Gy [153]. Particle radiation resistance has not only been tested on spores, but also on cells, although mainly using dried cells of the highly radiation resistant bacterium D. radiodurans [154]. It has been documented that one year of heavy ion beams (Helium and Argon) (1 Gy) had little effect on the viability of Deinocccus spp. The tested dose corresponds to one-year irradiation received on-board of the International Space Station ISS [155-157]. The dried cells of D. aetherius (D10: 8 000 Gy) showed higher resistance than dried D. radiodurans (D10: 6 700 Gy) and D. aerius (D10: 4 900 Gy) for 60Co gamma rays (dose rates of 370 - 1 450 Gy/h) [158]. Likewise, D. aetherius was also more resistant to Helium particles (4He2+ 150 MeV/u; LET: 2.2 KeV/µm) and Argon particals (Ar 500 Mev/u; LET: 90 Kev/µm) than D. radiodurans and D. aerius [159]. It seems that when a bacterium is relative resistant to high doses of electromagnetic radiation, it is also relative resistant to particle radiation. Next to Bacillus and Deinococcus, also some cyanobacteria have been tested with particle radiation. Cyanobacteria are studied as a photosynthetic model organism for space astrobiological research and were proposed to be used as photosynthetic pioneers for terraforming on Mars [160,161]. The cyanobacterium Chrococcidiopsis has been isolated from harsh environment, i.e. the Negev Desert and Atacama Desert, and was selected as model for testing the impact of cosmic radiation. It has been proven to withstand X rays ionizing radiation up to 15 000 Gy [126]. it has been characterized to withstand hot and cold desert environments, and X-rays ionizing radiation as high as 15 kGy, on Earth [126]. The free flying satellites and the international space station have provided unique opportunities for assessing the endurance of such microorganisms to life in space, and has given certainly more insights into the potential of life to survive beyond Earth [162]. Installed on the outside of the International Space Station (ISS), the EXPOSE facility allowed studying the effect of prolonged exposure (548 days) to space conditions in low Earth orbit. Natural

Introduction Chapter III: Susceptibility of cyanobacteria to ionizing radiation prototroph biofilms on rocks, augmented prior to flight with Chroococcidiopsis, showed post-flight survival of Chrococcidiopsis [163]. Chrococcidiopsis cells were irradiated with particle radiation in dried status, as reported for Deinococcus radiodurans [164,165] Only few studies investigated the impact of particle irradiation on bacteria on the molecular level. DSBs are the most severe type of damage induced by HZE particles in microorganisms, and were demonstrated in cells or spores of E. coli [166], B. subtilis TKJ 8431[167] and D. radiodurans R1 [168] after exposure to particle radiation. In addition, oxidative base damage, has been found in B. subtilis spores exposed to HZE particles (C and Fe ions) [169], which is probably caused by the indirect (ROS) effects of particulate radiation. Bacteria possess several mechanisms to repair DNA DSBs induced by HZE particles. These include the re-joining of broken ends, by homologous recombination (HR) with a sister strand molecule, or by non-homologous end joining (NHEJ) [170]. In spores of B. subtilis, NHEJ is the most efficient repair pathway during germination of spores exposed to ionizing radiation, such as X-rays or HZE particles [171]. These investigations on the biological effects of particle radiation were however only done on a few species and almost exclusively on dried cells. It is known that desiccation-tolerant organisms already exhibit a higher resistance towards UV and ionizing radiation. It has indeed been reported for Deinococcus radiodurans [172] and Chrococcidiopsis [164,165] that radiation resistance is enhanced when the cells are irradiated in dried status. Thus, there is truly a need to enlarge these studies towards also live planktonic cells, including cyanobacteria (e.g. Arthrospira), and explore much more the molecular responses of such cells to such high energetic particle irradiation.

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Part II: Thesis aims

Aims Chapter IV: Thesis aims

Chapter IV Thesis aims

The present study explored for the first time the radiation response of the edible cyanobacterium Arthrospira to ionising radiation, including both low-LET gamma rays and high-LET charged particles.

Our research plan was divided into three main chapters;

The first study was devoted to investigate the radiation tolerance of and the damaging impact of high doses of gamma rays on Arthrospira. For this, live cells of the cyanobacterium were exposed to 60Co gamma rays (Dose rate 527 Gyh-1) in RITA facility at BR2 of SCK•CEN (Mol, Belgium). The limits of radiation tolerance and the ability of Arthrospira to restore and resume photosynthetic growth, even after significant cell damage, were assessed. Pigments content (phycobilisomes and chlorophyll), and morphological changes were analysed and the assessment of Photosystem II functionality via chlorophyll fluorescence after irradiation was explored. Furthermore, different molecular techniques, including proteomic and transcriptomic, were used to investigate the response of Arthrospira after exposure up to 3200 and 5000 Gy. The results of this study are presented in chapter V (Paper 1) entitled “Molecular investigation of the radiation resistance of edible cyanobacterium Arthrospira sp PCC 8005”.

The second study aimed to understand the dynamic changes of gene expression and related metabolic pathways in response to ionising radiation over time. For this, live cells of Arthrospira were exposed very shortly to high dose rates of 60Co gamma rays (Dose rate 20000 Gy/h) in BRIGITTE facility at BR2 of SCK•CEN (Mol, Belgium), and immediately followed afterwards. The exposure of Arthrospira to 10000 Gy of 60Co gamma rays was lethal; this dose killed all the cells, after which no recovery was resumed. For the molecular study, non-damaging doses of 800 Gy and 1600 Gy, in addition to 3200 Gy, were selected, to allow the analysis of not only the damaging impact, but also the recovery of the full culture after irradiation. The response of Arthrospira to radiation exposure was followed by transcriptomic profiling immediately after irradiation and during recovery period. Meanwhile, total pigment content, and PSII quantum yield

Aims Chapter IV: Thesis aims were assessed. In addition, the anti-oxidative response of Arthrospira to gamma rays was investigated, including the monitoring of the total glutathione content over time. The results of this study are discussed in chapter VI (Paper 2) entitled “Temporal gene expression of the cyanobacterium Arthrospira”.

In the third part of this thesis, the impact of HZE particles, abundant in space radiation, was investigated. Active cells of cyanobacterium Arthrospira sp. PCC 8005, were irradiated with Iron and Helium particles at the Heavy Ion Medical Accelerator Chiba (HIMAC) at the National Institute for Radiological Sciences (NIRS) (Chiba-Shi, Japan), in the frame of STARLIFE project. Given the restrictions to sample size, not allowing molecular analysis, this study was focused on the physiological and morphological response of Arthrospira to such high LET particle radiation. The results are discussed and compared to the obtained findings with low LET gamma radiation in chapter VII (Paper 3) entitled "Response of the spaceflight-relevant cyanobacterium Arthrospira sp. PCC 8005 to high doses of charged-particle radiation".

With this study, we aimed to characterise the resistance of cyanobacteria to ionising radiation and to explore some of the molecular systems behind. The final goal is to improve our understanding of the cellular response to and protection from ionising radiation, which then can be the basis for future research studies and biotechnological applications.

Part III: Results

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

Chapter V Molecular investigation of the radiation resistance of edible cyanobacterium Arthrospira sp. PCC 8005

Hanène Badri, Pieter Monsieurs, Ilse Coninx, Ruddy Wattiez, and Natalie Leys

“Molecular investigation of the radiation resistance of edible cyanobacterium Arthrospira sp. PCC 8005” MicrobiologyOpen, In press.

Contributions: Hanène Badri performed all experiments and analysis, with the help of lab technician Ilse Coninx. Pieter Monsieurs performed the processing of the microarray data. Prof. Ruddy Wattiez and Dr. Natalie Leys have guided Hanene Badri towards the most optimal experiment design and data interpretation.

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Abstract The aim of this work was to characterize in detail the response of Arthrospira to ionizing radiation, to better understand its radiation resistance capacity. Live cells of Arthrospira sp. PCC 8005 were irradiated with 60Co gamma rays. This study is the first, showing that Arthrospira is highly tolerant to gamma rays, and can survive at least 6400 Gy (dose rate of 527 Gyh-1), which identified Arthrospira sp. PCC 8005 as a radiation resistant bacterium. Biochemical, including proteomic and transcriptomic, analysis after irradiation with 3200 or 5000 Gy showed a decline in photosystem II quantum yield, reduced carbon fixation, and reduced pigment, lipid, and secondary metabolite synthesis. Transcription of photo-sensing and signalling pathways, and thiol-based antioxidant systems was induced. Transcriptomics did show significant activation of ssDNA repair systems and mobile genetic elements (MGEs) at the RNA level. Surprisingly, the cells did not induce the classical antioxidant or DNA repair systems, such superoxide dismutase (SOD) enzyme and the RecA protein. Arthrospira cells lack the catalase gene and the LexA repressor. Irradiated Arthrospira cells did induce strongly a group of conserved proteins, of which the function irradiation resistance remains to be elucidated, but which are promising novel routes to be explored. This study revealed the radiation resistance of Arthrospira, and the molecular systems involved, paving the way for its further and better exploitation.

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.1 Introduction Why some cells are radiation sensitive and others are highly radiation resistant, is still intriguing and has been a matter of detailed investigation. The current knowledge describing the mechanisms or conditions that likely contribute to radiation resistance, indicates that radiation resistance correlates not exclusively with the induced radiation damage to DNA but rather with the susceptibility of the cellular proteins to radiation induced oxidation [1]. The ability of cells to protect their proteins from oxidation by scavenging the harmful reactive oxygen species (ROS) generated by ionizing radiation (IR) has been proposed as the key mechanism for survival of IR- resistant microorganisms. Due to the accumulation of small antioxidants molecules in Deinococcus radiodurans [2], Halobacterium salinarum [3], and the bdelloid invertebrate Adineta Vaga [4], they can protect their protein from oxidation and thereby preserve the function of enzymes needed to repair DNA and survive. Most studies however, have used non-photosynthetic test organism. Nevertheless, also some cyanobacteria were reported to be UV [5] and even X ray [6] and gamma ray radiation resistant [7,8], which makes them interesting study objects to further unravel the molecular principles of cellular radiation resistance of photosynthetic organisms. Arthrospira is a free-floating filamentous cyanobacterium that tends to aggregate and grows vigorously in water of alkaline lakes [9], and in regions with strong sunshine and high temperature [10]. Arthrospira is not pathogenic in nature and has been used for human consumption since 16th century, due to its high protein content and easy digestible property [11]. Its valuable nutritious components include essential fatty acids such as omega-3, and pigments, such as carotenes and phycocyanin [12,13]. The last decades, Arthrospira has gained increasing interest as health promoting food supplement, on Earth and for human space flight [14]. In specific, its strong anti-oxidant and anti-inflammatory properties are subject of investigation and seem promising for potential application in human radiation protection [15]. In fact, Arthrospira has been used in the nutraceutical “spirulina” to treat radiation sickness [16]. The aim of this work was to characterise in detail the response of Arthrospira to ionising radiation, to better understand its peculiar cellular protection against radiation. Therefore, the cellular and molecular response of Arthrospira strain PCC 8005 to high acute doses of gamma rays was investigated in detail, using transcriptomics and proteomics. Previously, the resistance and response of algea or cyanobacteria to radiation has been mainly investigated by morphological and

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 physiological analysis [7,8,17,18], but the molecular mechanisms remain to be elucidated. To our knowledge, this is the first study that investigates the tolerance of the edible cyanobacterium Arthrospira to ionising radiation, at molecular level. The complete genome sequencing of Arthrospira sp. PCC 8005 was recently defined [19]. Based on this genome sequence, a novel full- genome covering DNA-microarray, specific to Arthrospira sp. PCC 8005, was designed, and used in this study to monitoring expression genes in response to radiation. In addition, transcriptomic analyses were combined with proteomic and phenotypic analysis.

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.2 Materials and Methods

V.2.1 Strain and culture conditions The strain Arthrospira sp. PCC 8005 was obtained from The Pasteur Culture Collection. Three independent cultures (n = 3) of 600 ml each were used for irradiation. The three replicates were grown separately on a rotatory shaker in an incubator at 30°C (Binder KBW 400), in 600 mL

Zarrouk medium [20] to mid-exponential phase corresponding to an OD750nm ~1. Cultures were illuminated with a photon flux density of ~ 42µE m-2 sec-1 provided by three Osram daylight tubes. Each 600 ml culture was then divided in six aliquots of 100 ml into different flasks, which were further divided into 2, i.e. non-irradiated controls and samples for irradiation (CELLSTAR Filter cap cell, Greiner Bio-One, Vilvoorde, Belgium, 250 ml cell culture flasks). Irradiation was carried out on active planktonic filaments suspended in 40-mL aerobic liquid Zarrouk culture medium pH 9.5, at cell concentrations of ca. OD750 ~1.

V.2.2 Irradiation conditions The irradiation was performed using RITA facility at the Belgian Reactor N°2 (BR2). The irradiation was done inside a closed canister, surrounded by four sources of 60Co gamma rays (Energy of 1.33 Mev and 1.17 Mev). Different doses of gamma rays were given at a constant dose rate of 527 Gy h-1. (Figure S1) illustrate the different tested doses and the occurred irradiation time. The cultures were in the dark, and the temperature inside the irradiation canister was automatically monitored and ranged between 26-27°C. The time required for irradiation was dose-dependent and so respective controls were kept at the same conditions as irradiated samples. Samples were immediately put on ice after irradiation, at the irradiation facility, before transport to the lab for further processing. Some aliquots were used immediately after irradiation for regrowth and measurement of chlorophyll fluorescence. While the main part of the samples was centrifuged at 4 °C, and the obtained cell pellet was flash frozen in liquid nitrogen, and further conserved at -80 °C, for molecular and biochemical analysis, including mRNA, protein and pigment content.

V.2.3 Post-irradiation recovery and proliferation In order to investigate the ability of Arthrospira filaments to recover after irradiation, inoculation of 1% (v/v) from irradiated and non-irradiated samples was done in fresh medium, and incubated for growth at the same conditions as cited above. The growth was followed by absorbance measurement OD750nm (optical density) every five days using every 5 days using the

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 spectrophotometer AquaMate (Unicam, Cambridge, UK). The proliferation curves were made based on OD750nm versus time.

V.2.4 Photosynthetic potential measurement Chlorophyll A and phycobilisome fluorescence of PSII was determined using the DUAL PAM 100 device (Waltz-GmbH Effeltrich Germany). From 3 independent cultures for each test condition (n=3), aliquots of two ml of control cultures and irradiated samples were tested immediately after exposure, with DUAL PAM 100. All samples were dark adapted for 15 min. Then, the cells were exposed to a weak modulated red light (ML) (635 nm, 3 µE m-2s-1) (which is too low to excite and induce any photosynthetic activity or fluorescence), and minimum fluorescence was determined (F0). Next, the cells were exposed to a high red light excitation called saturating pulse (635 nm, 8000 µE m-2s-1) with short duration (0.8 s) and maximum fluorescence in dark adapted state (Fm) was determined. From those measurements, the ratio Fv/Fm was then calculated where the variable fluorescence Fv is equal to Fm – F0, and present the difference between maximum fluorescence from fully reduced PSII reaction centre (Fm) and the intrinsic fluorescence (F0) from the fully oxidized PSII. Healthy Arthrospira cells normally have a yield FV/FM of ca. 0.6 [21], while photo- synthetically non-functional (dead) cells have Fv/Fm of 0.

V.2.5 Pigments analysis From three independent cultures for each test condition (n=3), aliquots of one ml of irradiated and control cultures were collected immediately after exposure to gamma rays by centrifugation (5418R; Eppendorf Robelaar, Belgium) (10000 g, 15 min), and cell pellets were stored at -80°C until analysis (ca. 2 days). Later, frozen cell pellets were freeze-dried overnight using a freeze- dryer (Lyovac GT 2, Sweden), and the absolute dry weight was determined. Next, the pellet was resuspended in 1 ml of 0.05 M Na2HPO4 at pH=7, in order to extract the hydrosoluble fraction of phycobiliproteins containing phycocyanin and allophycocyanin pigments. To break the cells, five cycles of freezing in liquid nitrogen and thawing at 37°C were performed. In addition, in order to achieve total extraction, additional treatment with lysozyme at a final concentration of 100 mg ml- 1 was done. Next, the lysed fraction was centrifuged (10000 g, 10 min), and the supernatant, was measured at wavelengths 615 and 652 nm. The concentration of phycocyanin and allophycocyanin were calculated according to [22]. Then, the pellet remaining after extraction of the hydrosoluble fraction, was washed three times using 1 ml of 0.05 M Na2HPO4 at pH =7 , and then used for a

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Chlorophyll extraction with 100% methanol as organic solvent. Additional mechanic treatment by sonication (3 cycles of 10 s, amplitude 30%, 1 pulse rate, (Sonics Vibra cells, Newtown, USA)) was performed to allow total chlorophyll extraction. The lysed fraction was centrifuged (13000 g, 10 min) and the supernatant was measured via spectrophotometry at a wavelength 665 nm. Chlorophyll concentration was calculated then according to the procedure described by Benett [22].

V.2.6 RNA extraction RNA extractions were done on three independent cultures for each test condition (n = 3). The RNA extraction procedure had to be optimized. In total, 30 ml of irradiated and control Arthrospira cultures were put on ice immediately after irradiation, and were centrifuged (Avanti J- 26XP; Beckman Coulter, Suarlée, Belgium) for 20 min at 10000 g and 4°C, to collect the cell pellets (in falcon tubes of 15 ml). Cell pellets were then flash frozen in liquid nitrogen and stored immediately at -80°C, until analysis (ca. 5 days). Before extraction, the frozen cells were mixed with 1 ml Trizol (Invitrogen, Life Technologies Europe B.V, Ghent, Belgium) and, transferred into 2 ml Eppendorf tubes, so that the cells were already in the lysis solution (also preventing enzymatic activity for RNA degradation) during defrosting. The breakage of the cells was done by applying a heat shock procedure, i.e. cells suspended in Trizol were incubated at 95 °C for 5 min and then submerged immediately on ice for additional 5 min. Next, the released RNA was separated from the cell debris by centrifugation (Eppendorf 5418R) at 10000 g, for 10 min at 4°C. RNA purification was performed at 4 °C using the Direct-zol RNA miniprep 2050 (Zymo Research, by S.A Laborimpex NV, Brussels, Belgium) following the manufacturer's instructions. The volume ratio of the aqueous and organic phases was 1:1. RNA samples (150 µg) were then treated once for 30 min at 37°C using DNAse (Ambion TURBO DNA-freeTM, Life Technologies Europe B.V, Ghent, Belgium) following the manufacturer's instructions. The RNA was concentrated at 4 °C using RNA Clean & Concentrator™-25 (Zymosearch, by S.A Laborimpex NV, Brussels, Belgium). RNA quantity and purity was assessed by spectrophotometric analysis using NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Isogent Life science, Temse Belgium). The quality and integrity of RNA was assessed using the Bioanalyzer 2100 (Agilent Technologies, Diegem, Belgium) according to manufacturer's instructions. It is worthwhile to mention, that the Bioanalyser RNA profiles for Arthrospira do not allow to determine 'RNA integrity number' (RIN) values as the profiles are different from standard profiles obtained for most other bacteria. The rRNA profile for Arthrospira contains 3 fragments (3 peaks) instead of 2, representing 16S and 23S rRNA, as has also been

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 reported for Nostoc punctiforme [23]. Absence of DNA was confirmed by PCR with universal 16S primers.

V.2.7 Microarray design. The full genome of Arthrospira sp. PCC 8005 was sequenced by Genoscope (Team of Dr. Valerie Barbe) and Version 3 (692 contigs, ~6.8 Mbp) of this genome [19] was used as input for the microarray design by Nimblegen ( Madison,wI, USA). A tiling array 'Arthrospira HX12' was designed, with probes ranging from 50 up to 72 nucleotides and an average length of 53 nucleotides, and an average spacing of 34 nucleotides between 2 different probes. The 135 367 probes – excluding random and control probes – were mapped back to the improved version 5 of the genome (six ordered contigs), currently publically available at EMBL database (accession number : GCA_000176895, CAFN1000000 ) and imported in the Microbial Genome Annotation & Analysis Platform (MaGe) allowing private expert annotation of genes, which could be grouped to 5854 CDS and 3141 intergenic regions. For the production of Arthrospira HX12 microarray chips, the 12x135k array format of Nimblegen (Madison, WI, USA) was used.

V.2.8 RNA analysis via microarrays. For 2 radiation doses tested (i.e. 3200 and 5000 Gy), the RNA extracts of 3 irradiated cultures and their equivalent 3 non-irradiated cultures (n =3) were collected. At Institute for Research of Biomedicines in Barcelona (IRBB) in Barcelona, cDNA library preparation and amplification were performed on 25 ng of this total RNA, using the Complete Whole Transcriptome Amplification WTA2 kit (Sigma-Aldrich) and according to the instructions of the manufacturer with 17 cycles of amplification, resulting in microgram quantities of cDNA. Labelling and hybridization of the cDNA onto the new designed Arthrospira HX12 arrays (Nimblegen, wI, USA), were performed according to the Roche-Nimblegen expression guide v5p1. For each sample, 1µg cDNA was labelled by Cy3 nonamers primers and Klenow polymerization. Hybridization mixture with 2µg Cy3-labeled cDNA was subsequently prepared. Samples were hybridized to Arthrospira_ HX12 array (Nimblegen, wI, USA) for 18 hours at 42 ºC. The arrays were washed and scanned in a MS 200 scanner (Roche-Nimblegen). Raw data files (Pair and XYS files) were obtained from images using DEVA software (Roche-Nimblegen) and provide by IRBB to SCK•CEN.

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.2.9 Microarray data analysis Both for 3200 and for 5000 Gy, 3 microarrays of irradiated cultures and 3 microarrays of their equivalent non-irradiated cultures (n =3) were analysed. Raw data were pre-processed using the “Oligo” package (version 1.24) in BioConductor (version 2.12 / R version 3.0.1) as follows: i) background correction based on the Robust Multichip Average (RMA) convolution model [24], ii) quantile normalization to make expression values from different arrays more comparable [25], and iii) summarization of multiple probe intensities for each probe set to one expression value per gene using the median polish approach [24]. To test for differential expression between the different irradiated conditions and the reference conditions (no irradiation) the Bayesian adjusted t-statistics was used as implemented in the “LIMMA” package (version 2.18.0) [26]. P-values were corrected for multiple testing using the Benjamini and Hochberg’s method to control the false discovery rate [27]. Transcripts were considered significantly differentially expressed when the corresponding adjusted p-value was less than 0.05 and their absolute fold change (FC) was equal or larger than 2 for upregulated genes, and equal or miner than 0.5 for the down regulated ones. The fold change is the parameter measuring the change in the expression level of a gene between two conditions e.g. irradiated versus non-irradiated. Gene annotation was based on the manual expert annotation available in MaGe (ARTAN consortium) and further curated manually during this work.

V.2.10 Protein extraction and analysis Protein extractions were done on 3 independent cultures for each test condition (n = 3). Aliquots of two ml were centrifuged (Eppendorf, 5418R) at 10000 g for 10 min and cell pellets were stored at -80 °C immediately after irradiation. For protein extraction pellets were resuspended in approximately 100 µl of 6 M guanidine chloride solution pH 8.5 (Lysis buffer of ICPL Kit (SERVA, Germany)), and cells were lysed by sonication (U50 IKAtechnik, Boutersem, Belgium) (3 cycles of 10 s, amplitude 30%, 1 pulse rate) on ice. The samples were subsequently centrifuged (Eppendorf, 5418R) at 16 000 g at 4 °C for 15 min, to separate the soluble proteins from the insoluble cell debris. The total protein concentration was determined using the Bradford method with the Bio-Rad Protein Assay kit (Bio-Rad, Hertfordshire, UK) according to the manufacturer’s instructions, using bovine gamma globulin as a protein standard. Exactly 100 µg of proteins were treated to reduce their disulphide bounds using 0,5 µl of 5 mM Tris (2-carboxyethyl) phosphine (ICPL-SERVA Kit) at 60 °C for 20 min and then alkylated using 0.5 µl of 0.4 mM iodoacetamide (ICPL-SERVA Kit) at 25 °C for 20 min. The reaction was stopped by adding 0.5 µl of stop solution

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 (ICPL-SERVA Kit). Proteins were recovered by acetone precipitation during at least 2 h, using an acetone/protein ratio of 4:1 (V/V). Next, after a 15-min centrifugation at 16 000 g and an acetone evaporation, the resulting pellet was dissolved 800 µl of 50 mM ammonium bicarbonate containing 20 µg of trypsine (Promega V51.11). The enzymatic digestion of the proteins to peptides was performed overnight at 37 °C. The digestion was stopped then by adding formic acid (0.1% final v/v). Peptides were separated via reversed phase liquid chromatography and eluted with a gradient of acetonitrile from 10 to 35% during 120 min. Peptides were then ionized via Electron spray ionization source (ESI) at 150 °C and then analysed via Triple TOF 5600 (AB) SCIEX. Protein identification was performed against a local copy of the Arthrospira sp. PCCC 8005 genome version V5 using ProteinPilot Software v4.1 and the Paragon algorithm (4.0.0.0, 459). Search parameters included trypsin digestion and cysteine alkylation set to iodoacetamide. Processing parameters were set to “Biological modification” and a thorough ID search effort was used. Mass tolerance was set to 10 ppm in MS and 0.05 Da in MS/MS. Peptide FDR rate was set to 5% or less (p< 0.05) based on decoy database searching and all peptides included for analysis had a score representing ≤1% FDR (p≤ 0.01) in at least one of the search engine results. In addition, all peptides were manually inspected. For protein quantification, the protein needed to be represented by at least one unique peptide with 95% confidence (p< 0.05). MS1 chromatogram-based quantitation was performed in Skyline [28] (http://proteome.gs.washington.edu/software/skyline/). Details for MS1 Filtering and MS1 ion intensity chromatogram processing in Skyline were described recently in detail by Schilling et al. [29]. Briefly, comprehensive spectral libraries were generated in Skyline using the BiblioSpec algorithm [30] from database searches of the raw data files prior to MS1 Filtering. Subsequently, raw files acquired in data-dependent mode were directly imported into Skyline v1.3 and MS1 precursor ions extracted for all peptides present in the MS/MS spectral libraries. Quantitative analysis is based on extracted ion chromatograms (XICs) and resulting precursor ion peak areas for each peptide M, M+1, and M+2, the first, second, and third isotope peak of the isotopic envelope. ANOVA Test was performed to analyse the data and to define if protein quantity in irradiated and non-irradiated samples was significant different. Only quantitative data exhibiting a p-value < 0.05 and a fold change FC ≥ 1.25 or FC ≤ 0.8 were considered as biologically significant.

Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.2.11 Statistical analysis. For preparing data graphs and for statistical analysis the Prism software (version 5.00, GraphPad Software) was used, using a Paired T-Test with confidence interval 95% (p < 0.05).

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.3 Results

V.3.1 Recovery and proliferation In order to assess the ability of Arthrospira to recover after irradiation, the cells were allowed to regrow at the optimal conditions for photosynthetic growth (Figure V-1 and Table S 1). The growth curves showed that Arthrospira sp. PCC 8005 was able to regrow normally after exposure to 200 Gy until 1600 Gy. Regrowth was also observed after 3200 Gy, 5000 Gy and 6400 Gy of gamma radiation, although with a significant delay in time, up to 10-17 days (Table S1). With an increasing dose, the delay in post-irradiation growth increased.

Figure V-1: Growth curves of Arthrospira sp. PCC 8005 following exposure to different doses of gamma rays. Data represent mean of three independent biological replicates (n=3), and error bars present the standard error of the mean (SEM). V.3.2 Functionality of PSII system: Quantum yield To allow post-irradiation cell proliferation, cyanobacteria need to harvest light via the antenna on their membranes and generate cellular energy through photosynthesis, via photosystem II (PSII). Therefore, the functionality of PSII of Arthrospira was assessed immediately after irradiation by measuring Chlorophyll a (Chla) and phycobilisome fluorescence, to determine the PSII quantum yield FV/FM. Healthy Arthrospira cells normally have a yield FV/FM of ca. 0.6 [21], as was indeed measured for the non-irradiated control cultures, while cells without photosynthetic activity would

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 have a yield FV/FM of 0.0. The photosynthetic yield of the cells exposed to 200 Gy till 1600 Gy of gamma rays was not significantly affected by irradiation. Cells exposed 3200 Gy, 5000 Gy and

6400 Gy, showed a significant decrease of FV/FM to 0.5 or 0.4 (Figure V-2). These were also the cultures that displayed a delay in photosynthetic growth after irradiation.

Figure V-2: PSII quantum yield of Arthrospira sp. PCC 8005 after gamma irradiation. The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Data represent mean of three independent cultures (n= 3) and error bars present the standard error of the mean (SEM). An asterisk indicates a value for the irradiated sample which is significantly (p<0.05) lower than the value of the representative non-irradiated control culture.

V.3.3 Pigment content. To enable photosynthesis, cyanobacteria have large light-harvesting antennas on their membranes [31]. These antennas, also called phycobilisomes, are protein complexes which contain the photoactive pigments allophycocyanin and phycocyanin [32]. Phycobilisomes harvest and transmit the energy to PSII reaction centre containing chlorophyll (Chla) [33]. Here, the pigment content of Arthrospira sp. PCC 8005 was analysed immediately after irradiation. Data showed a significant decrease in allophycocyanin and phycocyanin after exposure to doses of 3200 Gy or higher (Figure V-3, A and B). No significant change occurred in the overall chlorophyll A content (Figure V-3, C). This reduction in light harvesting pigments coordinate well with our findings of PSII quantum yield that was also reduced at doses of 3200 Gy or higher.

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Figure V-3: Significant reduction in light harvesting antenna pigments (allophycocyanine and phycocyanine) while stable chlorophyll A pigment content of Arthrospira sp. PCC 8005 after irradiation. A: allophycocyanin content, B: Phycocyanin content; C: Chlorophyll A content. The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Data represent mean of three independent biological replicates, and error bars present the standard error of the mean (SEM). One asterisk indicates a value which is significant (p<0.05) different from the value of the non-irradiated control. Three asterisk indicate a value which is highly significant (p<0.001)

V.3.4 Photosynthesis and energy production

RNA analysis showed a major decrease in the expression of all components of the photosynthetic apparatus, which likely resulted in the impaired photosynthesis and proliferation after 3200 Gy and 5000 Gy irradiation discussed above. Several genes coding for phycobilisome pigment biosynthesis (cpc and apc genes) were down regulated and one gene specific for phycobilisome degradation was activated (nblA2) (Table V-2). Similarly, transcription of chlorophyll A pigment biosynthesis (chl genes) and PSII biosynthesis (psb genes) was reduced, while transcription of proteases for degradation of PSII-D1 proteins (ftsH) was induced (Table V-1, Table V-2). Likewise the significant upregulation of the psbI gene was seen. The exact role of PsbI in the assembly of PSII is unclear, nevertheless it has been shown that the loss of PsbI led to a dramatic destabilization of CP43 (PsbC) a core antenna protein of photosystem II , and suggest that PsbI might contribute to an early assembly partner for D1 protein (PsbA) [34]. In addition, the transcription of several genes involved in electron transfer from PSII to PSI, such as the ndh plastoquinone genes, cyd and

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 cox cytochrome genes, and hem haem biosynthesis genes, were reduced (Table V-1, Table V-2). Likewise, the transcription of several genes encoding the structural subunits of photosystem I (psa genes) was repressed (Table V-1). In addition, most of the genes involved in production and conversion of energy obtained from photosynthesis, such as the ferredoxin (FD) gene (petF), the ferredoxin:NADP+ oxidoreductase (FNR) gene (petH) and the ATP synthase coding operon (atp genes), showed a reduced expression.

Table V-1: Transcriptomic (microarray) results for genes known to be involved in photosynthesis. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for statistically not significant differentially expressed (p ≥ 0.05).

PHOTO- Accession number Gene Protein Function Fold Fold SYNTHESIS change change 3200 Gy 5000 Gy PSII ARTHROv5_10312 psbA1 Photosystem II reaction center D1 protein Q(B) NS NS ARTHROv5_10319 psbA2 Photosystem II reaction center D1 protein Q(B) NS NS ARTHROv5_40241 psbA3 Photosystem II reaction center D1 protein Q(B) 0,31 0,39 ARTHROv5_60197 psbA4 Photosystem II reaction center D1 protein Q(B) NS NS ARTHROv5_10245 psbB Photosystem II P680 chlorophyll A apoprotein (CP47 protein) 0,57 0,27 ARTHROv5_11994 psbC Photosystem II CP43 protein NS 0,58 ARTHROv5_11993 psbD1 Photosystem II reaction center D2 protein Q(A) NS NS ARTHROv5_60553 psbE Photosystem II reaction center subunit V (Cytochrome b559 NS 0,53 subunit alpha) ARTHROv5_60554 psbF Photosystem II reaction center subunit VI (Cytochrome b559, 0,58 0,35 subunit beta) ARTHROv5_60555 psbL Photosystem II reaction center protein L 0,53 0,32 ARTHROv5_40752 psbH Photosystem II reaction center protein H (PSII-H) NS 0,62 ARTHROv5_40753 psbN Photosystem II reaction center protein N (PSII-N) NS NS ARTHROv5_30303 psbI Photosystem II reaction center protein I 1,63 4,40 ARTHROv5_11112 psb28 Photosystem II reaction center protein W (13 kDa protein) NS NS ARTHROv5_61163 psb28 Photosystem II reaction center psb28-like protein NS 1,95 ARTHROv5_20093 psb27 Photosystem II 11 kD protein NS 0,51 ARTHROv5_40153 psbO Photosystem II Mn-stabilizing polypeptide precursor (MSP) NS 0,23 ARTHROv5_10852 psbP Photosystem II oxygen-evolving complex 23K protein NS NS ARTHROv5_40969 psbU Photosystem II extrinsic protein precursor (12 kDa protein) NS NS ARTHROv5_50093 Putative cytochrome c-550-like protein precursor 0,31 0,38 ARTHROv5_50094 psbV Cytochrome c-550 precursor 0,30 0,28 ARTHROv5_61031 psbY Photosystem II protein Y NS 0,46

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ARTHROv5_11396 psbZ Photosystem II reaction center protein Z (PSII-Z) NS NS ARTHROv5_60878 ftsH D1 protein specific ATP-dependent zinc-metallo protease 1,28 2,78 ARTHROv5_61180 isiA Iron stress-induced Photosystem II chlorophyll-binding 4,98 NS protein (CP43') Plastoquinone ARTHROv5_10689 ndhA NAD(P)H-quinone oxidoreductase, membrane subunit H 0,71 0,29 (PQ) ARTHROv5_10690 ndhI NAD(P)H-quinone oxidoreductase, subunit I NS 0,19 ARTHROv5_10691 ndhG NAD(P)H-quinone oxidoreductase, chain 6 NS 0,42 ARTHROv5_10692 conserved protein of unknown function 0,37 0,30 ARTHROv5_10693 ndhE NAD(P)H-quinone oxidoreductase, membrane subunit K NS 0,31 ARTHROv5_40057 ndhH NAD(P)H-quinone oxidoreductase, chain H 0,57 0,45 ARTHROv5_40540 ndhD1 NAD(P)H-quinone oxidoreductase chain 4 NS 0.53 ARTHROv5_40541 ndhF1 NAD(P)H-quinone oxidoreductase, chain 5 NS 0,42 ARTHROv5_40542 ndhD2 NAD(P)H-quinone oxidoreductase, chain 4 NS 0,35 ARTHROv5_60388 ndhF2 NAD(P)H-quinone oxidoreductase, chain 5 NS 0,38 ARTHROv5_60389 ndhD3 NAD(P)H-quinone oxidoreductase, chain 4 NS 0,35 ARTHROv5_60547 ndhJ NAD(P)H-quinone oxidoreductase, subunit J NS 0,41 ARTHROv5_60548 ndhK NAD(P)H-quinone oxidoreductase, subunit K NS 0,50 ARTHROv5_60549 ndhC NAD(P)H-quinone oxidoreductase, chain A NS 0,49 ARTHROv5_60715 ndhD4 NAD(P)H-quinone oxidoreductase, chain 4 0,53 0,17 ARTHROv5_60716 ndhF NAD(P)H-quinone oxidoreductase, subunit F NS 0,21 Cytochrome ARTHROv5_40397 cyoE Protohaem IX farnesyltransferase (haem O synthase) NS 0.32 b6f ARTHROv5_40398 Cytochrome oxidase assembly protein NS 0.29 ARTHROv5_40399 coxB Cytochrome c oxidase subunit II NS 0,54 ARTHROv5_40400 coxA Cytochrome c oxidase subunit I NS 0,41 ARTHROv5_40401 coxC Cytochrome c oxidase subunit III NS 0,32 ARTHROv5_61103 cydB Cytochrome bd ubiquinol oxidase, subunit II NS 0,47 ARTHROv5_60566 ccsB Cytochrome c biogenesis protein NS NS ARTHROv5_60567 Cytochrome c biogenesis protein transmembrane region NS NS ARTHROv5_60740 ccsA Cytochrome c biogenesis protein NS 0,45 ARTHROv5_50134 Cytochrome c, monohaem NS 0,46 ARTHROv5_40277 petJ Cytochrome c6 (Soluble cytochrome f) (Cytochrome c553) 0,35 0,33 ARTHROv5_40850 petA Cytochrome f NS NS Plastocyanin ARTHROv5_40851 petC Cytochrome b6-f complex iron-sulphur subunit 1 plastocyanin NS NS (PC) oxidoreductase PSI ARTHROv5_10984 psaA Photosystem I P700 chlorophyll a apoprotein A1 0,56 0,27 ARTHROv5_10985 psaB Photosystem I P700 chlorophyll a apoprotein A2 0,59 0,28 ARTHROv5_10235 psaC Photosystem I reaction center subunit VII, iron-sulphur center 0,54 0,37 ARTHROv5_30080 psaD Photosystem I reaction center subunit II (16 kDa polypeptide) NS NS ARTHROv5_30570 psaE Photosystem I reaction center subunit IV 2,12 2,21 ARTHROv5_30656 psaJ Photosystem I reaction center subunit IX 0,29 0,14 ARTHROv5_30657 psaF Photosystem I reaction center subunit III precursor 0,35 0,15 ARTHROv5_50157 psaL Photosystem I reaction center subunit V 0,50 0,24

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ARTHROv5_50163 psaK1 Photosystem I reaction center subunit X NS 0.51 ARTHROv5_40172 psaX Photosystem I reaction center subunit 0,31 0,27 ARTHROv5_11973 btpA Photosystem I biogenesis protein NS 0,36 ARTHROv5_11992 ycf4 Photosystem I assembly protein NS NS ARTHROv5_41247 ycf3 Photosystem I assembly protein NS NS Ferredoxin ARTHROv5_60106 petF1 Ferredoxin (2Fe-2S) 0,59 0,29 (FD) ARTHROv5_10430 petF2 Ferredoxin-2 0,27 0,18 FNR ARTHROv5_41386 petH Ferredoxin:NADPH reductase NS 0,57 ARTHROv5_50074 iscA1 FeS cluster assembly protein 0,51 0,58 ARTHROv5_60637 iscA2 FeS cluster assembly protein NS NS ARTHROv5_11765 sufR Iron-sulphur cluster biosynthesis transcriptional regulator NS 2,12 ATP synthesis ARTHROv5_60530 atpI ATP synthase protein I 0,33 0,18 ARTHROv5_60531 atpB ATP synthase A chain (ATPase protein 6) 0,35 0,17 ARTHROv5_60532 atpE ATP synthase C chain, membrane-bound, F0 sector 0,24 0,18 ARTHROv5_60533 atpG2 ATP synthase B chain (Subunit II) 0,07 0,14 ARTHROv5_60534 atpF ATP synthase B chain (Subunit I) 0,07 0,17 ARTHROv5_60535 atpH ATP synthase D chain; ATP synthase F1 0,12 0,15 ARTHROv5_60536 atpA ATP synthase, alpha subunit 0,23 0,17 ARTHROv5_60537 atpG1 ATP synthase, gamma subunit 0,33 0,33 ARTHROv5_12000 atpC ATP synthase epsilon subunit NS NS ARTHROv5_12001 atpD ATP synthase, beta subunit NS NS

Table V-2: Transcriptomic (microarray) results for genes known to be involved in pigment biosynthesis and degradation. The fold change (FC) values listed are only values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for statistically not significant differentially expressed (p ≥ 0.05).

PIGMENT Accession number Gene Protein Function Fold Fold Biosynthesis chan chan ge ge 3200 5000 Gy Gy C-phycocyanin ARTHROv5_11553 cpcB C-phycocyanin beta subunit NS NS ARTHROv5_11554 cpcA C-phycocyanin alpha subunit NS NS ARTHROv5_11555 cpcC1 Phycobilisome linker polypeptide, phycocyanin-associated, rod 1 NS NS ARTHROv5_11556 cpcC2 Phycobilisome linker polypeptide, phycocyanin-associated, rod 2 NS 0,62 ARTHROv5_11557 cpcD Phycobilisome linker polypeptide, phycocyanin-associated, rod NS 0,64 capping ARTHROv5_11558 cpcE Phycocyanin alpha subunit phycocyanobilin lyase NS NS ARTHROv5_11559 cpcF Phycocyanin alpha-subunit phycocyanobilin lyase NS NS

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ARTHROv5_40726 cpcG Phycobilisome rod-core linker protein 0,56 0,32 ARTHROv5_60720 cpcT Chromophore lyase NS 0,34 ARTHROv5_11397 nblB2 Phycocyanin alpha phycocyanobilin lyase related protein NS NS ARTHROv5_50028 nblB1 Phycocyanin alpha phycocyanobilin lyase related protein 1,86 1,80 ARTHROv5_61056 nblA1 Phycobilisome degradation protein NS NS ARTHROv5_61095 nblA2 Phycobilisome degradation protein NS 2,17 Allo- ARTHROv5_10637 apcA Allophycocyanin alpha subunit 0,59 0,19 phycocyanin ARTHROv5_10636 apcB Allophycocyanin beta subunit 0,27 0,13 ARTHROv5_10635 apcC Phycobilisome rod-core linker protein 0,29 0,16 ARTHROv5_60056 apcD Allophycocyanin alpha-B subunit NS 0.53 ARTHROv5_61214 apcE Phycobiliprotein NS 0.51 ARTHROv5_12132 apcF Allophycocyanin beta subunit 0,38 0,16 Chlorophyll ARTHROv5_30766 chlG Chlorophyll a synthase NS 0,28 ARTHROv5_30670 por chlorophyll synthase / NADPH-protochlorophyllide NS 0,31 oxidoreductase ARTHROv5_41143 chlL Light-independent protochlorophyllide reductase 0,35 0,22 ARTHROv5_40946 acsF Aerobic Mg-protoporphyrin IX monomethyl ester NS 0,32 ARTHROv5_11499 chlH Mg chelatase H subunit NS 0,22 ARTHROv5_40768 bchD Mg-protoporphyrin IX chelatase, subunit D NS 0,14 ARTHROv5_61176 bchI (Mg-protoporphyrin IX chelatase (38 kDa subunit) NS NS ARTHROv5_60718 GUN4 domain protein NS 0,36 ARTHROv5_11688 putative GUN4-like regulator 0,28 0,39 Carotenoid ARTHROv5_10189 putative Acyl-CoA dehydrogenase 0,40 0,30 ARTHROv5_10200 conserved hypothetical protein 0,41 0,26 ARTHROv5_10201 conserved hypothetical protein NS 0,31 ARTHROv5_10202 putative Beta-carotene ketolase 0,36 0,18 Isoprenoid ARTHROv5_40094 ispD1 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase NS 0,46 ARTHROv5_11117 ispE 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase NS 0,48 ARTHROv5_20267 ispF 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase NS 0,46 ARTHROv5_30478 ispD 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase NS 0,22 ARTHROv5_30479 hypothetical protein 0,11 0,13 ARTHROv5_60585 ispG 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase 0,22 0,17 Haem ARTHROv5_50123 hemC Porphobilinogen deaminase 0,41 0,38 ARTHROv5_60626 hemE Uroporphyrinogen decarboxylase 0,26 0,29 ARTHROv5_11660 hemF Coproporphyrinogen III oxidase 0,45 0,35 ARTHROv5_10139 hemG Protoporphyrinogen oxidase NS 0,27 ARTHROv5_50161 hemH Ferrochelatase 1,65 1,50 ARTHROv5_30029 hemL Glutamate-1-semialdehyde aminotransferase (aminomutase) 0,29 0,12

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.3.5 Photosensing and cell motility High doses of gamma radiation induced strongly the transcription of several genes coding for 'chromophore' proteins, i.e. photoreceptor pigments involved in photo-sensing and signalling for regulation of cyanobacterial photo-taxis (Table S 2). First, the biosynthesis of tetrahydrobiopterin (BH4)-containing proteins, i.e. pterin-like chromophores, was induced. Tetrahydrobiopterin is biosynthesized from guanosine triphosphate (GTP) by three chemical reactions mediated by the enzymes GTP cyclohydrolase I (GTPCH) (folE1 gene), 6-pyruvoyltetrahydropterin synthase (PTPS) (ygcM), and sepiapterin reductase (SR) [35]. Second, also the gene coding for the Cryptochrome-DASH protein (cry gene), which is a pterine-flavoprotein-type chromophore, was up-regulated. In addition, also several other genes coding for response regulatory proteins with photosensor domains (GAF, PAS), as well as the associated signal transduction and regulatory systems (i.e. histidine kinases and transcriptional regulators), and the genes for synthesis of the secondary messenger cyclic diguanylate (c-di-GMP) were induced. These are all systems typically involved in cell motility regulation. Based on COG enrichment analysis, the category of cell motility (N) proteins indeed showed a significant altered expression after irradiation. However, the expression of genes involved in swimming motility (pilin genes) and floating motility (gvp gas vacuole genes) was reduced by radiation.

V.3.6 Carbon fixation and secondary metabolite biosynthesis

The transcription analysis of genes related to carbon fixation showed a general repression after irradiation. The genes encoding the carbon dioxide fixation mechanism, i.e. the carboxysome (ccm and cch genes), and the genes related Calvin–Benson–Bassham (CBB) cycle (cbb, gpm, gap and glp genes) were repressed (Table V-3).

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table V-3: Transcriptomic (microarray) results for genes known to be involved in Carbon fixation. The fold change (FC) values listed are only values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not statistically significant differentially expressed (p≥0.05).

CO2 FIXATION Accession number Gene Function Fold Change Fold change 3200 Gy 5000 Gy Carboxysome ARTHROv5_60383 putative carbon dioxide concentrating NS 0,57 mechanism protein ARTHROv5_60384 ccmM Carbon dioxide concentrating mechanism 0,21 0,08 protein ARTHROv5_60385 cchB Putative carboxysome-like ethanolaminosome 0,32 0,12 structural protein, ethanolamine utilization protein ARTHROv5_60386 ccmK1 Carbon dioxide-concentrating mechanism 0,18 0,08 protein ARTHROv5_60387 cchA Putative carboxysome-like ethanolaminosome 0,34 0,15 structural protein, ethanolamine utilization protein

ARTHROv5_60714 CO2 hydration protein 0,44 0,16 ARTHROv5_61007 ccmK1 carboxysome shell protein NS 0,21 ARTHROv5_61008 ccmK2 carboxysome shell protein NS 0,19 RuBisCO ARTHROv5_50349 cbbS Ribulose bisphosphate carboxylase (RuBisCO), 0,19 0,08 small subunit ARTHROv5_50350 rbcX Chaperonin family protein 0,32 0,17 ARTHROv5_50351 cbbL Ribulose bisphosphate carboxylase (RuBisCO), 0,15 0,07 large subunit ARTHROv5_50352 cbbR1 Ribulose bisphosphate carboxylase (RuBisCO), 0,42 0,35 operon transcriptional regulator ARTHROv5_50129 rca Ribulose bisphosphate carboxylase/oxygenase NS NS activase ARTHROv5_10999 cbbR2 putative RuBisCO transcriptional regulator, 0,45 0,43 RbcR-like ARTHROv5_10998 cbbR3 putative RuBisCO transcriptional regulator, 0,39 0,41 RbcR-like Calvin cycle ARTHROv5_10997 spkF Ser/Thr protein kinase NS 0,36 ARTHROv5_20037 pgk phosphoglycerate kinase NS 0,47 ARTHROv5_60907 gpmB2 phosphoglycerate mutase NS 0,34 ARTHROv5_30667 gpmB1 phosphoglycerate mutase 0,46 0,21 ARTHROv5_30574 gpmI 2,3-bisphosphoglycerate-independent NS 0,38 phosphoglycerate mutase ARTHROv5_11456 gap1 Glyceraldehyde-3-phosphate dehydrogenase 1 NS 0,31 ARTHROv5_30613 gap2 Glyceraldehyde-3-phosphate dehydrogenase 2 0,53 0,25

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

ARTHROv5_41419 xfp D-xylulose 5-phosphate/D-fructose 6-phosphate 0,46 0,35 phosphoketolase ARTHROv5_20113 gnd gluconate-6-phosphate dehydrogenase, 0,44 0,36 decarboxylating ARTHROv5_10443 pgi Glucose-6-phosphate isomerase 0,45 0,31 ARTHROv5_30212 pfkB fructokinase 0,58 0,31 ARTHROv5_10198 pfkB putative pfkB family carbohydrate kinase 0,67 0,50 ARTHROv5_40143 putative ribulose-5-phosphate 4-epimerase 0,40 0,25 Glycogen ARTHROv5_41216 glgA1 Glycogen synthase 0,31 0,23 biosynthesis ARTHROv5_60979 glgA2 Glycogen synthase 0,59 0,59 ARTHROv5_61087 glgX2 Glycogen debranching enzyme NS 0.24

In line with reduced carbon capture, also genes involved in biosynthesis of intracellular carbon storage compounds such as glycogen (glg genes), and lipids such as fatty acids – including gamma- linolenic acid (GLA) synthesis – (fab and des genes), or intracellular solutes with a role in salt tolerance (stpA and ggpS genes) was transcriptionally reduced (Table S 3). Also genes involved in extracellular metabolite production, such as biosynthesis of the extracellular cyclic peptide pattelamide A (pat genes) were reduced in expression (Table S 3).

V.3.7 Stress response and antioxidants It is well-documented that cyanobacteria developed various antioxidant defence mechanism to cope with ROS damage, involving enzymatic and non-enzymatic ways [36].

Remarkably, the gene coding for the antioxidant enzyme catalase is absent in the Arthrospira sp. PCC 8005 genome, and the expression level of the gene coding for the antioxidant enzyme superoxide dismutase (sodB) was not significantly induced (even slightly reduced). Other known non-enzymatic antioxidants, are the pigments C-phycocyanin and β-carotene, but the genes involved in the synthesis of those compounds was down regulated, as explained above (Table V-2). In contrast, transcriptome data showed in Arthrospira sp. PCC 8005 mainly the up-regulation of the thiol-based antioxidant systems after irradiation, including glutathione, thioredoxin and peroxiredoxin systems. Several genes coding for glutathione synthesis and regeneration (i.e. reduction) were upregulated (gshB glutathione synthase, lactoylglutathione lyase, glutathionylspermidine synthase). Meanwhile the expression level of the glutaredoxin gene (gor), which is involved in glutathione oxidation, was reduced. Similarly, expression of genes coding for

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 thioredoxin was reduced, while the thioredoxin-reductase gene (trxB) for thioredoxin-regeneration (i.e. reduction), was induced. Moreover, our findings showed a significant elevation in the transcription level of gene coding for peroxiredoxin. It is reported that transcription of several key genes coding for proteins involved in redox homeostasis, such as glutaredoxin and a number of thioredoxins, is regulated by the Fur transcriptional regulator [37] whose expression was also found induced in Arthrospira sp. PCC 8005 (fur) after irradiation. Also other Fur regulated genes involved in iron homeostasis, such as the iron stress inducible isiA gene and the bacterioferritin genes, were differentially expressed.

V.3.8 Protein damage and recycling Irradiation induced expression of genes encoding heat shock proteins (HSP), known as molecular chaperones. These groups are a class of functionally related proteins involved in the folding and unfolding of other proteins. (Table S 5). Arthrospira sp. PCC 8005 contains five copies of the HSP70-type dnaK gene and the transcription level of 3 of them (dnaK1, dnaK2 and dnaK5) was significantly induced. Likewise, also the upregulation of dnaJ and cbpA (a dnaJ homologue) genes, which act synergistically with dnaK, was observed. The groL1 and groL2 genes, coding for the large subunit of the HSP60-type GroESL, were also found upregulated. However, groS, which generally act as a co-chaperone of GroEL, did not show a significant expression change. In addition, a set of protease and peptidase genes (e.g. HSP100-type clpS2) involved in the proteolytic degradation and removal of proteins that are damaged beyond repair, were upregulated after ionizing radiation (Table S 5). And as mentioned before, also the transcription of some very target specific proteases such as nblA and ftsH, involved in controlled phycobilisome or PSII-reaction center D1 protein degradation, were increased.

V.3.9 DNA-repair and genetic modifications Many of the differentially expressed genes in irradiated cells were involved in repair DNA damage (Table S 6). A few genes belonging to the dsDNA damage repair pathways such recJ, recQ, recG, holB, and gyrA were differentially expressed. However, the recA gene, which a key protein for DNA repair in bacteria, such as Deinococcus radiodurans [38], was not differential expressed. Surprisingly, the gene for the related LexA repressor that works together with RecA and activates the transcription of SOS system, is absent in Arthrospira sp. PCC 8005 genome. Microarray data did reveal differential expression of many genes involved in ssDNA-damage repair, either via

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Nucleotide excision repair (NER), such as uvr genes, or either via the ssDNA mismatch repair system (MMR), such as the mut genes. And non-repaired nucleotides were potentially removed by nudix hydrolase, which also displayed an up regulation pattern. Additionally, an interesting group of genes coding for Type I site specific deoxyribonucleases was highly upregulated. Some of those genes showed high homology with the R, S and M subunit of the Type I restriction modification system (RM) of Arthrospira platensis NIES39. Restriction- modification enzymes are used by many organisms to protect themselves against foreign DNA [39], and the role of RM systems in Arthrospira platensis has been discussed [40]. Type I restriction modification systems are composed of three subunits, encoded by three hsd genes: the hsdR gene required for restriction activity; the hsdS gene responsible for DNA specificity, and the hsdM gene which is required for methylation activity. These enzymes add a methyl-group to a DNA molecule at a specific site to protect the site from restriction endonuclease cleavage, and thus from DNA damage. Irradiation induced also a set of genes potentially involved in a toxin/antitoxin system and a number of mobile genetic elements (MGEs) (Tables S7, S8, and S9). In total, 37 genes coding for transposases (jumping genes) displayed an up-regulation pattern (Table S 7). Moreover, Arthrospira sp. PCC 8005 contains regions of phage immunity (CRISPR) genes (cas genes) and 7 phage-like genomic islands (fax genes) (Table S 8), of which 1 copy and 7 copies respectively exhibited a clear increase in their transcripts level after IR. This is the first observation that these genomic elements are responding to an exogenous stress in the cells environment, and thus might still be functional.

V.3.10 Differentially expressed conserved hypothetical proteins The genes coding for conserved hypothetical proteins (Table S 10) with unassigned function (COG: N/A) were the most abundant among all differentially expressed genes. A cluster of 7 genes, named arh (ARTHROv5_10466 to ARTHROv5_10472), showed a very high expression in a dose- dependent response to ionising radiation (Table V-4).

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table V-4: Transcriptomic and proteomic results for conserved hypothetical proteins, specifically expressed in response to ionising radiation. The fold change (FC) values listed are only values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5 for microarray data, FC > 1.25 or < 0.8 for proteomics data. 'NS' stands for statistically not significant differentially expressed (p≥0.05) as

RNA or as protein. 'ND' stands for not detected as protein. Bold are genes of which the proteins were also detected in Proteomics.

RNA RNA Protein Protein Fold Fold Fold Fold Accession number Gene Protein Function COG change change change change 3200 Gy 5000 Gy 3200 Gy 5000 Gy putative ABC-type phosphate transport ARTHROv5_10472 arhA P 14,58 9,24 ND ND system, periplasmic component, PstS-like ARTHROv5_10471 arhB conserved protein of unknown function D NS 22,35 1,75 NS conserved protein of unknown function ARTHROv5_10470 arhC (conserved domain involved in chromosome L 3,13 14,62 1,40 NS segregation) ARTHROv5_10469 arhD conserved hypothetical protein - 5,43 9,46 ND ND ARTHROv5_10468 arhE conserved protein of unknown function - 9,58 11,98 7,12 4,30 ARTHROv5_10467 arhF conserved hypothetical protein - 5,10 5,75 ND ND ARTHROv5_10466 arhG transcriptional regulator, XRE family K 2,55 2,78 ND ND

A number of these genes were in fact not only found to be highly up regulated at RNA level, but also at protein level (Table V-4, Table S 10, Table S 11). Three of the Arh proteins displayed high abundance after exposure to both 3200 Gy and 5000 Gy. It is well known, that the overlap between mRNA transcript and proteomics is minor, nevertheless proteomic results showed a clear correlation with respect to this new set of proteins. This is peculiar, as overall the proteomics analysis revealed only few differentially expressed proteins, i.e. 31 and 36 respectively after 3200 Gy and 5000 Gy, and with little similarity between 3200 Gy and 5000 Gy (Table S 10,Table S 11). Advanced bio-informatics analysis was carried out to assess the conservation and potential function of these proteins. Two proteins, ArhB and ArhC, were conserved within the cyanobacteria phylum. Arthrospira species showed the highest amino acids sequence similarity with PCC 8005 ranging from 95.12 %, 94.82 % and 94.21 % respectively for Arthrospira platensis C1, Arthrospira maxima CS328 and Arthrospira platensis NIES-39. Less nucleotide similarity was seen with members of the Nostocacea family such as Nostoc punctiforme PCC 73102 (49.38 %), Anabeana variabilis

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 ATCC 29413 (48.46 %) and Nostoc sp. PCC 7120 (47.84 %). Interestingly, the comparison of the two proteins with the Deinococcacae family, demonstrate amino acids sequence similarity with Deinococcus radiodurans R1 species. In addition, the analysis of conserved functional domains, showed only conserved domain involved in chromosome segregation for ArhC protein.

V.4 Discussion Arthrospira sp. PCC 8005 cells showed photosynthetic recovery and proliferation after all doses of 60Co gamma radiation (dose rate of 527 Gy h–1) tested, up to a total absorbed dose of 6400 Gy. This classifies this bacterium as 'radiation resistant' [41,42]. In general, for cyanobacteria, many studies focused on the tolerance to photo-synthetically active radiation (PAR)- and ultra violet (UV) radiation (photons in the wavelength range of 700nm to 400 nm, corresponding to photon energies from 1.5 eV to 3 eV; and photons of 400 nm - 1 nm with 3 eV - 1000 eV respectively), to understand its impact on the growth and biomass yield when they are cultivated for example on spirulina farms for diverse biotechnological applications [43-45]. Arthrospira was indeed also found to be tolerant to high fluxes of VIS and UV [43]. Most studies looked at the impact of UV-A (315-400 nm) or UV-B (280-315 nm). Few studies reports the effect of UV-C (100 nm-280 nm) or shorter wavelength (<100 nm) on cyanobacteria since theses wavelength does not reach the Earth surface at present owing to the absorption by ozone and losses through atmospheric scattering. One team investigated the effect of a Martian UV-flux (>200 nm) (e.g. UVC) on the cyanobacterium Chrococcidiopsis [46]. Only very few studies have investigated the tolerance of cyanobacteria to even more energetic radiation, such as X-rays (photons of 1 nm - 0,01 nm and 1000 eV -100 000 eV) [8] or gamma-rays (photons < 0,01 nm and > 100 000 eV) [6,47]. This study is the first, showing that Arthrospira is highly tolerant to gamma rays, and can survive at least 6 400 Gy (dose rate of 527 Gy h–1). A trait similar as the rock- dwelling coccoidal cyanobacterium Chroococcidiopsis able to survive X-ray as high as 15000 Gy [6]. Likewise, the planktonic filamentous cyanobacterium Anabaena, was tolerant up to 5000 Gy of acute 60Co gamma radiation (dose rate of 6250 Gy h–1) without adverse effect on diazotrophic growth and metabolism [8]. In addition the resistance of coccoidal cyanobacterium Synechocystis sp. PCC 6803 up to 30000 Gy 60Co gamma radiation (dose rate 10000 Gy h–1) was reported [48]. From an ecological point of view, one could wander why Arthrospira is gamma radiation resistant, as in its current natural habitat (soda lakes) it is not exposed to such types or such doses of ionising radiation. It is assumed that cyanobacteria have acquired an advanced defence mechanism against

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 radiation since they were exposed to high levels of ionising radiation on Earth during the Precambrian era [49]. On an early Earth, without complete atmosphere, cyanobacteria were exposed to high intensities of photosynthetic active light PAR or VIS, ultraviolet light UV and other types of electromagnetic waves such as X-rays and Gamma-rays. Such high energetic electromagnetic waves (photons) are strongly penetrating and can damage cells by interacting directly with cellular components (DNA, proteins, lipids) or indirectly with water molecules producing free radicals leading to cell damage [50]. As such, it is assumed that early on cyanobacteria have developed high effective mechanism to protect themselves and deal with detrimental effects of radiation [51], by combining multiple strategies such as avoidance (e.g. moving away), protection (e.g. shielding), detoxification (e.g. antioxidants), and repair [44]. Nevertheless, so far, no molecular investigations were performed, to understand this extraordinary radiation resistance property of cyanobacteria to such ionizing radiations. In this study, a new full genome tilling-array chip, specific for the filamentous Arthrospira sp. strain PCC 8005 was designed and constructed, in order to investigate the transcriptomic response of this cyanobacterium to high doses of gamma irradiation. Overall, a general reduction in the expression of genes, coding for the structural units of the light harvesting system (phycobilisomes), the photosynthesis systems (PSII and PSI), electron transfer systems (plastoquinones, cytochromes, ferredoxin), reduced carbon fixation and energy production systems (ATP synthase), was observed. Similar observations were reported in other studies [52,53], showing the impact of UV-B radiation on the cyanobacterium Synechosystis. This likely caused the reduction in photosynthetic activity and as such the delayed photosynthetic growth after exposure to ionizing radiation. The growth curves after exposure to 3200 Gy, 5000 Gy and 6400 Gy, did show a significant delay of 10 till 17 days, likely due to radiation-induced damage in the cells, which required longer repair and recovery. Similar delay in growth after irradiation has been reported for the fast growing Deinococcus radiodurans irradiated with 15000 Gy in which cell growth was restored only after 9 h [54]. The photosynthesis measurements confirmed that the PSII system of Arthrospira sp. PCC 8005 was still partially functional even after irradiation with 6400 Gy, but doses of 3200 Gy or more clearly had a significant effect on photosynthetic quantum yield. Photosynthesis and electron transport chains are the main source of reactive oxygen species (ROS) under physiological conditions. Thus shutting down photosynthesis seems a logic response in an effort to reduce production of oxidants, which is already enhanced by ionising radiation. Photosynthesis and carbon

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 fixation shutdown was also reported for Arthrospira in response to other stress factors such as nitrogen depletion stress [55] , but was never before demonstrated in such detail, at RNA level. Only physiological examinations were reported and there are no transcriptomic data available for Arthrospira in response to UV stress [43]. Hence, it was not possible to further explore such comparison. It is generally assumed that the photosynthetic pigments are potent antioxidants, and thus high intracellular concentrations of such pigments would be beneficial in ROS and radiation defense. Therefore, it was surprising to find reduced level of these pigments in irradiated Arthrospira cells. Indeed, analysis of the pigment cells contents showed a significant decrease in allophycocyanin and phycocyanin concentration exposed to 3200 Gy, 5000 Gy and 6400 Gy. Moreover, molecular data indicated specific down regulation of the enzymes involved in the biosynthesis of phycobilin and chlorophyll. The reduced pigment level is likely also the result of a controlled degradation guided by the enzyme NblA [56]. The observed increased transcription level of nblA gene would indeed suggest active phycobilisome degradation [57]. Such response has been explained as an effort of the cell to lower the proportion of light harvesting phycobilisomes, to minimize the effective exposure to harsh environment. For example, the nblA gene is also induced in expression during the desiccation of the filamentous cyanobacterium Microcoleus vaginatus [58]. A wide variety of enzymatic and non-enzymatic antioxidant systems has been reported to be involved in cellular radiation resistance. The primary scavenging defence system to neutralize the reactive oxygen species (ROS) is mediated by antioxidants enzymes such as (i) superoxide dismutase (Sod) to neutralize superoxide radicals (formed in the presence of dissolved oxygen), and (ii) catalase (Cat), glutathione peroxidase (Gpx), peroxiredoxin and other peroxidases to neutralize hydroperoxides (produced by the radiolysis of water) [59]. Similar enzymatic antioxidant systems have been reported in cyanobacteria. It has been shown that filamentous cyanobacterium Nostoc punctiforme ATCC 29133, induced mainly sod and cat to cope with oxidative stress generated by UVA exposure [60]. The unicellular Synechosystis sp. PCC 6803 increased the transcription level of sod and gpx upon UVB exposure [52]. Filamentous Anabeana sp. PCC7120 was able to cope with oxidative stress induced by salt and UVB by increasing the transcription levels of peroxiredoxin [45]. Regarding Anabeana sp PCC 7120, the Ahp alkylhydroperoxide reductase protein was reported to play an important role in combating multitude stresses: heat, copper, salt and ionizing radiations such as UVB [61]. While in Nostoc

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 punctiforme challenged by UVA radiations, the ahp gene was down regulated [60]. Also the highly gamma radiation resistant bacterium Deinococcus radiodurans relies on the enzymatic antioxidants, such as catalase, superoxide dismutase, glutaredoxines, thioredoxin reductase and alkylhydroperoxide reductase [62]. Arthrospira sp. PCC 8005, however, is catalase-negative, which is an exceptional trait for all Arthrospira species [40]. Arthrospira sp PCC 8005 does contain a number of genes for peroxiredoxin and other putative peroxidases but for most of these genes the expression was not changed or even reduced after irradiation. Either superoxide dismuatse (sod) in Arthrospira cells is not needed for radiation resistance, or either the unchanged level of expression could perhaps be due to a permanent high abundance, hence not needing additional induction. Either one of these hypothesises would need to be investigated more in detail and confirmed. Regarding non-enzymatic antioxidants molecules recent studies revealed that protecting protein from oxidation may allow the cells to survive high number of double strand breaks caused by ionizing radiation. In several studies [2] it was shown that small antioxidant molecules such the manganese and orthophosphate may play a key role in preventing protein oxidation, thereby protecting DNA-repair enzymes and increasing the efficiency in DNA repair. Our data indicate that Arthrospira sp. PCC 8005 seems to mainly rely on non-enzymatic thiol-based antioxidant systems such as glutathione (GSH) to cope with oxidative damage from ionising radiation comparing to other bacteria (Figure V-4). Following exposure to gamma rays Arthrospira sp. PCC 8005 expressed several genes involved in the synthesis and recycling of glutathione. Glutathione 1 is a potent scavenger of singlet oxygen O2, hydrogen peroxide H2O2, and the most harmful ROS hydroxyl radical OH•, [63,64]. The thiol group is very reactive, and quickly neutralizes radicals. Interesting to know, is that Deinococcus radiodurans in fact lacks the classical glutathione system, including gluthatione reductase [62]. A recent study [42], did however, reported the presence of thiol-based antioxidant in Deinococcus radiodurans, called Bacithiol and considered as a substitute for glutathione, with a role in its extreme resistance to gamma rays. In plants glutathione is one of the most crucial metabolites and is also considered as the most important intracellular defence system against ROS-induced oxidative damage [65]. The transcription of the fur regulator was induced after gamma irradiation. Increased transcription of fur in response to redox stress has been shown for many bacteria and in general suggests a cellular reorganisation to (i) increase the Fe2+-binding capacity, (ii) repress iron uptake and (iii) promote iron storage system via bacterioferritine, DPS and ferritine [66]. It has been documented

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 that in cyanobacteria, fur also contributes in the regulation of isiA which is an iron stress inducible gene coding for CP43’ proteins, presenting a pigment storage, a light harvesting ring structure surrounding PSI and energy dissipation for PSII [67,68]. The correlation between CP43’ and the core of PSII system, composed by CP34 and CP47, were reported [69]. It has been shown that hydrogen peroxide could enhance isiA expression as well. The transcriptome analysis of Synechocystis PCC6803 challenged by iron deficiency or hydrogen peroxide revealed a high overlap in the induction of isiA gene after both treatments [70]. Another strategy for cyanobacteria to cope with high doses of energetic photons (high light intensities, UV, IR) is to escape from stressing situation, by actively 'moving away' from the stress source towards a less stressful (more shielded) environment [49]. Thus as defence against high light, the cyanobacteria evolved sensory photoreceptors in the cell envelope, to monitor photon flux and to activate different cell motility systems when needed [71]. A significant transcriptional increase of Cry-DASH photoreceptor gene was observed in Arthrospira sp. PCC 8005 after irradiation. In Synechosystis sp. PCC 6803, Cry-DASH binds a pterine (Folate) derivate, which acts as light harvesting antenna sensor that absorb photons energy generated by UVA/Bleu light and transfers the energy to flavin molecule, to activate a negative phototaxis away from the light stress [72,73]. Despite the activation of antioxidant systems, the data suggest a significant protein, lipid, and DNA damage response. It is known, that ionizing radiation can cause oxidation or defolding of proteins that are needed for DNA repair [74]. However, Arthrospira cells seem to have several molecular chaperones and folding catalysts in place to prevent or deal with this damage. The increased transcription of heat shock proteins HSP, protease and peptidase coding genes by Arthrospira, was likely a response deal with the protein damage caused by the high doses of irradiation. HSP proteins such as the HSP70-type DnaK and GroEL/GroE are present in highly conserved forms in all bacteria, including cyanobacteria, and play crucial role in folding of newly synthesized proteins, preventing protein misfolding or aggregation and promoting protein degradation [75]. Similar observation was reported for the cyanobacterium Synechosystis sp. PCC 6803 which exhibit an increase in chaperonin GroEL and GrpE after exposure to UVB [52]. Arthrospira sp. PCC 8005 contains also five copies of HSP70-type dnaK gene, which is a lot comparing to Deinococcus which has only one copy, and the transcription level of 3 of them was significantly induced. Proteins that are oxidised beyond repair are dysfunctional and need to be removed and rapidly resynthesized

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 [76]. This requires efficient proteolytic degradation of the damaged proteins [2]. Indeed, it has been shown for some bacteria, including Deinococcus, that the level of intracellular proteolytic activity increased following radiation exposure [2,77]. Arthrospira sp. PCC 8005 induced the expression of a set of protease genes, including the nblA and ftsH genes which allow specific proteolysis of key components of the photosynthesis system [78]. The FtsH protein has been shown to be involved in repair of the PSII system in Synechocystis sp. PCC 6803 [70]. Therefore, it seems that the role of FtsH in PSII repair and D1 turnover might be conserved in both cyanobacteria and higher plants [79]. For DNA, as the dose of electromagnetic ionizing radiation (IR) increases, the linear density of bases damages and single strand breaks (SSBs) increases on both strands, which gives rise to double-strand breaks (DSBs). A specific dose of IR typically causes 40 times more SSBs than DSBs [38]. Our data showed, mainly, an activation of genes related to SSB, including the nucleotide excision repair (NER) and mismatch repair (MMR) system in Arthrospira after irradiation. The NER multi-enzyme complex UvrABC, is an exonuclease that recognize the structural changes in DNA, and is involved in the removal of many types of DNA lesions [80]. The mismatch repair (MMR) system, involves proteins such as MutS1, MutL, and UvrD, and was also activated in D. radiodurans after exposure to gamma rays [81]. Arthrospira sp. PCC 8005 lacks the RecBCD system, but it contains the RecFOR pathway for DSB repair via recombination, which is also the key pathway for post-irradiation genome reconstruction through extended synthesis- dependent strand annealing process (ESDSA) in Deinococcus radiodurans [82]. Transcriptome data also revealed the induction of a NUDIX gene in Arthrospira exposed to gamma rays. Proteins of the NUDIX family are abundantly present in the highly radiation resistant bacterium Deinococcus radiodurans and are involved in the housecleaning and fast recovery of the cells [83]. The major role of these enzymes is the degradation and the export of damaged DNA to purify the cells [62]. In addition to DNA repair genes, irradiated Arthrospira sp. PCC 8005 also overexpressed a large set of genes involved in the restriction modification mechanism, phage– immunity, and mobile genetic elements, possibly indicating radiation-induced genetic rearrangements. Mobile Genetic Elements (MGE's) have been shown to be important components of genomic rearrangements [84]. In most bacteria the expression of the DNA repair genes is under control of the SOS response system, which is usually silent and only activated in the case of DNA damage [85]. The induction

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 of the DNA repair system is regulated by two key SOS proteins, RecA and LexA. The coprotease RecA activates auto-cleavage of the transcriptional repressor LexA, which in turn then allows transcription of several genes involved in DNA damage repair, including RecA, which is also an essential DNA repair protein. This basic mechanism of LexA-dependent induction of DNA-repair in response to radiation seems to be conserved in E. coli and B. subtilis [86]. But LexA is not always required for the induction of RecA and DNA-repair in general, as in D. radiodurans for example it was demonstrated that an abundance in RecA protein following gamma radiation was detected in a lexA knock-out mutant [87]. Also in cyanobacteria LexA does not seem to play a key role in radiation resistance. Whereas a homolog of LexA exists in most cyanobacteria [88], it does not appear to be linked to DNA repair at least in Synechocystis sp. PCC 6803 [89]. Microarray analysis with a lexA mutant from Synechocystis revealed that LexA does not regulate typical DNA repair genes in this organism but, rather, might be important for genes involved in carbon metabolism [90]. Our findings actually show that the lexA repressor gene is absent in the genome of Arthrospira sp. PCC 8005. This has been also observed in other bacteria, such as Helicobacter pylori for example [91]. Hence, the DNA repair process seems to be independent of LexA in several organisms, including several radiation resistant organisms. Regarding the recA gene, our results showed no induction of this gene in Arthrospira after irradiation, neither at RNA level nor in protein abundance. In D. radiodurans recA gene was induced after irradiation [42], but absence of recA induction after irradiation was also reported in Synechocystis (Domain et al (2004) and Helicobacter pylori following UV and gamma radiation [91]. RecA is crucial for DNA repair and essential photosynthetic prokaryotes [92], thus probably also active in Arthrospira sp PCC 8005 after irradiation. But, it might be that Arthrospira cells constitutively produce large amounts of RecA protein at all times, even in the absence of radiation induced DNA damage. For Helicobacter pylori, this has been shown and suggested as explanation for the absence of recA induction [91]. Thus, this could also be a hypothesis to test for. The molecular analysis revealed also a new set of proteins that were induced seemingly in a dose- dependent manner following exposure to high doses of gamma rays. This set of genes was clustered in one genomic region, and annotated to code for 'conserved hypothetical proteins'. Although it is well known that mRNA expression profiles are not always causative but can be merely correlative and are not always easily correlated with proteomic abundance [93], these proteins were confirmed to be overexpressed both on RNA and protein level. Currently little can be said regarding the

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 function of this interesting series of genes but it does appear that they exhibit a specific response to high acute doses of gamma irradiation. As far as we are aware, these proteins were never been reported as significantly expressed in Arthrospira in response to any other stress condition tested such as light stress [94] or nitrogen deficiency [55], which makes their response to ionising radiation rather unique and peculiar. It is possible that this set of genes may play an important role in the high radiation resistance of Arthrospira sp. PCC 8005. In general, a way to demonstrate the function of such proteins and their role in radiation resistance should involve the knockout of the genes in Arthrospira and to investigate such mutants in details. Unfortunately, unlike other unicellular cyanobacteria (e.g. Synechococcus and Synechocystis) and some filamentous cyanobacteria (e.g. Anabaena), genetic transformation of Arthrospira had limited success to date, which is a drawback. Nevertheless, these results opened new horizons of research that involve deeper investigation of cellular radiation sensitivity or resistance and the role of these proteins therein, which is currently on-going in our team.

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

Figure V-4: A conceptual model describing the response of Arthrospira sp. PCC 8005 to gamma irradiation. The circular diagram represents a cell with the key genes and proteins up regulated after irradiation in Red and the down-regulated ones in Bleu. Gamma irradiation leads to ROS production and Redox imbalance. Glutathione molecules are used as key antioxidant molecules for ROS scavenging (the Glutathione cycle and the related enzymes involved in Glutathione synthesis were highlighted). Also enzymatic antioxidants such as Peroxiredoxin are used. ROS causes DNA damage, which activates DNA reparation, involving a number of different genes (uvr, rec, gyrA, mut, nud). Oxidative stress activates the Fur regulon which controls iron homeostasis and enhance isiA gene expression for protection of the photosystems PSI and PSII. In parallel, there is shutdown of all genes related to the synthesis of photo-pigments (cpc, apc, chl), the degradation of phycobilisomes by protein NblA is activated, and there is a coordinated repression of all the genes required for photosynthesis (psa, psb, atp). There is an induction of the photosensor Cry-DASH gene and the biosynthesis pathway of pterine, which are involved in signalling and phototactic

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 movement. Moreover, there is an activation of several new genes, coding for conserved hypothetical proteins (Arh) in response to gamma rays.

V.5 Conclusion In summary, this study demonstrated for the first time the resistance of the free-floating filamentous and edible cyanobacteria Arthrospira to high doses of gamma rays. Thanks to the newly designed DNA microarray specific to Arthrospira sp. PCC 8005, molecular analysis of the response to irradiation stress could be done in depth. Arthrospira cells exposed to ionising radiation shut down photosynthesis and carbon fixation while protein and DNA damage is repaired. Moreover, there was no significant induction of classical bacterial enzymatic antioxidant system such as SOD and peroxide reductase, and RecA a key protein for DNA repair. In contrast, a clear activation of thiol- based antioxidant systems, such the glutathione, was seen in Arthrospira, a system that is well known for plants but absent in many other radiation resistant bacteria such as Deinococcus. Beyond the response linked to genes with known functions, a novel set of seven conserved proteins of unknown function was identified. They were overexpressed in response to radiation exposure in a dose-dependent manner, providing new interesting targets for the future research. This first study was primarily observational in nature to screen for general cellular responses, but our basis for further detailed research.

V.6 Acknowledgements This work was supported through a PhD grant for Hanène Badri by SCK•CEN and ESA/Belspo via the ARTEMISS study, which is part of the MELiSSA project. The new microarray used in this study was designed based on the Arthrospira sp. PCC 8005 genome sequence (Version 3), produced in collaboration with the team of Dr. Valerie Barbe (Genoscope, Evry, France); and analysed and annotated via the MAGE platform provide by the team of Dr. Claudine Medigue (Genoscope, Every, France), with the help of the Dr. Paul Janssen (SCK•CEN, Mol, Belgium) and the ARTANN consortium; within the frame of the MELGEN-1&2&3 projects. This work was also partially financed by the FNRS under grant “grand equipement” N°2877824. Special thanks for Catherine Sheeren from UMONS for her help in sample preparation for protein analysis. We would like to thank also Annie Rodolosse from the Institute for Research of Biomedicines in Barcelona IRBB for hybridization steps and microarray processing.

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 V.8 Supplemental data

Doses Exposure time IR 200 Gy 21min47sec IR 800 Gy 1h27min7sec IR 1600 Gy 2h54min15sec IR 3200 Gy 5h48min30sec IR 5000 Gy 9h4min30sec IR 6400 Gy 11h36min

Figure S 1: RITA Facilty of the Belgian reactor (BR2) at SCK•CEN. RITA Facilty of the Belgian reactor (BR2) at SCK•CEN. The cultures were irradiated in the dark inside the cansiter, submerged in the water, surrounded by 4 sources of 60Co providing gamma rays with energy of ca. 1.33 Mev and 1.17 Mev, with a dose rate 527 Gy h-1. The time required for irradiation was dependent of the total received dose, as indicated in the table added to the figure. For each irradiation dose, an equivalent culture was kept for the same time in dark as representative non-irradiated control.

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For each time interval between 2 time points was calculated with following formula: µ = ( ) ( ) ln OD750 at t2 −ln 푂퐷750 푎푡 푡1 . The last row presents the maximum growth rate obtained for each 푡2−푡1 radiation dose. Data represent mean of three independent cultures (n= 3). An asterisk indicates a value for the irradiated sample which is significant (p<0.05) different from the value of the corresponding non-irradiated control. Three asterisk indicate a value which is highly significant (p<0.001).

CTR 200 Gy 800 Gy 1600 Gy 3200 Gy 5000 Gy 6400 Gy Time intervals (Days) (n=3) (n=3) (n=3) (n=3) (n=3) (n=3) (n=3) 1 -8 0,326 0,277 0,208 0,101 0 0 0 8-10 0,212 0,303 0,398 0,388 0 0 0 10-15 0,122 0,123 0,137 0,311 0,761 0,542 0 15-17 -0,002 0,085 0,204 0,148 0,282 0,003 0 17-21 0,002 0,004 -0,03 -0,012 0,045 0,232 0,505 21-24 -0,055 -0,073 -0,069 -0,055 -0,081 0,167 0,547 24-25 ------0,577 Lag time 0 0 0 0 10* 10 17 (µ = 0) (days) 0,326 0,303 0,398 0,388 0,761* 0,542 0,577 Maximum growth rate µmax ±0,135 ±0,02 ±0,096 ±0,130 ±0,012 ±0,092 ±0,272

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A : Total genes 3200 Gy 5000 Gy

B : Up-regulated C : Down-regulated 3200 Gy 5000 Gy 3200 Gy 5000 Gy

Figure S 2: Transcriptomic expression profile for the 5854 coding DNA sequences (CDS, or “genes”) of Arthrospira sp. PCC 8005 after exposure to 3200 Gy and 5000 Gy of gamma rays A: Represent all the differentially expressed genes (p<0.05), B: represent only the upregulated genes (p<0.05), and FC>2, and C: represent only the down regulated genes (p<0.05), and FC<0.05

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table S 2: Transcriptomic (microarray) results for genes known to be involved in photosensing, signalling, and motility. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

PHOTOSENSIN Accession number Gene Protein Function Fold Fold G & MOTILITY change change 3200 Gy 5000 Gy Chromophores ARTHROv5_40253 ygcM 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) 1,39 2,10 ARTHROv5_40925 folE GTP cyclohydrolase I (GTPCH) 7,31 12,54 ARTHROv5_20034 folD Bifunctional protein Methylenetetrahydrofolate NS 0,26 dehydrogenase ARTHROv5_40926 Putative metallo-dependent phosphatase 7,60 11,59 ARTHROv5_10963 cry Cryptochrome-DASH protein 2,22 3,36 ARTHROv5_20097 Putative diguanylate cyclase/phosphodiesterase 12,83 7,96 (GGDEF & EAL domains) with Phytochrome (GAF) ARTHROv5_20098 Putative diguanylate cyclase (GGDEF domain) NS NS ARTHROv5_20099 Sensor protein 2,58 2,70 ARTHROv5_30350 Signal transduction histidine kinase:Sensor with GAF 1,91 2,35 domain ARTHROv5_10439 Conserved protein of unknown function 3,43 7,79 ARTHROv5_10440 Response regulator receiver modulated PAS/PAC 1,32 3,60 sensor(S) Chemotaxis ARTHROv5_11061 cheY1 Putative response regulator receiver 1,16 2,18 ARTHROv5_11062 Putative ABC transporter, ATP-binding protein NS 2,51 ARTHROv5_11063 Hypothetical protein NS NS ARTHROv5_11064 cheY2 Response regulator receiver 1,72 3,01 ARTHROv5_60796 cheY3 Two-component response regulator 1,35 9,01 ARTHROv5_60571 cheC1 CheC inhibitor of MCP methylation 1,75 2,45 ARTHROv5_60572 cheB1 Fused chemotaxis regulator 3,13 3,44 ARTHROv5_60573 cheR1 CheR Chemotaxis protein methyltransferase, MCP NS NS methyltransferase, Protein-glutamate O- methyltransferase ARTHROv5_60574 Putative PAS/PAC sensor protein NS NS ARTHROv5_60575 Methyl-accepting chemotaxis protein NS NS ARTHROv5_60576 cheW1 CheW protein, purine-binding chemotaxis protein, NS 1,9 Chemotaxis signal transduction protein ARTHROv5_60577 cheA1 CheA signal transduction histidine kinase 1,64 2,05 ARTHROv5_11949 cheB2 Chemotaxis Response Regulator protein 1,94 2,37 Motility ARTHROv5_60996 Putative Pilin biogenesis protein 0,86 0,32 ARTHROv5_50098 Putative leader peptidase (Prepilin peptidase) 0,72 0,38 Gas vacuoles ARTHROv5_12032 gvpA Gas vesicle structural protein A 0,40 0,25 ARTHROv5_12033 gvpC1 Gas vesicle structural protein 0,18 0,16 ARTHROv5_12034 Conserved protein of unknown function 0,24 0,22 ARTHROv5_12037 gvpC2 Gas vesicle structural protein 0,23 0,18

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ARTHROv5_12038 Hypothetical protein 0,09 0,09 ARTHROv5_12039 gvpN Gas vesicle protein 0,06 0,08 ARTHROv5_12040 gvpJ Gas vesicle synthesis protein 0,23 0,32 ARTHROv5_11240 gvpW Putative gas vesicle protein 1,03 0,60

Table S 3: Transcriptomic (microarray) results for genes known to be involved in secondary metabolite production. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

METABOLITE Accession number Gene Protein Function Fold Fold biosynthesis change change 3200 Gy 5000 Gy Glycogen ARTHROv5_41216 glgA1 Glycogen synthase 1 0,30 0,22 ARTHROv5_60979 glgA2 Glycogen synthase 2 0,59 0,59 ARTHROv5_20114 glgP Glycogen/starch/alpha-glucan phosphorylases 0,36 0,23 ARTHROv5_61087 glgX2 Glycogen debranching enzyme 0,85 0,24 ARTHROv5_60834 glgB 1,4-alpha-glucan branching enzyme NS 0,56 Lipid ARTHROv5_10240 fabF1 3-oxoacyl-[acyl-carrier-protein] synthase 2 0,69 0,32 ARTHROv5_30260 fabG1 3-oxoacyl-[acyl-carrier-protein] reductase 0,58 0,24 ARTHROv5_30826 fabI Enoyl-[acyl-carrier-protein] reductase [NADH] 0,50 0,32 ARTHROv5_41232 fabZ (3R)-hydroxymyristoyl-[acyl-carrier-protein] 0,28 0,19 dehydratase ARTHROv5_40656 desD Delta-6 fatty acid desaturase 0,39 0,19 ARTHROv5_60707 desA Delta-12 fatty acid desaturase 0,59 0,31 Glucosylglycerol ARTHROv5_10080 stpA Glucosylglycerol 3-phosphatase 0,73 0,23 (GG) ARTHROv5_10816 suhB Inositol monophosphatase 0,51 0,49 ARTHROv5_30402 gpsA NAD+ dependent glycerol-3-phosphate dehydrogenase 0,55 0,41 ARTHROv5_30403 glpD sn-glycerol-3-phosphate dehydrogenase, aerobic, 0,43 0,36 FAD/NAD(P)-binding ARTHROv5_30404 ggpS Glucosylglycerol-phosphate synthase 0,22 0,36 Pattelamide A ARTHROv5_40574 patB conserved hypothetical protein 0,43 0,06 ARTHROv5_40575 patC conserved hypothetical protein 0,42 0,05 ARTHROv5_40587 patF conserved hypothetical protein 0,53 0,36 Toxin/AntiToxin ARTHROv5_12008 mazF mRNA interferase 2,57 7,52 ARTHROv5_12009 Toxin/ antitoxin 1,26 5,72 ARTHROv5_40682 putative antitoxin of toxin-antitoxin system, family Axe NS NS ARTHROv5_40683 putative toxin of toxin-antitoxin system, family 0,23 0,37 Txe/YoeB ARTHROv5_11210 putative antitoxin of toxin-antitoxin system, YefM-like NS 8,02

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ARTHROv5_11211 putative toxin of toxin-antitoxin system, YoeB-like NS 6,03 Polyhydroxy- ARTHROv5_10499 phaE Poly(R)-hydroxyalkanoic acid synthase, class III, subunit NS NS butyrate (PHB ) ARTHROv5_10500 phaC Poly(R)-hydroxyalkanoic acid synthase, class III, subunit NS 2,24 ARTHROv5_60059 phbA acetyl-CoA acetyltransferase with thiolase domain NS 0,20 (Acetoacetyl-CoA thiolase) ARTHROv5_10067 3-hydroxyisobutyrate dehydrogenase 2,81 3,29 Patatin ARTHROv5_30480 Patatin NS 0,50 ARTHROv5_10493 Putative patatin-like phospholipase NS NS ARTHROv5_10494 Hypothetical protein NS 2,03 ARTHROv5_10495 Putative patatin-like phospholipase NS 2,84 Haemolysin ARTHROv5_40115 Haemolysin-type calcium-binding toxin (secreted) 2,33 2,12 ARTHROv5_40224 Putative haemolysin-typecalcium-binding toxin, RTX- NS 0,38 like ARTHROv5_12129 Putative haemolysin A-like cytotoxin 2,04 2,08

Table S 4: Transcriptomic (microarray) results for genes known to be involved in stress response and antioxidant defense. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05). 'ND' stands for not detected.

ANTIOXIDANTS Accession number Gene Protein Function Fold Fold change change 3200 Gy 5000 Gy Catalase (CAT) Gene absent kat Catalase ND ND Superoxide ARTHROv5_50113 sodB Superoxide dismutase, Fe 0,84 0,68 dismutase Peroxiredoxin ARTHROv5_30341 Putative peroxiredoxin 2,20 1,66 ARTHROv5_20231 ahp Putative alkyl hydroperoxide reductase, AhpC-like 0,76 0,98 Glutathione (GSH) ARTHROv5_30647 gshB Glutathione synthase 2,13 3,80 ARTHROv5_60129 Putative Lactoylglutathione lyase (Glyoxalase I) 3,50 5,60 ARTHROv5_60820 Glutathionylspermidine synthase 1,87 2,80 ARTHROv5_11282 Hydroxyacylglutathione hydrolase 0,32 0,68 ARTHROv3_430028 gor Glutathione oxidoreductase 0,74 0,57 Glutaredoxin ARTHROv5_60735 grx Monothiol glutaredoxin NS NS (GRX) Thioredoxin (TRX) ARTHROv5_30047 trxA1 Thioredoxin-1 0,49 0,39 ARTHROv5_30261 trxA2 Thioredoxin-1 0,86 1,39 ARTHROv5_11001 trxA4 Thioredoxin-1 0,39 0,16 ARTHROv5_41074 trxB Thioredoxin-disulfide reductase 2,30 1,98

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ARTHROv5_10124 dbsA1 Putative disulfide oxidoreductase (fragment) 2,34 2,55 ARTHROv5_10131 dbsA2 Putative disulfide oxidoreductase NS 2,46 Fe-homeostasis ARTHROv5_30342 fur Transcriptional regulator, Fur family protein 2,68 1,94 ARTHROv5_11765 sufR Iron-sulphur cluster biosynthesis transcriptional NS 2,12 regulator ARTHROv5_61180 isiA Iron stress-induced chlorophyll-binding protein 4,98 1,81 (CP43') ARTHROv5_40087 bcp1 Bacterioferritin comigratory protein 0,59 0,21 ARTHROv5_10833 bcp4 Bacterioferritin comigratory protein 0,70 0,45 ARTHROv5_60045 Ferrous Iron(II) transporter 0,43 NS ARTHROv5_60046 Ferrous Iron(II) transporter, B domain protein NS NS ARTHROv5_60047 feoA Putative FeoA family protein NS NS Stress response ARTHROv5_10153 dps DNA protection during starvation protein NS NS ARTHROv5_11130 uspA Universal stress protein 1,55 4,72

Table S 5: Transcriptomic (microarray) results for genes known to be involved in protein repair and recycling. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

PROTEIN Fold Fold DAMAGE Accession number Gene Protein Function change change CLEAN-UP 3200 Gy 5000 Gy

HSP70-type ARTHROv5_10362 dnaK1 Chaperone protein, HSP70-type 1,99 3,97

ARTHROv5_11814 dnaK2 Chaperone protein, HSP70-type NS NS

ARTHROv5_11998 dnaK3 Chaperone protein, HSP70-type (fragment, part 2) NS NS ARTHROv5_30014 dnaK4 Chaperone protein, HSP70-type NS 4,66 ARTHROv5_30685 dnaJ Chaperone protein,, HSP70-type NS 2,25 ARTHROv5_30686 dnaK5 Chaperone protein, HSP70-type 2,20 3,49 ARTHROv5_61127 cbpA Curved DNA-binding protein, DnaJ homologue 0,51 5,35

HSP60-type ARTHROv5_30259 groL1 Cpn60 chaperonin, large subunit of GroESL 2,50 2,97

ARTHROv5_61181 groL2 Cpn60 chaperonin, large subunit of GroESL 2,34 2,35

ARTHROv5_61182 groS Cpn10 chaperonin GroES 1,49 1,48

HSP100-type ARTHROv5_11700 clpS2 ATP-dependent Clp protease adapter protein 1,32 2,40 ARTHROv5_60878 ftsH ATP-dependent zinc-metallo protease 1,27 2,77 ARTHROv5_61095 nblA2 Phycobilisome degradation protein NS 2,17

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table S 6: Transcriptomic (microarray) results for genes known to be involved in DNA repair. The fold change (FC) values listed are only values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05), 'ND' stands for not detected, NER for nucleotide excision repair, MMR for MisMatch Repair, BER and RM for restriction modification system.

Fold Fold DNA repair Accession number Gene Protein Function change change 3200 Gy 5000 Gy ARTHROv5_61151 radC RadC-like gene NS NS ssDNA- repair Excinulease of nucleotide excision repair UvrABC ARTHROv5_40732 uvrB 3,59 4,35 NER system, subunit B, DNA damage recognition component Excinuclease of nucleotide excision repair UvrABC ARTHROv5_60258 uvrC 3,51 2,86 system, subunit C ARTHROv5_41027 uvrD UvrD/REP DNA helicase (UvrD-RepA-PcrA like) 2,17 5,42 ssDNA- repair ARTHROv5_10136 mutS DNA mismatch repair protein 2,15 2,87 MMR ARTHROv5_40086 mutT hydrolase/pyrophosphatase, a NUDIX enzyme 3,74 3,45 ssDNA- repair formamidopyrimidine/5-formyluracil/ 5- ARTHROv5_30569 mutM 2,02 NS BER hydroxymethyluracil DNA glycosylase dsDNA repair Gene absent lexA SOS response transcriptional regulator (repressor) ND ND Recombination DNA strand exchange and recombination protein, with ARTHROv5_11364 recA NS NS protease and nuclease activity ARTHROv5_10396 recF DNA replication and repair protein RecF NS 0,42 ARTHROv5_60968 recO DNA repair protein RecO NS 0,41 ARTHROv5_10624 recR Recombination protein RecR NS NS ARTHROv5_20108 recJ DNA-specific exonuclease 2,76 2,31 ARTHROv5_41370 recQ Putative ATP-dependent DNA helicase, 1,84 2,31 ARTHROv5_40176 recG ATP-dependent DNA helicase 2,20 2,93 ARTHROv5_40244 holB DNA polymerase III, subunit delta prime 2,09 2,45

ARTHROv5_40734 gyrA DNA gyrase (type II topoisomerase), subunit A 1,97 3,60 DNA repair ARTHROv5_30623 hsdR1 Type I site-specific deoxyribonuclease, HsdR family 7,25 19,90 Restriction ARTHROv5_30624 hsdR2 Type I site-specific deoxyribonuclease, HsdR family 13,55 11,12 ARTHROv5_30625 hsdR3 Type I site-specific deoxyribonuclease, HsdR family 5,65 4,47 ARTHROv5_60699 hsdM Type I restriction-modificationsystem DNA methylase 3,95 1,88 ARTHROv5_60700 hsdS Type I restriction enzyme 4,00 4,27 ARTHROv5_50002 pvuIIM Type II restriction-modification DNA methylase 2,30 4,86 ARTHROv5_50004 pvuIIR Type II restriction enzyme 2,84 9,75 NUDIX ARTHROv5_61096 nudF ADP-ribose pyrophosphatase, a NUDIX enzyme NS 2,54

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table S 7: Transcriptomic (microarray) results for genes known to be involved in genetic rearrangement, in specific genes from “Transposases”. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

Fold Fold Transposases Accession number Gene Protein Function change change 3200 Gy 5000 Gy

ARTHROv5_10410 Transposase, IS630 family (fragment) 1,37 2,53 ARTHROv5_10411 Transposase (fragment) 1,31 4,75 ARTHROv5_10512 Transposase, IS630 family (fragment) 1,37 6,48 ARTHROv5_10564 Transposase, IS630 family (fragment) 1,31 4,56 ARTHROv5_10570 Transposase 1,32 3,31 ARTHROv5_10577 Transposase 1,35 2,78 ARTHROv5_10641 Transposase, IS630 family (fragment) 1,20 3,60 ARTHROv5_10881 Transposase (fragment) 1,66 5,66 ARTHROv5_10882 Transposase 1,33 2,99 ARTHROv5_11071 Transposase, IS605 family, OrfB (fragment) 0,85 2,48 ARTHROv5_11514 Transposase 1,21 2,18 ARTHROv5_11930 Transposase 1,49 2,32 ARTHROv5_11972 Transposase, IS605 family (fragment) 0,65 0,40 ARTHROv5_20017 Transposase, IS605 family, OrfB (fragment) 0,44 0,27 ARTHROv5_30018 Transposase (fragment) 2,80 2,38 ARTHROv5_30092 Transposase, IS630 family (fragment) 1,29 2,95 ARTHROv5_30093 Transposase, IS630 family (fragment) 1,33 5,87 ARTHROv5_30094 Transposase, IS630 family (fragment) 1,17 3,01 ARTHROv5_30127 Transposase, IS630 family (fragment) 0,44 0,30 ARTHROv5_30136 Transposase, IS4 family (fragment) 1,88 2,90 ARTHROv5_30141 Transposase 2,64 2,02 ARTHROv5_40367 Transposase, IS630 family (fragment) 1,32 2,88 ARTHROv5_40434 Transposase 1,26 2,72 ARTHROv5_40469 Transposase (fragment) 1,12 3,92 ARTHROv5_40511 Transposase (fragment) 1,39 2,25 ARTHROv5_40799 Transposase (fragment) 3,42 4,00 ARTHROv5_40800 Transposase, IS630 family (fragment) 1,76 2,78 ARTHROv5_40819 Transposase 1,22 2,08 ARTHROv5_41186 Transposase, IS630 family (fragment) 1,31 4,30 ARTHROv5_41194 Transposase (fragment) 1,26 2,45 ARTHROv5_41214 Transposase (fragment) 1,45 6,76 ARTHROv5_41266 Transposase, IS630 family (fragment) 1,36 4,35 ARTHROv5_41267 Transposase, IS630 family (fragment) 1,42 3,75 ARTHROv5_50016 Transposase (fragment) 3,43 5,25

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ARTHROv5_50128 Transposase, IS605 family, OrfB (fragment) 0,91 2,28 ARTHROv5_60487 Transposase 1,24 3,65 ARTHROv5_60945 Transposase 1,20 2,40

Table S 8: Transcriptomic (microarray) results for genes known to be involved in genetic rearrangement, in specific genes from phage-like (Fax) Elements. In total, there are 7 Fax clusters in the genome. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

Fold Fold FAX Accession number Gene Protein Function change change Elements 3200 Gy 5000 Gy

FAX1 ARTHROv5_10112 Putative phage tail sheath protein, Gp18-like NS 0,25 ARTHROv5_10113 Conserved Hypothetical protein NS 0,18 ARTHROv5_10114 Putative phage tail region protein NS 0,26 ARTHROv5_10115 Putative phage tail region protein NS 0,29 ARTHROv5_10116 Conserved Hypothetical protein 0,46 0,33 ARTHROv5_10117 Conserved Hypothetical protein NS 0,38 ARTHROv5_10118 Conserved Hypothetical protein NS 0,41 ARTHROv5_10119 Conserved Hypothetical protein NS NS ARTHROv5_10120 Conserved Hypothetical protein NS 0,46 FAX2 ARTHROv5_10166 Conserved Hypothetical protein NS NS ARTHROv5_10167 faxA2 Unknown phage of the genus Arthrospira, protein A 1,58 1,59 ARTHROv5_10168 faxB2 Unknown phage of the genus Arthrospira, protein B 2,17 1,71 ARTHROv5_10169 Conserved Hypothetical protein 5,94 NS ARTHROv5_10170 Conserved Hypothetical protein 6,18 3,65 ARTHROv5_10171 faxE2 Unknown phage of the genus Arthrospira, protein E 4,06 1,32 ARTHROv5_10172 Conserved Hypothetical protein NS NS ARTHROv5_10173 Conserved Hypothetical protein NS NS ARTHROv5_10175 Conserved Hypothetical protein NS NS ARTHROv5_10176 Conserved Hypothetical protein NS NS ARTHROv5_10177 faxJ2 Unknown phage of the genus Arthrospira, protein J 0,70 1,04 FAX3 ARTHROv5_10892 faxP3 Unknown phage of the genus Arthrospira, protein P 1,41 6,57 ARTHROv5_10893 Conserved Hypothetical protein 4,08 NS ARTHROv5_10894 Conserved Hypothetical protein NS NS ARTHROv5_10895 Conserved Hypothetical protein 2,28 3,10 ARTHROv5_10897 Conserved Hypothetical protein 8,69 5,63 ARTHROv5_10898 Conserved Hypothetical protein 5,21 5,36 ARTHROv5_10899 Conserved Hypothetical protein 4,64 4,22 ARTHROv5_10900 faxJ3 Unknown phage of the genus Arthrospira, protein J 1,16 1,74

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FAX4 ARTHROv5_20060 faxG4 Protein of fax element 2,94 0,87 ARTHROv5_20061 Conserved Hypothetical protein NS NS ARTHROv5_20062 Conserved Hypothetical protein NS NS ARTHROv5_20064 faxJ4 Unknown phage of the genus Arthrospira, protein J 0,80 1,80 ARTHROv5_20065 Conserved Hypothetical protein 8,11 NS ARTHROv5_20066 Conserved Hypothetical protein 6,49 7,38 ARTHROv5_20067 Conserved Hypothetical protein 10,42 10,44 ARTHROv5_20070 Conserved Hypothetical protein 2,35 2,28 ARTHROv5_20071 Conserved Hypothetical protein 5,91 10,79 ARTHROv5_20073 Conserved Hypothetical protein NS 3,80 FAX5 ARTHROv5_30540 Conserved Hypothetical protein NS NS ARTHROv5_30541 faxA5 Unknown phage of the genus Arthrospira, protein A 1,45 1,55 ARTHROv5_30544 Conserved Hypothetical protein NS NS ARTHROv5_30545 faxE5 Unknown phage of the genus Arthrospira, protein E 7,31 1,06 ARTHROv5_30546 Conserved Hypothetical protein NS NS ARTHROv5_30549 Conserved Hypothetical protein NS NS ARTHROv5_30551 faxJ5 Unknown phage of the genus Arthrospira, protein J 0,79 1,10 ARTHROv5_30552 Conserved Hypothetical protein 5,20 3,19 ARTHROv5_30553 faxK5f3 unknown phage of the genus Arthrospira, protein K (fragment) 10,49 4,11 ARTHROv5_30554 Conserved Hypothetical protein 8,32 5,03 ARTHROv5_30555 Conserved Hypothetical protein NS 5,36 ARTHROv5_30556 Conserved Hypothetical protein 4,93 4,73 ARTHROv5_30557 Conserved Hypothetical protein 5,10 7,00 ARTHROv5_30558 Conserved Hypothetical protein 7,60 5,83 ARTHROv5_30563 Conserved Hypothetical protein 5,45 6,99 ARTHROv5_30564 Conserved Hypothetical protein 5,84 8,42 ARTHROv5_30565 faxP5 Unknown phage of the genus Arthrospira, protein P 1,31 7,64 ARTHROv5_30566 Conserved Hypothetical protein NS 4,35 FAX6 ARTHROv5_30729 Conserved Hypothetical protein NS 2,10 ARTHROv5_30730 faxP6 Unknown phage of the genus Arthrospira, protein P 1,53 4,53 Unknown phage of the genus Arthrospira, protein O ARTHROv5_30731 faxO6f 4,12 7,19 (fragment) ARTHROv5_30732 Conserved Hypothetical protein 3,15 6,95 Unknown phage of the genus Arthrospira, protein M ARTHROv5_30734 faxM6f1 3,71 3,44 (fragment) ARTHROv5_30735 Conserved Hypothetical protein 5,06 NS ARTHROv5_30737 Conserved Hypothetical protein 6,57 6,63 ARTHROv5_30738 Conserved Hypothetical protein 6,10 6,15 ARTHROv5_30740 Conserved Hypothetical protein 12,77 4,50 ARTHROv5_30741 Conserved Hypothetical protein 5,09 NS ARTHROv5_30749 Conserved Hypothetical protein 7,07 NS ARTHROv5_30751 faxB6 Unknown phage of the genus Arthrospira, protein B 2,67 2,90 ARTHROv5_30752 faxA6 Unknown phage of the genus Arthrospira, protein A 2,49 3,54 ARTHROv5_30753 Conserved Hypothetical protein NS NS

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FAX7 ARTHROv5_40343 faxE7 Unknown phage of the genus Arthrospira, protein E 4,62 1,74 ARTHROv5_40344 Conserved Hypothetical protein NS NS ARTHROv5_40345 faxG7 Unknown phage of the genus Arthrospira, protein G 4,76 NS Unknown phage of the genus Arthrospira, protein K ARTHROv5_40353 faxK7f2 5,41 6,89 (fragment)

Table S 9: Transcriptomic (microarray) results for genes known to be involved in genetic rearrangement, in specific genes from “CRISPRs” elements. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

Fold Fold CRISPRs Accession number Gene Protein Function change change 3200 Gy 5000 Gy

CRISPR 1 ARTHROv5_40676 cas2 CRISPR-associated endoribonuclease Cas2 6,76 3,34 ARTHROv5_40678 cas1 CRISPR-associated endonuclease Cas1 3,69 2,24 ARTHROv5_40688 CRISPR-associated RAMP protein 2,12 2,90 ARTHROv5_40690 csm3 CRISPR-associated RAMP protein, Csm3 family 1,63 3,50 ARTHROv5_40694 csm2 CRISPR-associated RAMP protein, Crm2 family 1,83 2,71 ARTHROv5_40716 csm5 CRISPR-associated RAMP protein, Csm5 family 1,44 2,28 ARTHROv5_40717 csm4 CRISPR-associated RAMP protein, Csm4 family protein 1,56 2,65 ARTHROv5_40718 csm3 CRISPR-associated RAMP protein, Csm3 family 1,29 3,98

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table S 10: Transcriptomic (microarray) results for genes coding for conserved hypothetical proteins of “unknown function”. The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 2 or < 0.5. 'NS' stands for not significant differentially expressed (p>0.05).

onserved Fold Fold hypothetical Accession number Gene Protein Function change change proteins 3200 Gy 5000 Gy

ARTHROv5_10002 Conserved protein of unknown function 1,13 2,40 ARTHROv5_10012 Conserved hypothetical protein 0,90 0,41 ARTHROv5_10037 Conserved protein of unknown function 1,12 2,34 ARTHROv5_10048 Conserved hypothetical protein 2,66 3,96 ARTHROv5_10050 Conserved hypothetical protein 1,90 4,78 ARTHROv5_10055 Conserved hypothetical protein 1,70 2,67 ARTHROv5_10066 Conserved hypothetical protein 1,88 2,04 ARTHROv5_10068 Conserved hypothetical protein (membrane) 2,13 4,54 ARTHROv5_10069 Conserved hypothetical protein (secreted) 2,19 5,99 ARTHROv5_10070 Conserved membrane protein of unknown function 1,53 3,28 ARTHROv5_10075 Conserved hypothetical protein 2,13 2,62 ARTHROv5_10076 Conserved hypothetical protein 1,94 2,14 ARTHROv5_10088 Conserved exported protein of unknown function 0,56 0,40 ARTHROv5_10089 Conserved hypothetical protein 0,29 0,26 ARTHROv5_10090 Conserved hypothetical protein 0,28 0,13 ARTHROv5_10102 Conserved hypothetical protein 2,20 3,93 ARTHROv5_10107 Conserved hypothetical protein 3,30 13,57 ARTHROv5_10113 Conserved hypothetical protein 0,26 0,19 ARTHROv5_10116 Conserved hypothetical protein 0,46 0,33 ARTHROv5_10117 Conserved hypothetical protein 0,52 0,39 ARTHROv5_10122 Conserved hypothetical protein 0,35 0,28 ARTHROv5_10134 Conserved protein of unknown function 1,89 2,94 ARTHROv5_10145 Conserved protein of unknown function 3,68 2,63 ARTHROv5_10179 Conserved protein of unknown function 5,73 5,42 ARTHROv5_10180 Conserved protein of unknown function 6,49 6,33 ARTHROv5_10185 Conserved protein of unknown function 3,81 7,17 ARTHROv5_10186 Conserved protein of unknown function 1,54 5,63 ARTHROv5_10187 Conserved protein of unknown function 1,20 8,04 ARTHROv5_10199 Conserved hypothetical protein 0,74 0,41 ARTHROv5_10200 Conserved hypothetical protein 0,41 0,26 ARTHROv5_10201 Conserved hypothetical protein 0,45 0,31 ARTHROv5_10204 Conserved protein of unknown function 0,17 0,18 ARTHROv5_10208 Conserved hypothetical protein 0,49 0,22 ARTHROv5_10209 Conserved hypothetical protein (secreted) 0,34 0,18

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ARTHROv5_10212 Conserved hypothetical protein 1,40 4,00 ARTHROv5_10213 Conserved hypothetical protein (secreted) 0,58 0,45 ARTHROv5_10246 Conserved hypothetical protein 0,23 0,16 ARTHROv5_10252 Conserved hypothetical protein 0,70 0,37 ARTHROv5_10256 Conserved hypothetical protein (membrane) 3,98 5,78 ARTHROv5_10284 Conserved protein of unknown function 1,61 2,09 ARTHROv5_10286 Conserved protein of unknown function 3,87 2,14 ARTHROv5_10288 Conserved hypothetical protein 1,15 2,32 ARTHROv5_10290 Conserved hypothetical protein 1,95 2,04 ARTHROv5_10292 Conserved hypothetical protein 2,39 3,06 ARTHROv5_10295 Conserved hypothetical protein 0,21 0,25 ARTHROv5_10299 Conserved hypothetical protein 0,17 0,26 ARTHROv5_10300 Conserved protein of unknown function 0,53 0,33 ARTHROv5_10308 Conserved hypothetical protein 0,23 0,39 ARTHROv5_10338 Conserved exported protein of unknown function 0,09 0,10 ARTHROv5_10342 Conserved protein of unknown function 1,74 2,87 ARTHROv5_10343 Conserved protein of unknown function 1,25 3,83 ARTHROv5_10351 Conserved protein of unknown function 1,05 7,46 ARTHROv5_10358 Conserved protein of unknown function 1,83 3,84 ARTHROv5_10361 Conserved protein of unknown function 1,47 3,40 ARTHROv5_10371 Conserved protein of unknown function 0,99 3,54 ARTHROv5_10373 Conserved protein of unknown function 2,22 2,63 ARTHROv5_10374 Conserved protein of unknown function 1,43 2,58 ARTHROv5_10390 Conserved protein of unknown function 0,39 0,38 ARTHROv5_10391 Conserved protein of unknown function 0,74 0,37 ARTHROv5_10397 Conserved exported protein of unknown function 0,66 0,33 ARTHROv5_10417 Conserved protein of unknown function 3,82 5,81 ARTHROv5_10418 Conserved protein of unknown function 3,69 5,46 ARTHROv5_10419 Conserved protein of unknown function 1,95 2,77 ARTHROv5_10420 Conserved protein of unknown function 1,73 2,18 ARTHROv5_10427 Conserved protein of unknown function 0,81 0,41 ARTHROv5_10428 Conserved protein of unknown function 0,57 0,41 ARTHROv5_10429 Conserved protein of unknown function 0,49 0,34 ARTHROv5_10431 Conserved membrane protein of unknown function 0,50 0,17 ARTHROv5_10434 Conserved protein of unknown function 0,29 0,24 ARTHROv5_10438 Conserved protein of unknown function 1,21 2,40 ARTHROv5_10439 Conserved protein of unknown function 3,44 7,79 ARTHROv5_10451 Conserved hypothetical protein 2,60 3,08 ARTHROv5_10464 Conserved membrane protein of unknown function 1,42 2,74 ARTHROv5_10465 Conserved protein of unknown function 2,68 2,76 ARTHROv5_10467 arhF Conserved hypothetical protein 5,10 5,75 ARTHROv5_10468 arhE Conserved hypothetical protein 9,58 11,99 ARTHROv5_10469 arhD Conserved hypothetical protein 5,44 9,46

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ARTHROv5_10470 arhC Conserved hypothetical protein 3,14 14,62 ARTHROv5_10471 arhB Conserved hypothetical protein NS 22,36 ARTHROv5_10474 Conserved protein of unknown function 4,00 4,69 ARTHROv5_10480 Conserved hypothetical protein 0,64 0,39 ARTHROv5_10482 Conserved hypothetical protein (secreted) 0,56 0,27 ARTHROv5_10501 Conserved hypothetical protein 2,65 2,00 ARTHROv5_10585 Conserved hypothetical protein 1,97 2,37 ARTHROv5_10591 Conserved hypothetical protein (fragment part2) 1,25 3,07 ARTHROv5_10602 Conserved hypothetical protein 0,49 0,41 ARTHROv5_10660 Conserved protein of unknown function 2,59 4,42 ARTHROv5_10669 Conserved hypothetical protein (fragment part 2) 0,50 0,40 ARTHROv5_10677 Conserved hypothetical protein 0,81 0,45 ARTHROv5_10684 Conserved hypothetical protein 0,42 0,38 ARTHROv5_10692 Conserved protein of unknown function 0,37 0,30 ARTHROv5_10699 Conserved hypothetical protein 0,47 0,37 ARTHROv5_10721 Conserved hypothetical protein (membrane) 3,24 2,45 ARTHROv5_10743 Conserved hypothetical protein 4,64 10,15 ARTHROv5_10764 Conserved hypothetical protein 3,20 2,57 ARTHROv5_10765 Conserved hypothetical protein 1,37 3,17 ARTHROv5_10778 Conserved protein of unknown function 0,80 0,31 ARTHROv5_10782 Conserved exported protein of unknown function 0,26 0,22 ARTHROv5_10794 Conserved hypothetical protein 2,15 2,49 ARTHROv5_10802 Conserved hypothetical protein 0,91 2,01 ARTHROv5_10834 Conserved hypothetical protein 0,83 0,46 ARTHROv5_10841 Conserved hypothetical protein 0,43 0,23 ARTHROv5_10842 Conserved hypothetical protein 0,37 0,21 ARTHROv5_10843 Conserved hypothetical protein, CofD related 0,65 0,28 ARTHROv5_10861 Conserved hypothetical protein (membrane) 1,52 0,23 ARTHROv5_10926 Conserved protein of unknown function 1,03 5,22 ARTHROv5_10927 Conserved protein of unknown function 1,94 3,88 ARTHROv5_10928 Conserved hypothetical protein 2,09 3,06 ARTHROv5_10929 Conserved hypothetical protein 2,35 3,99 ARTHROv5_10930 Conserved hypothetical protein 2,26 3,49 ARTHROv5_10931 Conserved hypothetical protein 1,90 2,97 ARTHROv5_10933 Conserved protein of unknown function 7,49 6,42 ARTHROv5_10937 Conserved hypothetical protein (fragment) 1,89 3,97 ARTHROv5_10938 Conserved hypothetical protein 1,53 2,31 ARTHROv5_10940 Conserved hypothetical protein 1,38 2,11 ARTHROv5_10950 Conserved hypothetical protein (membrane) 3,04 3,69 ARTHROv5_10980 Conserved protein of unknown function 1,36 2,39 ARTHROv5_10983 Conserved hypothetical protein 0,55 0,28 ARTHROv5_10995 Conserved hypothetical protein (secreted) 0,81 0,45 ARTHROv5_11000 Conserved hypothetical protein, NnrU-like 0,77 0,20

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ARTHROv5_11002 Conserved hypothetical protein 0,63 0,32 ARTHROv5_11013 Conserved hypothetical protein 0,74 0,38 ARTHROv5_11024 Conserved protein of unknown function 1,37 8,59 ARTHROv5_11026 Conserved hypothetical protein (secreted) 2,40 3,45 ARTHROv5_11066 Conserved protein of unknown function 6,04 3,79 ARTHROv5_11076 Conserved hypothetical protein 0,81 0,34 ARTHROv5_11088 Conserved protein of unknown function 3,19 3,77 ARTHROv5_11089 Conserved protein of unknown function 3,95 7,44 ARTHROv5_11093 Conserved protein of unknown function 0,65 0,34 ARTHROv5_11104 Conserved hypothetical protein 2,17 4,68 ARTHROv5_11121 Conserved hypothetical protein (membrane) 0,46 0,21 ARTHROv5_11123 Conserved hypothetical protein 0,43 0,23 ARTHROv5_11190 Conserved hypothetical protein 2,19 3,00 ARTHROv5_11195 Conserved hypothetical protein (fragment) 1,05 0,47 ARTHROv5_11205 Conserved protein of unknown function 1,93 2,20 ARTHROv5_11207 Conserved protein of unknown function 5,48 2,78 ARTHROv5_11208 Conserved hypothetical protein (fragment) 2,96 2,13 ARTHROv5_11209 Conserved hypothetical protein 2,29 2,84 ARTHROv5_11215 Conserved protein of unknown function 2,30 2,39 ARTHROv5_11230 Conserved hypothetical protein 0,90 2,59 ARTHROv5_11233 Conserved hypothetical protein 0,76 2,49 ARTHROv5_11234 Conserved hypothetical protein 1,30 2,43 ARTHROv5_11250 Conserved protein of unknown function 0,45 0,37 ARTHROv5_11251 Conserved protein of unknown function 0,49 0,37 ARTHROv5_11283 Conserved hypothetical protein 0,64 0,26 ARTHROv5_11315 Conserved hypothetical protein (secreted) 0,16 0,15 ARTHROv5_11349 Conserved protein of unknown function 2,01 2,22 ARTHROv5_11374 Conserved protein of unknown function 1,00 2,77 ARTHROv5_11390 Conserved hypothetical protein 1,03 0,24 ARTHROv5_11404 Conserved protein of unknown function 1,45 2,42 ARTHROv5_11492 Conserved hypothetical protein 1,06 6,49 ARTHROv5_11510 Conserved hypothetical protein 0,70 0,37 ARTHROv5_11511 Conserved hypothetical protein (membrane) 0,61 0,26 ARTHROv5_11562 Conserved hypothetical protein 1,46 2,46 ARTHROv5_11564 Conserved hypothetical protein 0,41 0,44 ARTHROv5_11574 Conserved protein of unknown function 1,43 2,27 ARTHROv5_11581 Conserved protein of unknown function 1,30 2,00 ARTHROv5_11588 Conserved protein of unknown function 1,45 2,61 ARTHROv5_11599 Conserved protein of unknown function 0,90 2,25 ARTHROv5_11600 Conserved protein of unknown function 0,81 2,36 ARTHROv5_11610 Conserved hypothetical protein 0,76 0,38 ARTHROv5_11614 Conserved hypothetical protein 0,42 0,21 ARTHROv5_11621 Conserved hypothetical protein 0,66 0,28

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ARTHROv5_11635 Conserved hypothetical protein (secreted) 0,59 0,39 ARTHROv5_11653 Conserved hypothetical protein 0,56 0,34 ARTHROv5_11662 Conserved hypothetical protein 0,73 0,45 ARTHROv5_11664 Conserved hypothetical protein 1,78 2,15 ARTHROv5_11679 Conserved hypothetical protein (membrane) 1,02 0,45 ARTHROv5_11681 Conserved hypothetical protein 0,44 0,27 ARTHROv5_11695 Conserved hypothetical protein 2,84 2,53 ARTHROv5_11706 Conserved hypothetical protein 0,67 0,43 ARTHROv5_11732 Conserved protein of unknown function 0,50 0,39 ARTHROv5_11734 Conserved membrane protein of unknown function 3,43 2,73 ARTHROv5_11742 Conserved hypothetical protein 3,80 3,86 ARTHROv5_11754 Conserved hypothetical protein 1,34 2,45 ARTHROv5_11755 Conserved hypothetical protein 2,29 3,31 ARTHROv5_11807 Conserved hypothetical protein 0,07 0,19 ARTHROv5_11858 Conserved hypothetical protein 1,69 3,15 ARTHROv5_11879 Conserved hypothetical protein 0,09 0,08 ARTHROv5_11882 Conserved protein of unknown function 0,30 0,10 ARTHROv5_11892 Conserved hypothetical protein 0,59 0,32 ARTHROv5_11904 Conserved hypothetical protein 0,46 0,39 ARTHROv5_11913 Conserved hypothetical protein 0,29 0,39 ARTHROv5_11939 Conserved hypothetical protein (membrane) 1,04 0,33 ARTHROv5_11940 Conserved hypothetical protein (membrane) 1,04 0,39 ARTHROv5_11941 Conserved hypothetical protein (membrane) 0,62 0,47 ARTHROv5_11945 Conserved hypothetical protein 0,71 4,30 ARTHROv5_11957 Conserved hypothetical protein 0,51 0,32 ARTHROv5_12006 Conserved protein of unknown function 1,64 2,66 ARTHROv5_12009 Conserved protein of unknown function 1,26 5,72 ARTHROv5_12018 Conserved hypothetical protein 0,38 0,37 ARTHROv5_12026 Conserved hypothetical protein 0,51 0,43 ARTHROv5_12034 Conserved protein of unknown function 0,25 0,23 ARTHROv5_12051 Conserved membrane protein of unknown function 1,33 0,33 ARTHROv5_12064 Conserved hypothetical protein (membrane) 0,32 0,38 ARTHROv5_12065 Conserved exported protein of unknown function 0,20 0,16 ARTHROv5_12066 Conserved hypothetical protein (fragment) 0,12 0,13 ARTHROv5_12100 Conserved protein of unknown function 1,15 2,80 ARTHROv5_12102 Conserved protein of unknown function 0,94 0,38 ARTHROv5_12115 Conserved protein of unknown function 1,35 2,28 ARTHROv5_12137 Conserved hypothetical protein 0,41 0,34 ARTHROv5_20001 Conserved hypothetical protein (membrane) 0,85 0,28 ARTHROv5_20029 Conserved hypothetical protein 0,42 0,15 ARTHROv5_20030 Conserved protein of unknown function 0,58 0,25 ARTHROv5_20031 Conserved hypothetical protein 0,81 0,46 ARTHROv5_20035 Conserved hypothetical protein 0,37 0,27

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ARTHROv5_20042 Conserved protein of unknown function 2,73 2,65 ARTHROv5_20066 Conserved protein of unknown function 6,50 7,39 ARTHROv5_20073 Conserved protein of unknown function 1,31 3,81 ARTHROv5_20085 Conserved hypothetical protein 2,74 3,41 ARTHROv5_20100 Conserved hypothetical protein 5,15 6,84 ARTHROv5_20102 Conserved protein of unknown function 0,06 0,09 ARTHROv5_20106 Conserved protein of unknown function 2,31 2,13 ARTHROv5_20107 Conserved protein of unknown function 2,25 2,11 ARTHROv5_20109 Conserved protein of unknown function 1,22 2,15 ARTHROv5_20110 Conserved protein of unknown function 1,50 5,93 ARTHROv5_20119 Conserved hypothetical protein 0,89 0,47 ARTHROv5_20132 Conserved membrane protein of unknown function 0,86 0,49 ARTHROv5_20133 Conserved protein of unknown function 0,72 0,42 ARTHROv5_20134 Conserved protein of unknown function 0,70 0,34 ARTHROv5_20135 Conserved protein of unknown function 0,94 0,43 ARTHROv5_20137 Conserved protein of unknown function 0,38 0,30 ARTHROv5_20143 Conserved protein of unknown function 0,70 0,41 ARTHROv5_20161 Conserved protein of unknown function 0,92 2,20 ARTHROv5_20171 Conserved hypothetical protein 0,54 0,32 ARTHROv5_20188 Conserved exported protein of unknown function 2,80 2,44 ARTHROv5_20196 Conserved hypothetical protein 0,15 0,14 ARTHROv5_20216 Conserved hypothetical protein 0,28 0,40 ARTHROv5_20222 Conserved protein of unknown function 1,50 2,25 ARTHROv5_20235 Conserved protein of unknown function 0,20 0,40 ARTHROv5_20236 Conserved protein of unknown function 0,35 0,27 ARTHROv5_20238 Conserved hypothetical protein 0,42 0,30 ARTHROv5_20244 Conserved hypothetical protein 0,52 0,34 ARTHROv5_20251 Conserved protein of unknown function 1,93 2,62 ARTHROv5_30011 Conserved hypothetical protein (secreted) 1,26 2,18 ARTHROv5_30015 Conserved hypothetical protein 0,99 5,48 ARTHROv5_30019 Conserved hypothetical protein 5,58 6,74 ARTHROv5_30027 Conserved hypothetical protein 0,69 0,20 ARTHROv5_30045 Conserved hypothetical protein 0,54 0,29 ARTHROv5_30055 Conserved hypothetical protein 0,38 0,33 ARTHROv5_30063 Conserved hypothetical protein 1,08 2,37 ARTHROv5_30072 Conserved hypothetical protein 2,66 2,93 ARTHROv5_30075 Conserved protein of unknown function 0,27 0,20 ARTHROv5_30116 Conserved hypothetical protein 0,34 0,44 ARTHROv5_30180 Conserved hypothetical protein 0,70 0,39 ARTHROv5_30181 Conserved hypothetical protein 0,54 0,24 ARTHROv5_30201 Conserved protein of unknown function 1,33 0,17 ARTHROv5_30204 Conserved hypothetical protein 0,19 0,13 ARTHROv5_30208 Conserved membrane protein of unknown function 0,58 0,38

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ARTHROv5_30230 Conserved protein of unknown function 1,55 2,99 ARTHROv5_30234 Conserved hypothetical protein 0,52 0,30 ARTHROv5_30246 Conserved hypothetical protein 0,62 0,45 ARTHROv5_30251 Conserved hypothetical protein 0,47 0,24 ARTHROv5_30332 Conserved hypothetical protein 0,79 0,46 ARTHROv5_30388 Conserved hypothetical protein 1,00 0,28 ARTHROv5_30405 Conserved protein of unknown function 0,32 0,32 ARTHROv5_30406 Conserved hypothetical protein 0,55 0,48 ARTHROv5_30449 Conserved hypothetical protein (fragment) 3,08 6,21 ARTHROv5_30489 Conserved hypothetical protein 0,63 0,49 ARTHROv5_30512 Conserved hypothetical protein 2,80 2,19 ARTHROv5_30538 Conserved protein of unknown function 0,54 0,32 ARTHROv5_30552 Conserved protein of unknown function 5,21 3,19 ARTHROv5_30554 Conserved protein of unknown function 8,33 5,03 ARTHROv5_30555 Conserved protein of unknown function 3,62 5,37 ARTHROv5_30556 Conserved protein of unknown function 4,94 4,74 ARTHROv5_30558 Conserved protein of unknown function 7,60 5,83 ARTHROv5_30564 Conserved protein of unknown function 5,84 8,43 ARTHROv5_30566 Conserved protein of unknown function 1,50 4,36 ARTHROv5_30571 Conserved hypothetical protein (exported ) 2,32 2,47 ARTHROv5_30576 Conserved protein of unknown function 0,79 0,48 ARTHROv5_30582 Conserved hypothetical protein 0,61 0,36 ARTHROv5_30585 Conserved protein of unknown function 2,96 2,75 ARTHROv5_30588 Conserved hypothetical protein 1,94 2,70 ARTHROv5_30589 Conserved hypothetical protein 1,85 2,66 ARTHROv5_30595 Conserved protein of unknown function 3,86 3,85 ARTHROv5_30612 Conserved protein of unknown function 0,76 0,36 ARTHROv5_30621 Conserved hypothetical protein 3,39 28,21 ARTHROv5_30622 Conserved hypothetical protein (fragment) 4,63 38,06 ARTHROv5_30631 Conserved hypothetical protein 1,84 2,04 ARTHROv5_30632 Conserved hypothetical protein 0,67 0,29 ARTHROv5_30635 Conserved hypothetical protein 0,59 0,42 ARTHROv5_30649 Conserved hypothetical protein (bifunctional) 4,25 17,29 ARTHROv5_30655 Conserved protein of unknown function 0,51 0,29 ARTHROv5_30674 Conserved protein of unknown function 0,35 0,27 ARTHROv5_30702 Conserved protein of unknown function 2,90 2,63 ARTHROv5_30713 Conserved hypothetical protein 0,59 0,41 ARTHROv5_30737 Conserved protein of unknown function 6,58 6,64 ARTHROv5_30738 Conserved exported protein of unknown function 6,11 6,15 ARTHROv5_30762 Conserved protein of unknown function 0,70 0,41 ARTHROv5_30763 Conserved hypothetical protein 0,54 0,36 ARTHROv5_30768 Conserved hypothetical protein 0,73 0,37 ARTHROv5_30781 Conserved hypothetical protein 1,67 4,67

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ARTHROv5_30782 Conserved hypothetical protein (fragment) 1,64 4,85 ARTHROv5_30784 Conserved hypothetical protein (fragment) 1,76 5,55 ARTHROv5_30785 Conserved hypothetical protein 3,25 4,90 ARTHROv5_30786 Conserved hypothetical protein (fragment) 1,78 6,13 ARTHROv5_30824 Conserved hypothetical protein 1,69 5,32 ARTHROv5_30836 Conserved protein of unknown function 4,32 25,56 ARTHROv5_30856 Conserved hypothetical protein 3,82 7,45 ARTHROv5_30858 Conserved protein of unknown function 2,99 14,78 ARTHROv5_30859 Conserved hypothetical protein 2,23 16,62 ARTHROv5_30860 Conserved protein of unknown function 1,96 9,23 ARTHROv5_30861 Conserved protein of unknown function 2,42 3,87 ARTHROv5_40002 Conserved hypothetical protein 0,60 0,46 ARTHROv5_40029 Conserved hypothetical protein 0,66 0,45 ARTHROv5_40038 Conserved hypothetical protein 2,78 4,55 ARTHROv5_40039 Conserved hypothetical protein 2,25 4,10 ARTHROv5_40043 Conserved hypothetical protein 1,82 5,42 ARTHROv5_40073 Conserved exported protein of unknown function 0,87 0,42 ARTHROv5_40111 Conserved protein of unknown function 1,15 5,06 ARTHROv5_40118 Conserved hypothetical protein (membrane) 1,13 2,52 ARTHROv5_40136 Conserved protein of unknown function 0,63 0,41 ARTHROv5_40154 Conserved hypothetical protein 0,66 0,33 ARTHROv5_40156 Conserved hypothetical protein 0,72 0,41 ARTHROv5_40159 Conserved hypothetical protein 0,76 0,26 ARTHROv5_40179 Conserved hypothetical protein 0,77 0,48 ARTHROv5_40189 Conserved hypothetical protein (secreted) 0,66 0,31 ARTHROv5_40216 Conserved hypothetical protein 1,65 2,09 ARTHROv5_40226 Conserved hypothetical protein 0,94 0,44 ARTHROv5_40242 Conserved hypothetical protein 0,33 0,26 ARTHROv5_40245 Conserved hypothetical protein 1,79 10,4 ARTHROv5_40246 Conserved hypothetical protein 1,08 10,9 ARTHROv5_40247 Conserved hypothetical protein 0,81 9,49 ARTHROv5_40248 Conserved hypothetical protein 0,96 10,0 ARTHROv5_40249 putative [Myosin heavy-chain] kinase NS 3,44 ARTHROv5_40250 Conserved hypothetical protein 4,69 9,01 ARTHROv5_40251 Conserved hypothetical protein 2,21 4,12 ARTHROv5_40256 Conserved protein of unknown function 1,89 2,26 ARTHROv5_40285 Conserved exported protein of unknown function 0,59 0,37 ARTHROv5_40289 Conserved protein of unknown function 0,48 0,24 ARTHROv5_40306 Conserved hypothetical protein 1,22 5,67 ARTHROv5_40308 Conserved hypothetical protein (fragment) 1,35 3,11 ARTHROv5_40333 Conserved membrane protein of unknown function 0,31 0,18 ARTHROv5_40334 Conserved protein of unknown function 0,16 0,15 ARTHROv5_40339 Conserved protein of unknown function 2,17 3,91

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ARTHROv5_40364 Conserved hypothetical protein 0,69 0,31 ARTHROv5_40384 Conserved hypothetical protein (secreted) 2,20 2,63 ARTHROv5_40424 Conserved hypothetical protein 3,41 4,74 ARTHROv5_40427 Conserved hypothetical protein 1,74 2,98 ARTHROv5_40430 Conserved hypothetical protein (membrane) 0,42 0,42 ARTHROv5_40431 Conserved hypothetical protein (membrane) 0,37 0,30 ARTHROv5_40447 Conserved hypothetical protein (membrane) 2,39 3,96 ARTHROv5_40448 Conserved hypothetical protein 2,81 3,10 ARTHROv5_40455 Conserved hypothetical protein 0,10 0,17 ARTHROv5_40465 Conserved hypothetical protein 1,86 4,03 ARTHROv5_40477 Conserved hypothetical protein (fragment) 0,21 0,23 ARTHROv5_40480 Conserved hypothetical protein 1,62 2,12 ARTHROv5_40526 Conserved hypothetical protein 1,81 2,71 ARTHROv5_40534 Conserved hypothetical protein 0,24 0,31 ARTHROv5_40543 Conserved protein of unknown function 0,97 0,36 ARTHROv5_40544 Conserved hypothetical protein 0,88 6,35 ARTHROv5_40546 Conserved hypothetical protein 1,42 2,88 ARTHROv5_40547 Conserved hypothetical protein (membrane) 3,13 2,44 ARTHROv5_40550 Conserved protein of unknown function 1,57 3,71 ARTHROv5_40553 Conserved protein of unknown function 0,81 0,41 ARTHROv5_40574 Conserved hypothetical protein, PatB-like 0,43 0,06 ARTHROv5_40575 Conserved hypothetical protein, PatC-like 0,42 0,05 ARTHROv5_40576 Conserved hypothetical protein 0,53 0,10 ARTHROv5_40577 Conserved protein of unknown function 0,74 0,10 ARTHROv5_40578 Conserved protein of unknown function 0,63 0,12 ARTHROv5_40579 Conserved protein of unknown function 0,84 0,08 ARTHROv5_40584 Conserved protein of unknown function 0,67 0,37 ARTHROv5_40585 Conserved protein of unknown function 0,71 0,43 ARTHROv5_40586 Conserved protein of unknown function 0,95 0,43 ARTHROv5_40587 Conserved hypothetical protein 0,53 0,37 ARTHROv5_40588 Conserved protein of unknown function 0,68 0,38 ARTHROv5_40589 Conserved protein of unknown function 0,73 0,39 ARTHROv5_40595 Conserved hypothetical protein 2,03 2,80 ARTHROv5_40596 Conserved hypothetical protein 2,50 2,97 ARTHROv5_40598 Conserved hypothetical protein 1,55 4,13 ARTHROv5_40604 Conserved hypothetical protein 2,61 4,62 ARTHROv5_40605 Conserved hypothetical protein 1,71 2,49 ARTHROv5_40614 Conserved hypothetical protein 1,22 3,66 ARTHROv5_40615 Conserved hypothetical protein 1,41 11,19 ARTHROv5_40623 Conserved hypothetical protein 1,52 2,02 ARTHROv5_40637 Conserved hypothetical protein 2,05 2,98 ARTHROv5_40638 Conserved hypothetical protein 1,30 4,60 ARTHROv5_40650 Conserved hypothetical protein 0,75 3,31

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

ARTHROv5_40651 Conserved hypothetical protein 2,16 2,78 ARTHROv5_40654 Conserved hypothetical protein 2,64 2,29 ARTHROv5_40655 Conserved hypothetical protein 4,52 3,19 ARTHROv5_40666 Conserved hypothetical protein 1,51 3,82 ARTHROv5_40667 Conserved protein of unknown function 2,23 3,10 ARTHROv5_40671 Conserved protein of unknown function 0,85 0,21 ARTHROv5_40684 Conserved protein of unknown function 2,68 4,64 ARTHROv5_40685 Conserved hypothetical protein 1,70 7,09 ARTHROv5_40686 Conserved protein of unknown function 2,65 3,73 ARTHROv5_40687 Conserved hypothetical protein 2,75 2,68 ARTHROv5_40689 Conserved protein of unknown function 1,43 3,33 ARTHROv5_40700 Conserved hypothetical protein 1,43 5,02 ARTHROv5_40702 Conserved hypothetical protein 1,81 4,72 ARTHROv5_40705 Conserved hypothetical protein 2,05 3,96 ARTHROv5_40719 Conserved hypothetical protein 1,09 7,21 ARTHROv5_40731 Conserved protein of unknown function 1,94 2,70 ARTHROv5_40735 Conserved hypothetical protein 1,40 2,15 ARTHROv5_40736 Conserved hypothetical protein 0,85 2,14 ARTHROv5_40745 Conserved hypothetical protein 1,98 5,24 ARTHROv5_40787 Conserved hypothetical protein 0,22 0,19 ARTHROv5_40788 Conserved hypothetical protein 0,90 0,45 ARTHROv5_40811 Conserved protein of unknown function 3,48 6,09 ARTHROv5_40934 Conserved protein of unknown function 0,63 0,31 ARTHROv5_40936 Conserved protein of unknown function 0,63 0,19 ARTHROv5_40952 Conserved exported protein of unknown function 0,26 0,15 ARTHROv5_40957 Conserved protein of unknown function 1,81 3,92 ARTHROv5_40958 Conserved protein of unknown function 3,06 3,34 ARTHROv5_40977 Conserved hypothetical protein 1,42 2,00 ARTHROv5_40982 Conserved hypothetical protein 3,45 4,61 ARTHROv5_41037 Conserved hypothetical protein (secreted) 0,65 0,32 ARTHROv5_41044 Conserved hypothetical protein 1,34 5,22 ARTHROv5_41045 Conserved hypothetical protein 2,10 2,76 ARTHROv5_41051 Conserved hypothetical protein 1,57 2,64 ARTHROv5_41056 Conserved hypothetical protein (exported) 2,33 3,99 ARTHROv5_41064 Conserved protein of unknown function 0,49 0,30 ARTHROv5_41081 Conserved hypothetical protein 3,81 6,03 ARTHROv5_41094 Conserved hypothetical protein 1,27 3,48 ARTHROv5_41097 Conserved protein of unknown function 3,02 6,81 ARTHROv5_41098 Conserved protein of unknown function 2,91 6,10 ARTHROv5_41115 Conserved protein of unknown function 3,53 3,10 ARTHROv5_41129 Conserved protein of unknown function 5,80 4,17 ARTHROv5_41132 Conserved protein of unknown function 1,36 3,10 ARTHROv5_41138 Conserved protein of unknown function 0,45 0,25

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ARTHROv5_41144 Conserved hypothetical protein 0,33 0,37 ARTHROv5_41154 Conserved exported protein of unknown function 0,14 0,28 ARTHROv5_41176 Conserved protein of unknown function 0,11 0,23 ARTHROv5_41220 Conserved protein of unknown function 0,35 0,44 ARTHROv5_41237 Conserved hypothetical protein (fragment part 2) 3,45 6,29 ARTHROv5_41238 Conserved hypothetical protein (fragment part 3) 3,16 4,23 ARTHROv5_41281 Conserved hypothetical protein (fragment) 0,79 0,32 ARTHROv5_41282 Conserved hypothetical protein (fragment) 0,61 0,15 ARTHROv5_41330 Conserved hypothetical protein 0,31 0,44 ARTHROv5_41337 Conserved hypothetical protein 0,54 0,36 ARTHROv5_41366 Conserved hypothetical protein 0,97 0,23 ARTHROv5_41371 Conserved hypothetical protein 1,01 4,67 ARTHROv5_41372 Conserved hypothetical protein 2,05 4,20 ARTHROv5_41395 Conserved protein of unknown function 0,28 0,25 ARTHROv5_41410 Conserved hypothetical protein 0,52 0,43 ARTHROv5_41429 Conserved hypothetical protein (membrane) 2,23 3,20 ARTHROv5_41432 Conserved hypothetical protein 1,25 2,78 ARTHROv5_41439 Conserved protein of unknown function 1,40 2,71 ARTHROv5_41441 Conserved exported protein of unknown function 3,06 5,43 ARTHROv5_50005 Conserved hypothetical protein 2,39 11,19 ARTHROv5_50019 Conserved protein of unknown function 1,36 2,14 ARTHROv5_50047 Conserved hypothetical protein 3,54 4,68 ARTHROv5_50048 Conserved hypothetical protein (fragment) 3,46 4,00 ARTHROv5_50049 Conserved hypothetical protein (fragment) 4,12 9,63 ARTHROv5_50050 Conserved hypothetical protein (fragment) 3,03 5,52 ARTHROv5_50051 Conserved hypothetical protein (fragment) 3,36 4,08 ARTHROv5_50059 Conserved hypothetical protein 1,24 2,76 ARTHROv5_50076 Conserved hypothetical protein (secreted) 1,10 0,39 ARTHROv5_50112 Conserved hypothetical protein (secreted) 0,92 0,39 ARTHROv5_50120 Conserved protein of unknown function 0,57 0,45 ARTHROv5_50122 Conserved hypothetical protein 0,55 0,43 ARTHROv5_50137 Conserved protein of unknown function 1,62 2,05 ARTHROv5_50145 Conserved hypothetical protein 0,52 0,39 ARTHROv5_50147 Conserved hypothetical protein 0,88 0,43 ARTHROv5_50175 Conserved hypothetical protein 3,63 9,50 ARTHROv5_50182 Conserved hypothetical protein 1,56 2,20 ARTHROv5_50183 Conserved hypothetical protein (fragment) 1,34 2,26 ARTHROv5_50185 Conserved hypothetical protein (fragment) 2,13 3,69 ARTHROv5_50186 Conserved hypothetical protein 2,14 5,09 ARTHROv5_50187 Conserved hypothetical protein 1,54 4,44 ARTHROv5_50199 Conserved hypothetical protein 1,05 2,87 ARTHROv5_50247 Conserved protein of unknown function 0,38 0,38 ARTHROv5_50273 Conserved hypothetical protein 0,47 0,36

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

ARTHROv5_50292 Conserved hypothetical protein 2,10 2,78 ARTHROv5_50296 Conserved hypothetical protein (fragment) 3,74 3,13 ARTHROv5_50300 Conserved protein of unknown function 6,58 5,93 ARTHROv5_50301 Conserved protein of unknown function 1,93 9,66 ARTHROv5_50313 Conserved protein of unknown function 2,73 3,45 ARTHROv5_50314 Conserved protein of unknown function 3,00 3,76 ARTHROv5_50315 Conserved protein of unknown function 2,95 4,78 ARTHROv5_50326 Conserved protein of unknown function 0,81 0,43 ARTHROv5_50330 Conserved membrane protein of unknown function 0,51 0,44 ARTHROv5_50341 Conserved protein of unknown function 0,80 0,44 ARTHROv5_50345 Conserved protein of unknown function 1,43 3,23 ARTHROv5_50346 Conserved exported protein of unknown function 1,08 3,17 ARTHROv5_50348 Conserved membrane protein of unknown function 0,68 0,35 ARTHROv5_60009 Conserved protein of unknown function 0,76 0,38 ARTHROv5_60032 Conserved exported protein of unknown function 0,47 0,23 ARTHROv5_60039 Conserved protein of unknown function 0,63 0,41 ARTHROv5_60052 Conserved protein of unknown function 1,06 2,04 ARTHROv5_60060 Conserved protein of unknown function 1,57 0,43 ARTHROv5_60066 Conserved hypothetical protein 0,42 0,38 ARTHROv5_60068 Conserved hypothetical protein 0,90 2,40 ARTHROv5_60070 Conserved hypothetical protein 0,44 0,25 ARTHROv5_60103 Conserved hypothetical protein 0,35 0,21 ARTHROv5_60116 Conserved protein of unknown function 0,76 0,22 ARTHROv5_60128 Conserved hypothetical protein 3,39 10,28 ARTHROv5_60177 Conserved hypothetical protein (membrane) 0,04 0,04 ARTHROv5_60195 Conserved protein of unknown function 1,44 2,55 ARTHROv5_60200 Conserved hypothetical protein 1,71 2,49 ARTHROv5_60201 Conserved hypothetical protein 1,26 2,37 ARTHROv5_60207 Conserved hypothetical protein 0,60 0,36 ARTHROv5_60260 Conserved protein of unknown function 2,23 2,47 ARTHROv5_60261 Conserved protein of unknown function 2,71 4,93 ARTHROv5_60263 Conserved protein of unknown function 3,50 2,07 ARTHROv5_60270 Conserved membrane protein of unknown function 3,92 5,54 ARTHROv5_60273 Conserved exported protein of unknown function 1,30 2,27 ARTHROv5_60290 Conserved hypothetical protein 0,76 0,36 ARTHROv5_60305 Conserved protein of unknown function 0,40 0,29 ARTHROv5_60319 Conserved hypothetical protein 2,45 2,81 ARTHROv5_60328 Conserved protein of unknown function 1,12 0,46 ARTHROv5_60329 Conserved protein of unknown function 4,20 3,23 ARTHROv5_60330 Conserved protein of unknown function 4,47 3,90 ARTHROv5_60331 Conserved protein of unknown function 2,14 2,04 ARTHROv5_60346 Conserved protein of unknown function 4,09 2,19 ARTHROv5_60347 Conserved protein of unknown function 8,50 4,07

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ARTHROv5_60348 Conserved protein of unknown function 5,36 4,67 ARTHROv5_60367 Conserved protein of unknown function 3,39 4,45 ARTHROv5_60377 Conserved protein of unknown function 0,83 2,83 ARTHROv5_60379 Conserved protein of unknown function 1,91 2,80 ARTHROv5_60381 Conserved protein of unknown function 0,96 4,49 ARTHROv5_60390 Conserved protein of unknown function 0,62 0,29 ARTHROv5_60406 Conserved protein of unknown function 1,62 2,30 ARTHROv5_60408 Conserved protein of unknown function 1,47 2,04 ARTHROv5_60411 Conserved protein of unknown function 0,84 0,37 ARTHROv5_60418 Conserved protein of unknown function 0,52 0,47 ARTHROv5_60460 Conserved protein of unknown function 1,21 4,85 ARTHROv5_60504 Conserved protein of unknown function 3,09 4,78 ARTHROv5_60507 Conserved protein of unknown function 3,61 3,22 ARTHROv5_60508 Conserved protein of unknown function 6,94 8,08 ARTHROv5_60509 Conserved protein of unknown function 6,84 7,57 ARTHROv5_60510 Conserved protein of unknown function 4,59 8,05 ARTHROv5_60511 Conserved protein of unknown function 2,73 4,83 ARTHROv5_60512 Conserved protein of unknown function 1,34 3,34 ARTHROv5_60514 Conserved protein of unknown function 0,63 18,28 ARTHROv5_60515 Conserved protein of unknown function 1,49 10,60 ARTHROv5_60516 Conserved protein of unknown function 2,47 6,74 ARTHROv5_60517 Conserved protein of unknown function 1,35 6,37 ARTHROv5_60518 Conserved protein of unknown function 2,36 5,36 ARTHROv5_60519 Conserved protein of unknown function 2,07 3,15 ARTHROv5_60520 Conserved protein of unknown function 1,45 4,16 ARTHROv5_60521 Conserved protein of unknown function 1,58 7,45 ARTHROv5_60546 Conserved hypothetical protein 0,64 0,28 ARTHROv5_60614 Conserved protein of unknown function 0,62 0,46 ARTHROv5_60616 Conserved protein of unknown function 2,06 2,74 ARTHROv5_60621 Conserved protein of unknown function 1,68 2,68 ARTHROv5_60623 Conserved protein of unknown function 2,89 3,03 ARTHROv5_60628 Conserved protein of unknown function 1,55 10,05 ARTHROv5_60631 Conserved protein of unknown function 0,34 0,40 ARTHROv5_60647 Conserved protein of unknown function 1,60 2,09 ARTHROv5_60648 Conserved protein of unknown function 1,49 2,27 ARTHROv5_60652 Conserved protein of unknown function 0,55 0,44 ARTHROv5_60688 Conserved protein of unknown function 2,99 2,35 ARTHROv5_60689 Conserved exported protein of unknown function 3,67 3,09 ARTHROv5_60697 Conserved exported protein of unknown function 1,53 3,05 ARTHROv5_60708 Conserved protein of unknown function 0,22 0,10 ARTHROv5_60717 Conserved protein of unknown function 0,41 0,14 ARTHROv5_60739 Conserved hypothetical protein 0,66 0,39 ARTHROv5_60741 Conserved hypothetical protein 0,49 0,47

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

ARTHROv5_60755 Conserved hypothetical protein 5,86 10,81 ARTHROv5_60766 Conserved protein of unknown function 3,57 5,22 ARTHROv5_60767 Conserved hypothetical protein (fragment) 2,39 6,82 ARTHROv5_60768 Conserved hypothetical protein 2,85 8,99 ARTHROv5_60769 Conserved hypothetical protein 2,26 4,71 ARTHROv5_60770 Conserved hypothetical protein 1,25 2,64 ARTHROv5_60771 Conserved hypothetical protein (fragment) 4,67 3,70 ARTHROv5_60772 Conserved hypothetical protein 4,99 4,23 ARTHROv5_60773 Conserved hypothetical protein 6,07 7,21 ARTHROv5_60774 Conserved hypothetical protein 1,99 3,30 ARTHROv5_60775 Conserved protein of unknown function 5,80 5,44 ARTHROv5_60776 Conserved hypothetical protein 0,95 3,31 ARTHROv5_60778 Conserved hypothetical protein (fragment) 1,85 4,81 ARTHROv5_60779 Conserved hypothetical protein 1,89 9,32 ARTHROv5_60780 Conserved hypothetical protein 1,75 15,73 ARTHROv5_60781 Conserved hypothetical protein 4,21 19,69 ARTHROv5_60786 Conserved hypothetical protein 2,68 3,69 ARTHROv5_60788 Conserved hypothetical protein 1,00 4,43 ARTHROv5_60818 Conserved hypothetical protein 2,13 14,71 ARTHROv5_60819 Conserved hypothetical protein 3,63 8,69 ARTHROv5_60823 Conserved hypothetical protein 0,43 0,35 ARTHROv5_60824 Conserved hypothetical protein 0,29 0,16 ARTHROv5_60831 Conserved protein of unknown function 0,46 0,30 ARTHROv5_60838 Conserved hypothetical protein 1,53 2,03 ARTHROv5_60840 Conserved hypothetical protein 1,35 6,50 ARTHROv5_60841 Conserved hypothetical protein (fragment) 4,38 2,83 ARTHROv5_60845 Conserved hypothetical protein 8,97 14,03 ARTHROv5_60846 Conserved hypothetical protein 8,05 10,21 ARTHROv5_60847 Conserved hypothetical protein(fragment) 1,45 4,30 ARTHROv5_60848 Conserved hypothetical protein (fragment) 11,31 18,79 ARTHROv5_60849 Conserved hypothetical protein (fragment) 14,92 23,44 ARTHROv5_60850 Conserved protein of unknown function 2,72 4,93 ARTHROv5_60855 Conserved protein of unknown function 3,49 5,24 ARTHROv5_60859 Conserved hypothetical protein 3,93 3,20 ARTHROv5_60879 Conserved protein of unknown function 1,27 3,02 ARTHROv5_60890 Conserved hypothetical protein 4,03 2,99 ARTHROv5_60894 Conserved protein of unknown function 1,40 2,19 ARTHROv5_60901 Conserved hypothetical protein 0,48 0,41 ARTHROv5_60915 Conserved hypothetical protein 1,40 2,12 ARTHROv5_60925 Conserved hypothetical protein 1,38 0,45 ARTHROv5_60928 Conserved hypothetical protein 0,83 0,49 ARTHROv5_60944 Conserved membrane protein of unknown function 1,01 0,19 ARTHROv5_60946 Conserved protein of unknown function 3,77 4,76

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005

ARTHROv5_60990 Conserved hypothetical protein 1,52 2,41 ARTHROv5_60997 Conserved protein of unknown function 0,11 0,11 ARTHROv5_60998 Conserved hypothetical protein (secreted) 0,31 0,18 ARTHROv5_61015 Conserved hypothetical protein (membrane) 0,64 0,43 ARTHROv5_61021 Conserved hypothetical protein 2,52 3,39 ARTHROv5_61024 Conserved protein of unknown function 1,95 2,84 ARTHROv5_61038 Conserved hypothetical protein 2,05 4,02 ARTHROv5_61042 Conserved hypothetical protein 2,77 5,54 ARTHROv5_61043 Conserved hypothetical protein 2,25 3,81 ARTHROv5_61046 Conserved hypothetical protein 3,16 5,12 ARTHROv5_61049 Conserved protein of unknown function 2,36 6,26 ARTHROv5_61050 Conserved protein of unknown function 5,07 5,55 ARTHROv5_61051 Conserved hypothetical protein (fragment) 2,32 4,10 ARTHROv5_61067 Conserved hypothetical protein 1,23 3,40 ARTHROv5_61071 Conserved hypothetical protein 0,50 0,49 ARTHROv5_61073 Conserved hypothetical protein (membrane) 0,33 0,17 ARTHROv5_61111 Conserved protein of unknown function 0,47 0,43 ARTHROv5_61138 Conserved hypothetical protein 2,99 9,05 ARTHROv5_61167 Conserved hypothetical protein (membrane) 0,70 0,49 ARTHROv5_61170 Conserved hypothetical protein 0,34 0,23 ARTHROv5_61196 Conserved hypothetical protein 0,84 0,19 ARTHROv5_61202 Conserved protein of unknown function 2,55 3,14 ARTHROv5_61205 Conserved exported protein of unknown function 0,30 0,29 ARTHROv5_61207 Conserved hypothetical protein (membrane) 0,70 0,19 ARTHROv5_61208 Conserved hypothetical protein (fragment) 0,77 0,29 ARTHROv5_61217 Conserved protein of unknown function 0,53 0,26

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table S 11: Proteomics results for 3200 Gy The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 1.25 or < 0.8. 'NS' stands for not significant differentially expressed (p>0.05).

Fold Proteomics Peptides Accession number Gene Protein Function change 3200 Gy coverage 3200 Gy

ARTHROv5_10213 Conserved hypothetical protein (secrete) 0,73 4 ARTHROv5_10468 arhE Conserved hypothetical protein 7,12 4 ARTHROv5_10470 arhC Conserved hypothetical protein 1,40 10 ARTHROv5_10471 arhB Conserved hypothetical protein 1,75 9 ARTHROv5_10600 Hypothetical protein 0,67 3 ARTHROv5_10939 Putative bacterioferritin 1,44 2 ARTHROv5_10941 Hypothetical protein (secreted) 0,44 2 ARTHROv5_10970 Conserved hypothetical protein 2,04 1 ARTHROv5_10983 Conserved hypothetical protein 1,30 3 ARTHROv5_11338 Peptidase C14 caspase catalytic subunit p20 0,60 4 ARTHROv5_11504 Conserved hypothetical protein 0,78 2 Putative membrane-associated zinc metallopeptidase, M50 ARTHROv5_11981 1,77 4 family ARTHROv5_30042 Putative extracellular nuclease (fragment) 0,56 12 ARTHROv5_30080 psaD Photosystem I reaction center subunit II 1,45 10 ARTHROv5_30816 Conserved hypothetical protein 0,70 6 ARTHROv5_30849 Type II protein secretion system protein 0,44 2 ARTHROv5_40132 purM Phosphoribosylaminoimidazole synthetase 1,64 1 ARTHROv5_40888 Putative serine protease inhibitor family protein 0,56 1 ARTHROv5_41069 ilvB Acetolactate synthase large subunit 0,73 4 ARTHROv5_41076 Putative hydrolase 1,84 12 ARTHROv5_41179 Conserved hypothetical protein (membrane). 1,41 3 ARTHROv5_41229 Conserved hypothetical protein 0,55 1 Hydroxylamine reductase, hybrid-cluster [4Fe-2S-2O] ARTHROv5_41296 hcp 0,69 1 protein in anaerobic terminal reductases ARTHROv5_60030 Conserved exported protein of unknown function 0,67 3 ARTHROv5_60064 Na-Ca exchanger/integrin-beta4 0,73 12 ARTHROv5_60608 Conserved exported protein of unknown function 0,45 6 ARTHROv5_60622 nifU Nitrogen-fixing protein 0,73 2 ARTHROv5_60737 Conserved hypothetical protein 0,55 1 D-alanyl-D-alanine carboxypeptidase/D-alanyl-D-alanine- ARTHROv5_60836 0,69 7 endopeptidas ARTHROv5_61026 thiC Thiamine biosynthesis protein 1,33 4 ARTHROv5_61150 putative haemolysin-type calcium-binding toxin, RTX-like 2,13 8

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Results Chapter V: Molecular investigation of the radiation resistance of edible cyanobaterium Arthrospira sp. PCC 8005 Table S 12: Proteomics results for 5000 Gy The fold change (FC) values listed are values for which p-value is p<0.05, and are only considered biologically significant if FC > 1.25 or < 0.8. 'NS' stands for not significant differentially expressed (p>0.05).

Fold Proteomics Peptides Accession number Gene Protein Function change 5000 gy coverage 5000 Gy

ARTHROv5_10064 leuC 3-isopropylmalate dehydratase large subunit 1,37 5 ARTHROv5_10148 rpsA1 30S ribosomal protein S1 0,77 10 ARTHROv5_10208 Conserved hypothetical protein 0,47 2 Pyridine nucleotide transhydrogenase, alpha subunit, soluble ARTHROv5_10269 pntAA 0,78 4 domain ARTHROv5_10468 arhE Conserved hypothetical protein 4,30 3 ARTHROv5_10752 dapB Dihydrodipicolinate reductase 1,81 3 ARTHROv5_10984 psaA Photosystem I P700 chlorophyll a apoprotein A1 (PsaA) 1,71 9 ARTHROv5_11019 Hypothetical protein 0,61 8 ARTHROv5_11549 ssb Single-stranded DNA-binding protein 0,74 5 ARTHROv5_11629 Putative glycosyl transferase, family 2 0,36 1 ARTHROv5_11670 gmd GDP-D-mannose dehydratase, NAD(P)-binding 0,62 5 ARTHROv5_11794 Putative phytoene dehydrogenase / carotene isomerase 1,52 3 ARTHROv5_11902 Conserved hypothetical protein 1,88 1 ARTHROv5_11966 Macrophage migration inhibitory factor family protein 1,43 1 ARTHROv5_11987 Peptidase C11 clostripain 0,14 1 ARTHROv5_11991 Putative Peptidyl-prolyl cis-trans isomerase, cyclophilin family 0,54 3 ARTHROv5_11993 psbD1 Photosystem II reaction center D2 protein Q(A) 1,64 8 ARTHROv5_30042 Putative extracellular nuclease (fragment) 0,41 10 ARTHROv5_30080 psaD Photosystem I reaction center subunit II 2,07 4 ARTHROv5_30191 pheT Phenylalanine tRNA synthetase, beta subunit 0,65 2 ARTHROv5_30386 Conserved hypothetical protein (secreted) 0,73 6 ARTHROv5_30727 hisF Imidazole glycerol phosphate synthase subunit 0,66 1 ARTHROv5_30849 Type II protein secretion system protein 0,27 1 ARTHROv5_40785 Conserved hypothetical protein 0,73 5 ARTHROv5_41102 purM Putative short-chain dehydrogenase/reductase 1,61 2 ARTHROv5_41150 Putative Methylenetetrahydrofolate reductase [NAD(P)H] 0,67 1 ARTHROv5_50101 Putative structural maintenance of chromosomes (SMC) protein 0,60 2 ARTHROv5_50156 Putative Haemolysin-type calcium-binding toxin, RTX-like 0,43 3 ARTHROv5_50171 pgl 6-phosphogluconolactonase (6PGL) 0,78 6 ARTHROv5_60256 hup3 Histone-like bacterial DNA-binding protein, HU-like 0,75 3 ARTHROv5_60301 Conserved protein of unknown function 0,43 1 ARTHROv5_60318 Putative acireductone dioxygenase 0,68 1 ARTHROv5_60608 Conserved exported protein of unknown function 0,61 4 ARTHROv5_60992 ndk Nucleoside diphosphate kinase 1,31 6 ARTHROv5_60999 Conserved hypothetical protein 0,63 1

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ARTHROv5_61122 Conserved hypothetical protein 0,58 1

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays

Chapter VI Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays

Hanène Badri, Pieter Monsieurs, Ilse Coninx, Robin Nauts, Ruddy Wattiez, Natalie Leys ‘Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays’ Submitted to PlosOne

Contributions: Hanène Badri performed all experiments and analysis, with the help of lab technician Ilse Coninx. Robin Nauts helped with the glutathione measurements. Pieter Monsieurs performed the processing of microarray data. Prof. Ruddy Wattiez and Dr. Natalie Leys have guided Hanène Badri towards the most optimal experiment design and data interpretation.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays Abstract The edible cyanobacterium Arthrospira is resistant to ionising radiation. The cellular mechanisms underlying this radiation resistance are, however, still largely unknown. Therefore, additional molecular analysis was performed to investigate how these cells can escape from, protect against, or repair the radiation damage. Arthrospira cells were shortly exposed to different doses of 60Co gamma rays and the dynamic response was investigated by monitoring its gene expression and cell physiology at different time points after irradiation. The results revealed a fast switch from an active growth state to a kind of 'survival modus' during which the cells put photosynthesis, carbon and nitrogen assimilation on hold and activate pathways for cellular protection, detoxification, and reparation. The higher the radiation dose, the more pronounced this global emergency response is expressed. Genes repressed during early response suggested a reduction of photosynthesis and reduced tricarboxilic acid (TCA) and Calvin-Benson-Bassham (CBB) cycles, combined with an activation of the pentose phosphate pathway. In parallel, also biosynthesis of additional carbon storage molecules, compatible solutes, and vitamins was stopped, and only reactivated during recovery. For ROS detoxification and restoration of the redox balance in the cell, Arthrospira relied mainly on metal homeostasis processes and the powerful antioxidant molecule glutathione. The repair mechanisms of Arthrospira cells that were immediately switched on, involve mainly proteases for removal of damaged proteins, and single strand DNA repair and restriction modification system. Additionally, the exposed cells showed significant increased expression of arh genes, coding for a group of novel proteins of unknown function, also seen in our previous irradiation studies. This observation confirms our hypothesis that arh genes are key element in radiation resistance of Arthrospira, requiring further investigation. This study provides new insights into phasic response and the cellular pathways involved the radiation resistance of microbial cells, in particularly for photosynthetic organisms as the cyanobacterium Arthrospira.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays VI.1 Introduction

Arthrospira is a motile filamentous planktonic non N2-fixing cyanobacterium. They grow naturally in alkaline lake water environments in regions with strong sunlight and high temperature [1]. Arthrospira are also cultivated on industrial scale for animal and human consumption and has gained considerable popularity as natural microbial dietary supplement in the health food industry [2]. Its valuable nutritious components include essentially fatty acids such as omega-3, pigments such as carotenes and phycocyanin, and minerals such as iron [3,4]. Due to its high nutritive value and claimed anti-oxidant and anti-inflammatory activities [5], Arthrospira is considered as a promising nutraceutical with applications on Earth and in space [6]. The resilience of Arthrospira cells to extreme environmental conditions is impressive: it has the ability to grow in highly alkaline environments [7], environments with high salt concentration [8], at extreme temperatures (high and low) [9], and at high light intensities [10]. It is also able to resist ionising radiation from UV and gamma rays, as well as charged particles [11]. The response of Arthrospira to such environmental stresses has mainly been investigated based on morphological and physiological traits [12]. Recently, we were the first to study the damage of radiation stress (i.e. high doses of gamma rays) at molecular level, using a combination of genomic, transcriptomic and proteomic data [13]. Cells exposed to high doses of 3200 or 5000 Gy of gamma rays, causing significant cell damage, showed organised shutdown of photosynthesis and carbon fixation, decreased pigment, lipid and secondary metabolite synthesis; and induced thiol-based antioxidant systems, photo-sensing and signalling pathways. Here we extended our molecular research of the radiation resistance of Arthrospira, to lower and less lethal doses of gamma rays (800 Gy and 1600 Gy, in addition to 3200 Gy) received in short exposure time, thereby allowing gene expression profiling immediately after the exposure, and at different time points during recovery after irradiation. This approach allowed us to map the dynamics of the response and related metabolic pathways in function of time. Thus, this study aims to elucidate the molecular mechanisms of Arthrospira cells to resist to and recover from radiation exposure.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays VI.2 Materials and methods

VI.2.1 Strain and culture conditions The strain of Arthrospira sp. PCC 8005 was originally obtained from the Pasteur Culture Collection (2009), sub-cultured, and maintained as active cultures in the lab. Three independent lab cultures (i.e. three biological replicates, n=3) were propagated and used separately for irradiation. They were grown aerobically in 1000 ml Zarrouk-UBP medium in 2000 ml Erlenmeyers [14] on a rotatory shaker 120 rpm in an incubator at 30°C illuminated with a photon flux density of ~ 42 µE m-2 s-1 provided by three Osram daylight tubes (Binder KBW 400), to mid-exponential growth phase corresponding to an optical density of OD750nm ~1. Each of the 3 separate 1000 ml cultures was then divided in three aliquots (ca. 300 ml each) to be used for the three different irradiation experiments (three doses : 800 Gy, 1600 Gy and 3200 Gy). And each 300 ml aliquot, was further divided into two (ca. 150 ml each), to split the cultures into irradiation samples and non-irradiated controls in different flasks (500 ml CELLSTAR® Filter cap cell, Greiner Bio-One, Vilvoorde, Belgium). Thus for each irradiation dose, three independent cultures were irradiated, and in parallel of each irradiated sample, an equal non-irradiated sample was kept. Irradiation was done on aerobic liquid cultures, containing active planktonic filaments suspended in ca. 150 ml liquid Zarrouk-UBP medium, in plastic culture flasks (CELLSTAR® Filter Cap Cell; 500 ml Cell Culture Flasks).

VI.2.2 Irradiation conditions The irradiation was performed using BRIGITTE facility at the Belgian Reactor N°2 (BR2) of SCK•CEN. The irradiation was done inside a closed canister under water, surrounded by ten 60Co gamma rays sources (Photon energy of 1.33 MeV and 1.17 MeV). Arthrospira cultures were irradiated with different doses of gamma rays, i.e. 800 Gy, 1600 Gy and 3200 Gy, using a constant dose rate of 20 000 Gy h-1. The time required for irradiation was dose-dependent, i.e. 2.4 minutes for 800 Gy, 4.8 minutes for 1600 Gy and 9.6 minutes for 3200 Gy. During irradiation, the cultures were in the dark, and the temperature inside the irradiation canister was automatically monitored and ranged between 26-27°C. In parallel, three other cultures were maintained at the same conditions (also in the dark) but outside the irradiation facility, as non-irradiated controls. Samples were immediately put on ice after irradiation, at the irradiation facility, before transport to the lab for further processing. Some aliquots were used immediately after irradiation at T(0H) for regrowth and measurement of chlorophyll fluorescence. While some part of the samples was centrifuged at

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays 4°C (10000g, 20 min) and the obtained cell pellet was flash frozen in liquid nitrogen, and further conserved at -80 °C, for molecular and biochemical analysis, including mRNA profiling, glutathione and pigment content quantification. Another part of the sample was put back into the incubator (at the same conditions as mentioned above) after irradiation for recovery and was harvested 2h and 5h after irradiation.

VI.2.3 Post-irradiation recovery and proliferation analysis In order to investigate the ability of Arthrospira filaments to recover after irradiation, inoculation of 1% (v/v) from irradiated and non-irradiated samples (at OD750 ~1), including three independent biological replicates per test condition (n=3), was done in fresh Zarrouk-UBP medium, and incubated for growth at the same conditions as cited above. The growth was followed by optical density OD750nm measurements, i.e. absorbance measurement at 750 nm, every three days using a spectrophotometer (AquaMate, Unicam, Cambridge, UK). The proliferation curves were plotted as OD750nm increase versus time.

VI.2.4 Photosystem II quantum yield Immediately after irradiation, a 2 ml aliquot was taken from all the irradiated cultures and their respective controls (at OD750 ~1), including three independent biological replicates per test condition (n=3), and further dark adapted at room temperature for 15 min before photosynthetic potential measurements. Photosynthetic potential was measured by Phycobilisome and chlorophyll A fluorescence of Photosystem II (PSII) using the DUAL PAM 100 (Waltz-GmbH Effeltrich Germany). First, the cells were exposed to a weak modulated red light (ML) (635 nm, 3 µE m-2s-1) (which is too low in intensity to induce any photosynthetic activity), and minimum fluorescence was determined (F0). Next, the cells were exposed to a high intensity light flash or saturating pulse of red light (635 nm, 8000 µE m-2s-1) with short duration (0.8 s) (which is too sudden and short to induce any photosynthetic activity) and maximum fluorescence in dark adapted state (Fm) was determined. From those measurements, the ratio Fv/Fm was then calculated where the variable fluorescence Fv is equal to Fm – F0. Fv/Fm represents the maximum potential quantum efficiency of Photosystem II. An Fv/Fm value around of 0.6 is the approximate optimal value for Arthrospira species [15], with lower values indicating cell stress [16].

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays VI.2.5 Pigments analysis Immediately after exposure to gamma rays, aliquots of two ml were taken from all irradiated and control cultures (at OD750 ~1), including three independent biological replicates per test condition (n=3), for pigment analysis. Cells were collected by centrifugation (10000 g, 15 min, RT) (Microcentrifuge 5418R, Eppendorf), and cell pellets were stored at -80°C until analysis (ca. 2 days). Later, frozen cell pellets were dried overnight using a freeze-dryer (Lyovac GT 2, GEA Lyophil GmbH), and the absolute dry weight was determined. Next, the pellets were re-suspended using 1 ml of 0.05 M Na2HPO4 at pH 7, and, five cycles of freezing in liquid nitrogen and thawing at 37°C were performed, to crack the cells and release the phycobiliproteins containing phycocyanin and allophycocyanin pigments. In order to achieve total extraction, an additional treatment of 30 min at 37 °C with 100 µl of lysozyme at a final concentration of 100 mg ml-1 was performed. Next, the suspension was centrifuged (13000 g, 10 min, RT) (Microcentrifuge 5418R, Eppendorf), the pellet was kept apart for further analysis and the supernatant containing the water soluble pigments phycocyanin (PC) and allophycocyanin (APC), was measured for absorbance at wavelengths 615 and 652 nm, using a photospectrometer (Unicame, Aquamat). The concentration (OD615)−0.474(OD652) of PC and APC were calculated according to following formula: 푃퐶 = and 5.34 (OD652)−0.208(OD615) 퐴푃퐶 = [17]. Then three washing steps were done on the remaining pellet 5.09 using 1 ml of 0.05 M Na2HPO4 at pH 7, followed by a chlorophyll extraction step using 1 ml of 100 % methanol as organic solvent. Additional mechanic treatment by sonication (3 cycles of 10 s, amplitude 30 %, 1 pulse per second; Vibra cells) was performed to enhance total chlorophyll extraction. The suspension was centrifuged (13000 g, 10 min, at 4°C) (Microcentrifuge 5418R, Eppendorf) and the supernatant was measured via absorbance spectrophotometry at a wavelength of 665 nm, using a photospectrometer (Unicame, Aquamat). Chlorophyll (Chl) concentration was OD665 calculated then according to following formula : 퐶ℎ푙 = [17], where 74.5 ml mg-1 cm-1 is the 74.5 extinction coefficient of Chl A at 665nm in absolute methanol. The pigment concentrations were recalculated to pigment weight (mg) per biomass dry weight (g) (w/w) and the results from irradiated samples were plotted as percentage of their representative non-irradiated control (which was put at 100%).

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays VI.2.6 Glutathione measurement Aliquots of fifteen ml were taken from all irradiated and control samples (at OD750 ~1), including three independent biological replicates per test condition (n=3), and cells were collected via centrifugation (10000g, 20 min) using (Avanti J- 26XP; Beckman Coulter, Suarlée, Belgium). The cells were transferred into 1.5 ml collection tubes and the wet biomass weight was determined for each sample. The cells were put in liquid nitrogen and shredded via bead-beating during 3.5 min at 30 Hz using two tungsten carbide beads (Ball Mill Mixer MM400, Retsh, Germany). Further extraction was performed by adding 400 µl of 200 mM HCl solution to the cells. Then, the samples were centrifuged at 4 °C (15 min, 13200 rpm) (Microcentrifuge 5418R, Eppendorf). Aliquots of

300 µl from each sample were mixed to 30 µl NaH2PO4 buffer (pH 5.6). The pH was adjusted in the range of 3.5-5.0 by adding 200 mM NaOH. During the entire procedure the samples were kept on ice. The final reactions and measurements were done at room temperature. Total glutathione concentration (including the reduced form GSH and oxidized form GSSG) was measured by quantifying the reduction of Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB), a chemical to quantify the concentration of thiol groups in a sample, in the presence of glutathione reductase (GR). Reactions were performed in the presence of 100 μl phosphate buffer (200 mM

NaH2PO4, 10 mM EDTA (pH 7.5)), 60 μl dH2O, 10 μl 10 mM NADPH and 10 μl 12 mM DTNB, in multi-well plates. After addition of 10 µl GR, 10 µl of extract was added, after which DTNB reduction was monitored by measuring absorbance at 415 nm using the plate reader (PowerWave XS2, Bioteck). To measure only the oxidized form GSSG, the same measurement was done after blocking the reduced form GSH present with 2-Vinyl-pyridine (1 µl). The final GSH and GSSG concentration were expressed in nmolg-1 of biomass-wet weight, and the results from irradiated samples were normalized versus their representative non-irradiated controls and plotted as percentage.

VI.2.7 RNA extraction Thirty ml of all irradiated and control Arthrospira cultures (at OD750 ~1), including three independent biological replicates per test condition, were centrifuged (20 min at 10 000 g, at 4°C) (Avanti J- 26XP; Beckman Coulter, Suarlée, Belgium) to collect the cells. Cell pellets were flash frozen in liquid nitrogen and then immediately stored at -80°C, until analysis (ca. 5 days). Frozen cells were resuspended in 750 µl RNA Wiz solution (Ambion), and were disrupted mechanically by bead- beating using Zirconia Beads for 10 min, at room temperature, in the Vortex-Genie® 2

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays apparatus (MoBio), using a vortex adapters (13000-V1, Mo Bio). Next, the released RNA was separated from the cell debris by centrifugation (10000 g, 10 min, 4°C) (Microcentrifuge 5418R, Eppendorf). RNA purification was performed using the RiboPure™-Bacteria Kit (Ambion) following the manufacturer's instructions. The RiboPure™-Bacteria Kit (Ambion) allowed the collection of larger RNA (e.g. 16S and 23S ribosomal and messenger mRNA), but not the collection of small RNA (e.g 5S ribosomal RNAs and tRNAs). Hence the flow-through form the purification with RiboPure™-Bacteria Kit (Ambion) was carefully collected and further purified with a Direct-zol RNA MiniPrep 2050 (Zymo Research) to recover the small RNAs. The final RNA products obtained from both purification processes were combined and used for preparing the RNA for microarray analysis. RNA samples (ca. 500 ng/µl, in a total volume of 70 µl) were then treated once with DNAse (Ambion TURBO DNA-freeTM, Life Technologies Europe B.V, Ghent, Belgium) for 30 min at 37°C following the manufacturer's instructions to remove DNA. Absence of DNA was confirmed by PCR with universal 16S primers. Finally, the RNA was concentrated using RNA Clean & Concentrator™-25 (Zymosearch) to a concentration (ca; 250 ng/µl, in a total volume of 20 µl. This extraction method was found to yield sufficient quantity and superior quality of RNA, compared to the method based on heat shock lysis and hot Trizol (Invitrogen) extraction and Direct-zol RNA MiniPrep 2050 (Zymo Research) purification previously used [13]. RNA quantity and purity was assessed by spectrophotometric analysis using the NanoDrop ND-1000 Spectrophotometer (Thermo scientific). Samples had to meet the following minimum basic criteria: 260/280 > 2.0 and 260/230 > 1.8. The quality and integrity of RNA was assessed using the Bioanalyzer 2100 (Agilent) according to manufacturer's instructions. RNA selected for microarray had an RNA integrity number (RIN value) above 7.

VI.2.8 RNA analysis via microarrays RNA extracts were analysed using our own designed Arthrospira HX12 microarray chips, having a 12x135k array format (Nimblegen). The 'Arthrospira HX12' (Nimblegen, wI, USA) tiling array spans the full Arthrospira sp. PCC 8005 genome (version 5, available at EMBL, http://www.ebi.ac.uk/ena/data/view/GCA_000176895.2), with 135.367 probes ranging from 50 up to 72 nucleotides and an average length of 53 nucleotides, and an average spacing of 34 nucleotides between 2 different probes, which could be mapped back to 5854 CDS and 3141 intergenic regions. Microarray analysis was done at Institute for Research in Biomedicine (IRB) in Barcelona, Spain (IRBB). cDNA library preparation and amplification were performed on 25 ng total RNA, obtained

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays from three independent irradiated and control samples per test condition (n=3), using the Complete Whole Transcriptome Amplification (WTA2) kit (Sigma-Aldrich), according to the instructions of the manufacturer, with 17 cycles of amplification, resulting in microgram quantities of cDNA. Labelling and hybridization of the cDNA onto the microarray were performed according to Roche- Nimblegen expression guide v5-p1. For each sample, 1 µg cDNA was labelled by Cy3 nonamer primers and Klenow polymerization. Subsequently, a hybridization mixture was prepared with 2 µg Cy3-labeled cDNA. Samples were hybridized to Arthrospira HX12 array (Nimblegen) for 18 hours at 42 ºC. The arrays were washed and scanned in a Roche-Nimblegen MS 200 scanner. Raw data files (Pair and XYS files) were obtained from images using DEVA software (Roche- Nimblegen) and provide by IRBB to SCK•CEN for further data analysis. Per irradiation dose and per time point, 3 microarrays of irradiated cultures and 3 microarrays of their equivalent non- irradiated cultures (n =3) were analysed, resulting in 6 arrays per condition. Thus, for all 3 irradiation doses (800 Gy, 1600 Gy and 3200 Gy) and for all 3 time points after irradiation (0H, 2H and 5H) tested, this resulted in 36 arrays in total.

VI.2.9 Microarray data analysis At SCK•CEN raw microarray data were pre-processed using the “Oligo” package (version 1.24) in BioConductor (version 2.12 / R version 3.0.1) as follows: i) background correction based on the Robust Multichip Average (RMA) convolution model [18], ii) quantile normalization to make expression values from different arrays more comparable [19], and iii) summarization of multiple probe intensities for each probe set to one expression value per gene using the median polish approach [18]. To test for differential expression between the different irradiated conditions and the reference conditions (no irradiation) the Bayesian adjusted t-statistics was used as implemented in the “LIMMA” package (version 3.16.4) [20]. P-values were corrected for multiple testing using the Benjamini and Hochberg’s method to control the false discovery rate (FDR) [21]. Transcripts were considered significantly differentially expressed when the corresponding adjusted p-value was lower than 0.05 and their Log2FC was equal or higher than 1 for up-regulated genes, and equal or lower than -1 for the down regulated ones, meaning fold changes (FC) of >2 or <0.5 respectively. The gene annotation was based on the expert annotation privately available in the 'Arthroscope' database on the MaGe platform [22], which is a manually curated annotation. Genes having an absolute Log2 fold change higher than 1 and a corrected p value <0.05, in each separate test condition, were displayed on a scatter plot. The total number, of differentially

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays expressed genes after 800 Gy are respectively: 1485 at T0H, 181 at T2H and 203 at T5H. These numbers were higher after exposure to 1600 Gy resulting respectively in 2153 genes at T0H, 252 at T2H and 249 at T5H. Finally, the strongest expression and response was seen after exposure to the highest dose of 3200 Gy, showing 2585 expressed genes at T0H, 414 genes at 2H and 452 genes at T5H. In order to group genes that are co-expressed over the different time points and irradiation doses, the unsupervised soft clustering implementation Mfuzz was used (Mfuzz version 2.18.0; R-version 3.0.1) [23]. As input the fold changes of those genes having an absolute fold change higher than 2 and a p-value corrected for multiple testing lower than 0.05 in either one of the 9 conditions, were selected, i.e. 1838 genes in total. A fuzzier value of 1.38 was applied, as calculated based on the method proposed by Schwammle and Jenssen [24]. The number of predefined clusters was arbitrarily set to 9. One gene can potentially belong to multiple clusters. Additionally, a Principal Component Analysis (PCA) plot was created, to visualize the similarity between two clusters by looking at the proximity of the cluster centres in the PCA plot on the one hand, and the overlap between the averages in expression profile between different clusters is visualized by the line width between the different cluster centres on the other hand. Gene set enrichment analysis (GSEA) based on the Cluster of Orthologous Groups (COG) was obtained using the hypergeometric distribution. This analysis allowed deriving whether a gene cluster is containing a higher number of representatives of a specific COG then would be expected by chance. Visualization is performed in R (version 3.0.1) using the heatmap.2 command in the gplots package (version 3.4.1).

VI.2.10 Statistical analysis The Graph Pad Prism software (version 5.00, GraphPad Software) was used, for preparing data graphs and for statistical analysis using One way ANOVA followed by "Dunnett Multiple Comparison Test" with confidence interval 95% (p < 0.05).

VI.3 Results

VI.3.1 Global Gene expression Dynamics For all 3 different doses tested (800 Gy, 1600 Gy and 3200 Gy), the gene expression of Arthrospira in response to radiation exposure was monitored immediately (0H), two hours (2H) and five hours (5H) after irradiation, The transcription profiles showed an intense 'general emergency' response immediately after irradiation (0H), with a high number of differentially expressed genes (both up

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays and down), while this number decreases significantly in following two hours and five hours recovery period. There was one specific set of genes, however, the arh genes, which remained highly expressed throughout the full recovery period. The higher the radiation dose, the more pronounced this global emergency response was expressed (Figure VI-1).

Figure VI-1: Scatter plot showing the differentially expressed genes of Arthrospira sp. PCC 8005 in response to gamma irradiation plotted accordingly to their change in mRNA concentration (Log2 fold change values), for 3 radiation doses (800, 1600 and 3200 Gy) and 3 time points after radiation (0 hours, 2 hours, 5 hours).

Genes having an absolute Log2 fold change higher than 1 and a corrected p value <0.05, are displayed. The total number, of differentially expressed genes after 800 Gy are respectively: 1485 at T0H, 181 at T2H and 203 at T5H. These numbers were higher after exposure to 1600 Gy resulting respectively in 2153 genes at T0H, 252 at T2H and 249 at T5H. Finally, the strongest expression and response was seen after exposure to the highest dose of 3200 Gy, showing 2585 expressed genes at T0H, 414 genes at 2H and 452 genes at T5H. The circles highlight the top ranked expressed gene set, i.e. the arh genes, which were highly overexpressed by Arthrospira for all irradiation doses and throughout enriched during recovery period after irradiation. 169

Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays To gain an overview of the major transcriptional response patterns, cluster analysis was performed on the differentially expressed genes and the expression profiles of the different clusters over time (0H-2H-5H) were plotted, for each of the 3 doses (Figure VI-2). The selected genes were those having a Log2 fold change higher than one and a p-value corrected for multiple testing lower than 0.05 in either one of the nine conditions.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays

Figure VI-2: Dynamic changes in gene expression using the Mfuzz clustering software, according to their gene expression profile during recovery time (0 hours, 2 hours, 5 hours), for 3 radiation doses (800, 1600 and 3200 Gy). Based on the PCA plot (Figure VI-3), the clusters were assembled into two main different groups. The first group (E) contains clusters 3, 5, 6 and to a minor degree cluster 4, involved in the Early or Emergency response. In general, these four clusters group genes, which display the highest expression at, time point T0H while their expression is fading out at later time points. The second group (R) includes clusters 1, 2, 7, 8 and to a lesser extent 9, involved in the Recovery response.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays In general, these five clusters group genes with lower expression at T0H, that increases significantly thereafter (T2H), with a clear impact of the radiation dose.

Phase 1 : Emergency

Phase 2 : Recovery

Figure VI-3: Principal Component Analysis (PCA) of the cluster centres. This PCA biplot – plotting the first two principal components – gives a general idea on how the average expression patterns of the clusters are similar to each other. The closer the different cluster centres to each other, the more similar their average expression profiles are. Additionally, on top of the PCA plot, the overlap between different clusters is visualized by lines with variable width between different clusters: the wider the line between two clusters, the higher the overlap. The

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays graph generally shows a clustering in two groups, representing two distinct phases. Phase one - “Emergency response”: Clusters 3, 5 and 6 (blue ellipse) display the highest up-regulation at time point 0h and this effect fades out at later time points. To a lesser extent, also cluster 4 (red ellipse) can be categorized in the Phase1 response clusters, as genes belonging to this cluster display a significant high expression at 0H, and a strong repression after 2 hours and again a normal expression level after 5 hours. Phase 2 -“Recovery response”: Clusters 1, 2, 7, and 8 (blue ellipse) grouping genes with lower expression at T0H that increased during recovery; and in minor degree cluster 9 (red ellipse) showing significant increase in expression for all doses during recovery.

Thus the analysis of the global gene expression response revealed 2 'phases' in the dynamic transcriptional pattern: an Emergency response, activated immediately upon radiation exposure, followed by a Recovery response, in which genes that were silenced are reactivated activated later after irradiation.

VI.3.2 Gene specific response patterns Emergency (E) - Activation of protection, detoxification, and repair systems Clusters 3 (460 genes), 5 (274 genes) and 6 (122 genes) contain genes which are strongly induced immediately after irradiation and which decreased in expression intensely after 5 hours. The transcriptomic profile of the cluster 5 showed the strongest induction for the highest dose of 3200 Gy, for cluster 6 this was the opposite with a stronger induction for the lowest dose of 800 Gy, and for cluster 3, induction levels were similar for all doses. These clusters contain genes involved in the activation of protection, detoxification, and repair mechanisms in Arthrospira during irradiation. Immediately after irradiation, there was an up-regulation of gene huyA, involved in the synthesis of glutathione, an important intracellular metabolite for repair of ROS induced damage in plants, but rare in bacteria (Table VI-S 2). In line with glutathione, also a set of genes involved in proline synthesis (proA1, putA, pep) was expressed, recognized as non-enzymatic antioxidants of microbes and plants to mitigate the adverse effects of ROS [25]. Radiation led also to the induction of the synthesis of the enzymatic antioxidant peroxiredoxine. Other genes from these clusters contributing to cell detoxification were involved in homeostasis and active transport of various redox active metals which are cofactors for many enzymes, such as iron (feoA, feoB, fur), magnesium (mtgC), copper (copA1, cutA), nickel (corA), cobalt (cbiQ1, cbiQ2), zinc (znuA), and potassium (Table VI-

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays S 2). The expression of genes coding for outer membrane OmpA-like porins and permeases may have helped for the efficient diffusion of small and hydrophilic solutes over the membrane (Table VI-S 2). Exposure to radiation also strongly induced the transcription of genes involved the clean- up of damaged proteins, peptides and amino acids, such as proteinases and peptidases (cbpA, clpB2, patG) that remove dysfunctional proteins, and some chaperones (hspA, dnaK1, dnaJ). Several genes involved in production of amino acids under nitrogen starvation, such as glutamate generated via aspartate aminotransferase (aat1), amino acid transport (lysE) and degradation of urea (ureABCDF), were significantly induced in a dose dependent manner (Table VI-S 2). In addition, the transcriptional regulator (sufR) for Fe-S cluster regeneration was highly expressed (Table VI-S 2). Surprisingly, also genes with a predicted function in nitrogen fixation (nifU, devA) were expressed (Table VI-S 2), although Athrospira is described as non-heterocyst forming and non- nitrogen fixating. The cells strongly expressed several genes involved in the clean-up of nucleotides released from DNA damage repair. This includes the ADP-ribose pyrophosphatase, and the NUDIX hydrolases (mutT) (Table VI-S 2). The DNA repair system of Arthrospira involved the activation of nucleotide excision repair system (uvrBCD), mismatch repair (mutT), gene ruvB involved in resolving holiday junctions, and other helicases and DNA repair genes (dnaG, mod and recJ) (Table VI-S 2). In addition, several systems for DNA modification and protection, such as the endonuclease genes (pvuIIR) and the Enzymatic Restriction Modification genes (hsdR), were highly induced (Table VI-S 2). In parallel, there was a high induction of phage-like genomic islands (fax genes), the phage immunity system (CRISPR) (cas genes) and a Toxin/antitoxin system (Table VI-S 2). The synthesis of nucleotides and nucleic acids was rerouted via the pentose phosphate pathway (ribokinase gene). The carbon metabolism seemed to be oriented towards the degradation of glycogen (sigE), the synthesis of intracellular C-storage molecules such as trehalose (ARTHROv5_41060) and polyhydroxyalkanoates (phaC) (Table VI-S 2). Interestingly, the cluster also includes a set of genes involved in cobalamin (precursor of vitamin B12) biosynthesis (cbiM2, cbiQ2, cobW) and xanthine/uracil/vitamin C transport and sugar transport. Cells expressed also genes involved polyamine transport (potBC and potA) (Table VI-S 2) which may participate in export or import of putrescine/spermidine, well known as vital compounds for cell survival for plants and eukaryotes. The activation of lipase and esterase lipase genes suggested lipid degradation.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays Cluster 5 was significantly enriched with genes belonging to the category related to the signal transduction system (COG – T) (Figure VI-4). Genes coding for response regulatory proteins with photosensor domains (GAF, PAS), as well as the genes for synthesis of the secondary messenger cyclic diguanylate (c-di-GMP), and the associated signal transduction and regulatory systems (i.e. histidine kinases and transcriptional regulators) were induced (below). In addition, the induction of the photo-sensor Cry DASH (cry gene) was observed, well known as photosensor to control UV-A induced phototaxis [26].

B Chromatin structure and dynamics C Energy production and conversion 4 3 2 1 0 D Cell cycle control, cell division, chromosome partitioning E Amino acid transport and metabolism F Nucleotide transport and metabolism G Carbohydrate transport and metabolism H Coenzyme transport and metabolism I Lipid transport and metabolism J Translation, ribosomal structure and biogenesis K Transcription L Replication, recombination and repair M Cell wall/membrane/envelope biogenesis N Cell motility O Posttranslational modification, protein turnover, chaperones P Inorganic ion transport and metabolism Q Secondary metabolites biosynthesis, transport and catabolism R General function prediction only S Function unknown T Signal transduction mechanisms U Intracellular trafficking, secretion, and vesicular transport V Defense mechanisms

Figure VI-4: Gene Set Enrichment Analysis (GSEA) in the clusters of differentially expressed genes based on the Clusters of Orthologs Groups (COG) functional categories. This plot visualises whether a certain gene cluster (1-9) is containing a higher number of representatives of a specific COG then would be expected by chance. The COG functional category is shown in the vertical direction, the clusters of differentially expressed genes in the horizontal direction. The colour code is according to the 10log value of the corresponding p-value of the GSEA analysis: a p-value of smaller than 1.10-4 (10log-value of 4) results in a colour code red, a p-value of 1 (10log value 0) results in colour code white.

Recovery (R) – Restart of photosynthesis, carbon fixation, and nitrogen assimilation systems

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays Clusters 1, 2, 7, 8 and 9 responded in the opposite way compared with clusters 3, 5, and 6 mentioned above. They group those genes with lowered expression upon irradiation that increased again gradually during the recovery period.

Clusters 2 and 8, were very similar in expression profiles, and showed the strongest response for the highest dose (Figure VI-2). Genes from Cluster 1 genes responded in a similar way as Cluster 2 and 8 for 800 Gy and 1600 Gy, but had a less pronounced response to 3200 Gy. Clusters 1, 2 and 8 were significantly enriched with genes belonging to the category of Energy production and conversion (C) and/or Coenzyme transport and metabolism (H) (Figure VI-4). The genes of cluster 7 were also strongly repressed after irradiation, mainly at the highest dose (3200 Gy), but slightly induced after two hours recovery period, and back to original expression after five hours (Figure VI-2). Cluster 7 was significantly enriched with genes involved in the category of amino acid biosynthesis and transport (E) (Figure VI-4). Cluster 9 grouped genes which were slightly repressed after irradiation and induced during the recovery period, but required up to 5 hours to reach normal expression levels. Cluster 9 was significantly enriched with genes belonging to the category of Inorganic ion transport and metabolism (P) (Figure VI-4). The genes grouped in these clusters were indeed mainly involved in photosynthesis, carbon fixation and nitrogen assimilation. These clusters contain genes involved in (i) the synthesis of structural proteins of the phycobilisome and their linker polypeptides (apcAB, apcF, apcC, cpcC1, cpcE), the PSII (psbBNO) and PSI (ycf4, psaE) complexes, (ii) the biosynthesis of pigments and cofactors such as porphyrin and haem (hemCE, hemG, hemL, hemN1, cobA), phycobilin, and chloropyll (bchD, chlP, chlGH), and (iii) the electron transport chain (ndh, coxABC, cydB). Also genes involved ATP production (atpF, atpG2, atpH) were silenced immediately after irradiation (Table VI-S 3). The expression of the hydrogenase genes (hypA1, hypB1) was not really reduced upon radiation, but strongly induced during recovery.

The energy collected from photosynthesis in the form of NADPH and ATP is normally used for

CO2 fixation and synthesis of carbohydrate molecules via the Calvin-Benson-Bassham (CBB) cycle and also the genes for this process (cbbR, pgI, pgk, xfp) were strongly reduced in expression immediately after irradiation (Table VI-S 3). In line with the reduced carbon capture, also genes involved the Krebs cycle (also called tricarboxylic acid TCA cycle) providing precursors of certain amino acids (sdhA, sucD, sucC), and in biosynthesis of lipids such as fatty acids (fabF2, fabG1,

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays fabZ, fabG2, fabH, des) showed a reduced expression (Table VI-S 2). Also the expression of genes for biosynthesis of intracellular solutes with a role in salt tolerance (stpA, ggpS) was significantly reduced (Table VI-S 3). Cluster 1 and 8 contain in addition genes involved in cell envelope synthesis (mrdA1, murBCE, lpxA, yfbF), cell division (minC, minD) and motility (pilT1), which were all also reduced in expression.

Carbon metabolism is tightly correlated with nitrogen metabolism, and many genes related to nitrogen assimilation and metabolism, were immediately repressed (glnB, ntcA) upon irradiation. Many genes of cluster 9 are involved in transport and assimilation of nitrogen sources, including for example genes for nitrate and nitrite uptake (nrtABDC, nrtP, narB), cyanate uptake (cynBD), and amino acid uptake, transport and biosynthesis (livGMHJ, aapJ, aapP, aapQ, iaaA, aroB, argG, argH, argJ) (Table VI-S 3). Also the genes for the assimilation of ammonium in glutamate amino acids via the glutamine synthetase pathway (glnA, glsF), the uptake and degradation of organic N- polymers, such as nitrile (nthA1, nthB2), the hydrolysis of the polyamine agmatine to putrescine and urea, and the biosynthesis of the extracellular cyclic peptide patellamide A (patABC) were immediately shut down (Table VI-S 3). In contrast, the nitrogen metabolism seemed to be rerouted to urea hydrolyses, polyamide transport, and ammonium assimilation and glutamate amino acids production via the aspartate aminotransferase pathway, which were highly induced upon irradiation, as was mentioned above (Table VI-S 2).

Cluster 1 and 8 contain also the arh genes, a gene set coding for proteins with unknown function which we recently discovered to be specifically produced in Arhtrospira sp. PCC 8005 in response to lethal doses of gamma rays (3200 & 5000 Gy) [13]. This gene cluster, composed of five genes named arhABDEF, showed again a very high and dose-dependent transcription in response to ionising radiation (Table VI-S 4). This gene cluster was among the top ranked differentially expressed genes identified immediately after irradiation, and remained highly expressed during the recovery (Figure VI-1). Next to the gene involved in the biosynthesis and regeneration of glutathione (gshB) which were also clearly expressed during recovery, it was the only gene set displaying such strong expression profile (Table VI-S 2).

VI.3.3 Glutathione measurement The intracellular glutathione concentration of Arthrospira sp. PCC 8005, was assessed immediately after and during the five hour recovery period following irradiation. While no significant change

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays was noted for cells exposed to the lowest dose (800 Gy), there was a significant increase of total glutathione concentration in the cells exposed to higher doses (1600 and 3200 Gy), two hours and five hours post irradiation (Figure VI-5). This is consistent with the expression profile of the genes involved in the biosynthesis and regeneration of glutathione, described above. The extra glutathione was mainly present in the reduced form, as the level of the oxidized glutathione did not change significantly.

Figure VI-5: Intracellular glutathione concentrations of Arthospira sp. PCC 8005 after irradiation. A, B and C shows respectively concentrations of total glutathione (GSH+GSSG), reduced glutathione (GSH) and oxidized glutathione (GSSG) immediately (T0H), 2 hours (T2H) and 5 hours (T5H) after irradiation. The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Data represent the mean of three independent biological replicates, and error bars present the standard error of the mean (SEM). The statistical analysis was calculated on the raw data, before normalisation to percentages. One asterisk indicates a value which is significantly (p<0.05) different from the value of the non-irradiated control

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays VI.3.4 Photosynthesis efficiency and Pigments For photosynthetic growth, Arthrospira requires photosynthetic pigments and an active photosystem. The analysis of the photosynthetic pigments, showed very little change in the overall pigment content in the cells after irradiation, Nevertheless, there seemed to be a trend showing that phycocyanin and allophycocyanin content slightly decreased in the 2H after radiation, but was restored to a normal level during recovery 5 hours later (Figure VI-7). For chlorophyll this slight decrease was only observed 5 hours after irradiation. This trend was more pronounced for the higher dose of 3200 Gy. The functionality of the photosystem was assessed by measuring the PSII quantum yield via fluorescence, at 3 different time points after irradiation. The results showed no adverse effect of irradiation on the photosynthetic potential of the cells, even after the highest dose 3200 Gy (Figure VI-6). The PSII quantum yield of irradiated cells was stable immediately, 2 hours and 5 hours after irradiation and similar to non-irradiated healthy Arthrospira cells displaying a

FV/FM yield of ca. 0.6 [15].

Figure VI-6: Photosynthetic capacity of Arthrospira sp. PCC 8005 after gamma irradiation. The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Data represent the mean of three independent cultures (n= 3) and error bars display the standard error of the mean (SEM). The statistical analysis was calculated on raw data.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays

Figure VI-7: Pigment content of Arthrospira sp. PCC 8005 after irradiation. The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Allophycocyanin content, Phycocyanin content; and Chlorophyll A content were shown at T0H, T2H and T5H respectively. Data represent the mean of three independent biological replicates, and error bars present the standard error of the mean (SEM). The statistical analysis was calculated on raw data. One asterisk indicates a value which is significantly differing (p<0.05) from the non-irradiated control.

VI.3.5 Survival, Recovery and proliferation The Arthrospira sp. PCC 8005 cultures were indeed able to fully recover normal photosynthetic growth after exposure to all 3 different doses tested, i.e. 800 Gy, 1600 Gy and 3200 Gy (Figure VI- S 1). Growth curves showed total recovery of photosynthetic growth after all exposures, although with significant delay at 1600 Gy and 3200 Gy (upto 8 days for 3200 Gy) (Figure VI-S 1). Non-

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays irradiated samples and samples irradiated with 800 Gy obtained a maximum specific growth rate between day 1 and 3 respectively (Table VI-S 1). For cells exposed to 1600 Gy, maximum growth rate was only seen between day 3 and 6, and cell exposed to 3200 Gy needed 14-17 days to reach maximum growth rate respectively (Table VI-S 1). The exposure of Arthrospira sp. PCC 8005 in this set-up to 10 000 Gy of 60Co gamma rays was lethal, this dose killed all the cells, after which no recovery was resumed (data not shown).

VI.4 Discussion Arthrospira is highly resistance against gamma radiation [13]. In this study, the cells maintained photosynthetic capacity and were capable to recover photosynthetic growth after exposure, for all doses tested up to 3200 Gy. The radiation had little effect on the morphology, pigmentation, or photosynthetic capacity of the cells. Despite these little physiological changes, there was however, a strong gene-expression reprogramming in the cells exposed to radiation. The higher radiation dose, the more pronounced the transcriptional and physiological response of the cells, and the longer the delay in restart of active photosynthetic growth after irradiation. The dynamic transcriptomic response showed to two waves: an early 'emergency-type' response that occurred immediately upon irradiation and a recovery response during the two and five hours after irradiation. The acute exposure to high doses of radiation, induced a clear 'shock' in the cells, which showed the largest changes in transcription immediately after irradiation. Arthrospira sp. PCC 8005 cells, immediately expressed and supressed a high number of genes, even without direct correlation to physiological changes. During early response, the transcriptome showed a significant repression of genes involved in photosynthesis, carbon, and nitrogen assimilation; and the higher the radiation dose, the longer the reduced expression was maintained. Although overall pigment content and the photosynthesis capacity (PSII quantum yield) measurements did not reveal a drastic irradiation effect, the active renewal of the proteins involved in photosynthesis was temporarily put-on hold during the irradiation and only switched back-on gradually after irradiation during the recovery. The short irradiation time (minutes), the long half-life of the proteins involved, and the lack of an active phycobilisome degradation via NblA enzyme (whose expression was not altered) [27], may explain why photosynthesis measurement revealed intact and functional photosystem even after exposure to the highest dose 3200 Gy. The reduced transcription for photosynthesis in response to radiation was combined with a reduced transcription of the photosynthetic energy production pathway, i.e. the ATP production systems.

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays Our results did show, however, the active transcription of a bidirectional hydrogenase, a Ni-Fe metalloenzyme, for reversible oxidation of di-hydrogen (H2) [28] [29]. The physiological role of this enzyme, however, has been a matter of speculation and is still unclear. Nevertheless it has been proposed that this enzyme functions as a valve for low potential electrons generated during the light reaction of photosynthesis, thus preventing slowing down the electron transport chain under stress conditions [30]. This enzyme is known to stimulate metabolic processes in darkness, with reduced supply of bicarbonate, nitrate or sulphate and other environmental stresses [31]. High rates of hydrogen production have been obtained in Arthrospira sp PCC 8005 cells adapted to nitrogen and sulphur deprived medium supplemented with iron and beta-mercapto-ethanol [32]. With the reduced transcription for photosynthesis and energy production, cells reprogrammed also the expression of genes for carbon metabolism during irradiation. For instance, several genes involved in de Calvin-Benson-Bassham-cycle (CBB-cycle) that fixes carbon dioxide into glyceraldehyde-3-phosphate (G-3-P) and finally hexose sugars such as glucose, and the Krebs cycle (also known as the tricarboxylic acid (TCA) or the citric acid cycle) providing the precursors for amino acids biosynthesis, were significantly repressed in early response. This is consistent with reported findings that showed the repression of TCA pathway cycle immediately after irradiation of Deinocccus radiodurans as well as during the first hours of the recovery period [33,34]. The carbon metabolism gene expression profile showed that irradiation caused a re-routing of the metabolic flux from glycolysis to the pentose phosphate pathway, in favour for NADPH and pentoses generation [35]. NADPH and pentose molecules are essentially required for d’NTP synthesis, and may acts as cofactor for glutathione and thioredoxin reductases [36]. In addition, Arthrospira sp. PCC 8005 cells rerouted their carbon metabolism to synthesis of intracellular compatible solutes and storage molecules in the form of trehalose and polyhydroxyalkanoate (PHA). Several reports suggest the contribution of trehalose in response to high salt stress [8], and in desiccation tolerance by protecting the cells and proteins from oxygen radicals [37]. Recently Kimberley and co-worker suggested a possible role of trehalose as an efficient protectant of protein activity (enzymes) against irradiation, either alone or in combination with Mn2+. The addition of trehalose resulted in a significant increase in enzyme protection, up to 6 000 Gy (given at a dose rate 3 200 Gy hr-1) of 60Co gamma rays [38]. Trehalose can also stabilize membranes and maintain their integrity and fluidity [39]. The intracellular store of carbon in the form of polyhydroxybutyrate (PHB) enables most cyanobacteria to survive stress conditions, such as

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays micronutrient starvation (e.g. nitrogen) and to recover rapidly [40]. It has been reported that during desiccation Micrococcus vaginatus induces the transcription level of its PHA biosynthesis genes [41]. Furthermore, our findings showed an altered nitrogen uptake, assimilation and metabolism. Nitrogen is, however, an essential component for synthesis of amino acids for structural proteins and enzymes, nucleotides for DNA and RNA, and amino sugars for lipopolysaccharide and peptidoglycan in the cell envelope [42]. In general, genes for nitrogen uptake and assimilation were strongly reduced in expression after irradiation. This includes nitrate, nitrite, cyanate, and amino- acid uptake, nitrile hydrolysis and pattelamide biosynthesis. It is well known that cyanophycin (L- Arginine and L-Aspartic acid) is thought to represent a dynamic reservoir of nitrogen accumulated in both non-nitrogen fixing and filamentous or unicellular N2-fixers, responding to the N regime [43]. Under nitrogen limiting conditions cyanophycin is catabolized as an internal nitrogen source, balancing the nitrogen deficiency. However, our findings did show no related genes responsible for this process namely cyanophycinase (cphA, cphB genes), confirming that the cells did not seem to experience N-limitation. At the contrary, the nitrogen metabolism seemed to be also adjusted to deal with high ammonium concentrations, possibly released from radiation damaged proteins, nucleotides and amino-sugars, which are known to be highly toxic to the cell. The ammonia produced from degradation of aminoacids, is usually quickly neutralised via urea synthesis [44]. There was after irradiation indeed also an increased transcription of genes in Arthrospira for the decomposition of urea to CO2 and ammonium. There was also a clear deactivation of genes involved in polyamine degradation, i.e. agmatine degradation to putrescine and urea, and a clear transcriptional activation of polyamines transporters (e.g spermidine and putrescine). This is possibly a mechanism to prevent even more urea and ammonium production. Polyamines are a group of nitrogen (amine) containing compounds that received recently considerable attention owing to its possible role in abiotic stress resistance [45]. Studies on polyamine transport in cyanobacteria have been scarce, but the implication of polyamine transport to protect Synechosystis against salt stress has been reported [46]. The regulation of C and N nutrient stress is typically under the control of the metabolic signal 2- oxoglutarate (in TCA cycle), which interacts with the sensor regulator PII protein (glnB), a transcriptional regulator, which in its turn induces the expression of genes involved in the signalling cascade controlling nitrogen metabolism, such as nblA. The nblA gene was induced significantly

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays after exposure of Arthrospira sp. PCC 8005 to high acute doses of gamma rays in our previous studies (5000 Gy) [13], but not significant at the doses tested here (800 Gy, 1600 Gy, 3200 Gy).

The transcription of the PII protein was indeed significantly altered after radiation. The mechanism of the PII protein is well known and conserved for many organisms. It has been reported that the cellular carbon, nitrogen, energy and redox status which are sensed through ATP and 2- oxoglutarate. It is likely that the PII protein and the global transcription factor ntcA are involved in glycogen degradation [47]. The PII protein induces global transcriptional regulators such NtcA, the RNA polymerase sigma factor SigE and other response regulators such OmpR in Synechosystis sp PCC 6803 [48] and NrrA in Anabeanna sp PCC 7120 [49]. The growth of cyanobacteria cells depends on a tight balance in Carbon/Nitrogen ratio. Overall, this temporarily reduced expression of proteins involved in photosynthesis, carbon and nitrogen assimilation immediately after radiation, are likely responsible for the observed delay in growth and the lower maximum specific growth rate achieved in cultures after exposure to 1600 and 3200 Gy. Similarly, it was shown that the growth rate of Synechocystis sp. PCC 6803 was retarded after 1 W m−2 UV-B radiation due to the reduction in amino acid biosynthesis [50]. Furthermore, our results suggest that while photosynthesis and growth was temporarily put on hold, Arthrospira sp. PCC 8005 counteracts the cell damage caused by gamma rays via an increased expression of a wide range of detoxification, repair, and protection systems. The classical enzymatic way for anti-oxidative defense relies mainly on catalase and superoxide dismutase (SOD), well reported to be highly expressed and essential for survival of cyanobacteria exposed to various stresses [51]. However, the catalase gene is absent in the Arthrospira sp. PCC 8005 genome and the single SOD gene was not induced in Arthrospira sp. PCC 8005 after exposure to gamma radiation, not in our previous studies and not in this study. The lack of significant expression of the SOD gene in response to irradiation might be due to their continuous high expression during normal conditions (i.e. in absence of irradiation), but would need to be confirmed, or could mean that other (non-enzymatic) antioxidant systems are used. Arthrospira indeed seemed to rely on different ROS detoxification and redox balancing methods to resist ionizing radiation. Arthrospira used essentially thiol-based anti-oxidant enzymes and molecules, such as peroxiredoxine and glutathione. Transcription in Arthrospira showed the activation of different genes involved in biosynthesis and regeneration of glutathione: the enzyme 5-oxoprolinase (hyuA) catalyses the generation of glutamate from 5-oxoproline, enzyme Glutamate Cysteine Ligase (gcL) converts

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays glutamate to gamma-glutamylcystein ,and the enzyme glutathione synthetase (gshB) converts the precursor gamma-glutamylcystein to glutathione. Proline is the main precursor for glutathione synthesis and our findings suggest also a synthesis of proline in response to radiation. In addition, considered as potent antioxidant. It has been proposed that free proline can act as hydroxyl and singlet oxygen scavenger, inhibitor of lipid peroxidation and osmoprotectant [52,53]. Our results also showed the up-regulation of the putA gene contributing in glutamate synthesis from proline. PutA is a flavoprotein with mutually exclusive functions as membrane-associated enzyme and a transcriptional repressor. The switch between the two activities is due to conformational changes triggered by proline binding. In the presence of proline, PutA is associated with the cytoplasmic membrane and acts a bifunctional enzyme catalyzing both reactions of the proline degradation pathway: the oxidation of proline to Pyrroline-5-carboxylate (P5C) by proline dehydrogenase and subsequent oxidation to glutamate by pyrroline-5-carboxylate (P5C) dehydrogenase. In the absence of proline, PutA is cytoplasmic and functions as a transcriptional repressor of the put regulon. Glutamate synthesis was also induced via asparte-aminotransferase catalysing the formation of glutamate from aspartate and 2-oxoglutarate (also called α-ketoglutarate) from the TCA cycle. Glutamate can also synthesised from 2-oxoglutarate, via the GS-GOGAT pathway or the dehydrogenase (GLDH) pathway [54]. The GS-GOGAT pathway (also called glutamine synthetase (GS) or glutamate synthase (GOGAT) pathway) was immediately shut-down; while the glutamate dehydrogenase (GLDH) pathway was induced. Both pathways provide glutamate synthesis from

NH3 and 2-oxoglutarate, but normally the GS-GOGAT pathway is used at low ammonium concentrations and when the cell is not under energy limitation (i.e. with sufficient reduced ferrodoxin or NAD(P)H), while the GLDH pathway is used when the cell is limited for energy and carbon but ammonium and phosphate are present in excess [55]. Synechocystis sp. strain PCC 6803, for example, utilizes the GS-GOGAT pathway as the primary pathway of ammonia assimilation, but the presence of GDH appears to offer a selective advantage for the cyanobacterium under nonexponential growth conditions [56]. It is known that high ammonia availability (as discussed above) leads to repression and deactivation of GS-GOGAT and induction of GDH [57]. The actual production of glutathione, which increased significantly during recovery, was confirmed via glutathione metabolite concentration analysis. Glutathione molecules are well known to play an important anti-oxidative role in the defense system of plants [58], and thus seem to provide the same benefits for cyanobacteria. Also the radiation resistant bacterium Deinococcus relies on a

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays thiol-based antioxidant, called Bacithiol and considered as a substitute for glutathione, for its extreme resistance to gamma rays [34]. The thiol group is very reactive, and quickly neutralizes radicals, such hydrogen peroxide, and singlet oxygen and hydroxyl radicals. Several studies report a significant role of the such small antioxidants molecules in protecting proteins against oxidation after irradiation and as such preserving the enzymatic functions needed for DNA repair [59-61] (Figure VI-8). In line with the detoxification systems described above, gene expression results demonstrate an increase in trans-membrane transport of redox-active metals. It is well known that metals such as iron (Fe), manganese (Mn), magnesium (Mg), and copper (Cu) are essential cofactors for the operation of the oxygenic photosynthetic electron transfer apparatus [62]. Higher levels of these metals are found in cyanobacteria compared to non-photosynthetic bacteria [63]. Despite the fact that metals play a key role in oxygenic photosynthesis, they can pose at the same time a major risk via the generation of free radicals (ROS). Therefore, metal transport and storage are tightly regulated to ensure adequate supply and to protect against oxidative damage, a process called metal homeostasis. The proliferation of all photosynthetic organisms depends on this delicate balance between the metal requirements and oxidative damage [64]. The activation of such ROS detoxification response normally involves different sensors, signal transduction systems and transcriptional regulators. Molecules with PAS and GAF domains serve as specific sensors that react to oxidative stress, light, oxygen and many other signals [65,66]. Gene expression showed also the induction of the cry-DASH gene well known as photo-sensor for UV- A light induced photo-tactic movement [26]. Where a recent study reported that Syn-cry, the cry- DASH gene from the cyanobacterium Synechosystis sp. PCC 6803, was required for efficient restoration of photosystem activity following UV-B and PAR induced photo-damage [67]. Also, the transcriptions of the genes for synthesis of the secondary messenger cyclic diguanylate (c-di- GMP) were induced. The aconitase enzyme (in TCA cycle), which showed an increased transcription upon irradiation (3200 Gy), has been reported to play a role in transcriptional regulation of ROS detoxification processes [68]. It has been proposed that aconitase, containing an iron sulphur cluster [4Fe-4S] as ROS sensor, can mediate the response to oxidative stress via transcription regulation [69]. The regeneration of Fe-S cluster is under the transcriptional regulation sufR, a gene which was also up regulated upon irradiation (Figure VI-8).

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays Despite antioxidant systems, radiation usually still damages lipids, proteins and DNA, which needs to be cleaned up or repaired within the cell to allow survival and proliferation. In cyanobacteria, lipids present in the thylakoids contain a high percentage of polyunsaturated fatty acid (PUFA) residues and are thus susceptible to peroxidation [70]. Gene expression showed induction of esterase/lipase activity that might be involved in degradation of damage lipids. In the meantime, desaturase genes (des) were significantly repressed. Proteolysis and the import of exogenous peptides and amino acids are among the metabolic properties reported for Deinococcus to overcome oxidative stress [36]. Also Arthrospira seemed to increased protease transcription during early response which was maintained in the recovery period. Together with proteases, also heat shock protein and chaperone genes were overexpressed, well known to be involved in stress response [71]. In bacteria such as E. coli and B. subtilis repair of radiation-induced DNA-damage is activated via the SOS system. Two key proteins regulate the SOS system; the protease RecA and transcriptional repressor LexA were the former activates auto-cleavage of the latter to induce SOS response. However, in the study of Narumi et al. [72] the authors demonstrate the non-involvement of LexA in the RecA induction in D. radiodurans following gamma radiation. Similar observations were discussed for the cyanobacterium Synechocystis sp. PCC 6803 [73,74]. Arthrospira sp. PCC 8005 genome lacks the gene of the lexA repressor. In addition, the cells did not induce the recA gene in this study. As for SOD, it might be possible that a large amount of RecA protein is constantly present in the cell, even in the absence of DNA damage, and therefore is not induced by radiation. A similar observation was reported Helicobacter pylori [75], showing no activation of the RecA protein following exposure to UV (10 J/m2) or gamma radiation (75 Gy). Thus, this might suggest the existence of a rather high constitutively expressed DNA damage repair system. The only DNA- repair pathway activated in Arthrospira sp. PCC 8005 in a dose dependent manner at early response was the nucleotide excision and repair (NER) mechanism (uvrBCD genes), responsible for the repair of single strand breaks. [76]. In addition, Arthrospira sp. PCC 8005 activated several genes involved in DNA Restriction and Modification. These findings would suggest that the main effort is going to single strand break repair, but that there is double strand damage and repair or that the true mechanism of double strand DNA repair systems of this organism are yet to be characterised. The most pertinent finding is the high expression of the gene cluster arhABCDEF, the only set of genes that was highly expressed from start and throughout the full recovery. As reported before,

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Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays the cluster was overexpressed both on RNA and protein level in response to irradiation. The gene set was induced in dose dependent manner after exposure of Arthrospira sp. PCC 8005 to lethal doses (e.g: 3200 Gy and 5000 Gy) of 60Co gamma rays. So far the reported gene set was not differentially expressed in Arthrospira sp. PCC 8005 in response to other stresses, such as nitrogen deprivation [77] or light/dark growth transition [78]. The clear correlation between this gene set and the resistance of Arthospira sp. PCC 8005 to gamma rays cannot be denied. Although, no clear function could be assigned to theses genes yet, it cannot be excluded that they are involved in DNA repair. Two of the five genes (i.e. arhC and arhB) show a significant homology with the conserved domain of chromosome segregation proteins (SMC). In eukaryotic cells SMC proteins are responsible for chromosome condensation, segregation, cohesion and DNA recombination repair [79]. SMC-like proteins are also present in Bacteria and Archea and perform essential functions in a variety of chromosome dynamics, such as chromosome compaction, segregation, and DNA repair [80]. SMC-like genes that exist in bacteria include recN and sbcC, which catalyse protein assembly at replication forks, and which are present in PCC 8005 in X copies, but not differentiall expressed. These proteins may act in early stage upon induction of DNA damage. This finding urge for additional studies, to further dissect the clear function of these genes.

VI.5 Conclusion The response of the cyanobacteria Arthrospira to an acute exposure to gamma rays involves a fast switch from an active growth state to a kind of 'survival state' during which the cells put photosynthesis and carbon and nitrogen assimilation on hold and activate pathways for cellular detoxification, reparation and protection. The cellular detoxification Arthrospira is mainly based on glutathione and metal homeostasis. DNA damage and DNA repair or protection based on classical systems seems surprisingly minimal. However, the radiation resistance of Arthrospira likely involves also some genes unique for Arthrospira with unknown functions and which are highly and specifically expressed in response to radiation, in a dose dependent manner. The higher the radiation dose, the more pronounced this shock response and acclimation to survival state is expressed. This probably makes it for the cells more difficult to revert from survival to active growth state, resulting in a longer delay in photosynthetic recovery and slower growth rates after exposure to higher radiation doses.

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A B

Figure VI-8: General overview of the main Transcriptional response events of Arthrospira sp PCC 8005 after exposure to different doses of 60Co gamma rays. Schemes represent a global gene expression response (a) immediately after irradiation; (B) after 2H and 5H in recovery period. Blue color, stand for down-regulated genes. Red color stand for up-regulated genes, Green color stand for restored expression of the initial silenced genes.

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(A): The largest changes in transcription occurred upon irradiation: “Emergency Response” displayed a reduced transcription for photosynthesis and energy production, cells reprogrammed also the expression of genes for carbon and nitrogen metabolism during irradiation. TCA cycle was repressed. Induced transcription of sigE regulator acting as nitrogen-dependent activator for catabolic genes towards glycogen degradation was seen. A re-routing of the metabolic flux from glycolysis to the pentose phosphate pathway (PPP) was seen. A synthesis of carbon storage molecules (PHB) and compatible solutes (trehalose) was seen. Induced polyamines transporters potABC, well known as group of nitrogen containing compounds. Urease (ureABC) activity was induced.

The cellular detoxification, repair and protection were enhanced immediately after irradiation. Reactive oxygen species generated via indirect damage has to be removed. ROS detoxification was maintained via the tight control of metal transport to protect against oxidative damage. In addition as antioxidant molecules, peroxiredoxine enzyme and glutathione synthesis genes were observed. The generation of glutathione starts at T0H via the formation of glutamate from proline by huyA or aspartate by aspartate aminotransferase (aat1), and from 2-oxoglutarate via GDH. Then the synthesis of glutathione occurred via glutathione synthase during recovery. Glutamate synthesis via the GS/GOGAT cycle was repressed, but glutamate synthesis was induced GDH pathway and via asparte-aminotransferase catalysing the formation of glutamate. In parallel Arthrospira may enhance some genes related to DNA repair system (uvrBCD for nucleotide excision and repair, ruvB resolving holiday junction, and recJ genes). The DNA-repair mechanism of Arthrospira included also Enzymatic Restriction Modification (hsdr) and endonucleases molecules. Chaperones and proteases were also significantly present during this stage.

(B) During the late phase Arthrospira cells try to recover slightly, minor (but not significant) induction of genes related to Energy production /Conversion / Coenzyme transport and metabolism: photosynthesis, Calvin cycle, TCA cycle was observed. Hydrogen formation occurred in this stage.

In parallel slight activation of amino acid transport occurred. The inorganic molecules such Taurine (via transporters tauABC) known as nitrogen source were highly expressed. ROS detoxification was maintained efficiently via glutathione molecule. Few genes related to protease, DNA repair

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VI.7 Supplemental data

Figure VI-S 1: Growth curves of Arthrospira sp. PCC 8005 following exposure to different doses of gamma rays. Data represent mean of three independent biological replicates (n=3), and error bars present the standard error of the mean (SEM).

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Table VI-S 1: Specific growth rate for the cultures grown after irradiation

ln(OD750 at t2)−ln(푂퐷750 푎푡 푡1) For each time interval between 2 time points was calculated with following formula: µ = . 푡2−푡1 The last row presents the maximum growth rate obtained for each radiation dose. Data represent mean of three independent cultures (n= 3). An asterisk indicates a value for the irradiated sample which is significant (p<0.05) different from the value of the corresponding non-irradiated control. Three asterisk indicate a value which is highly significant (p<0.001).

Time intervals (Days) CTR 800 Gy 1600 Gy 3200 Gy (n=3) (n=3) (n=3) (n=3) 1-3 0,956 0,686 0,284 -0,591 3-6 0,463 0,440 0,406 0,326 6-8 0,255 0,286 0,319 -0,168 8-10 0,186 0,212 0,279 0,231 10-14 0,128 0,150 0,212 0,435 14-17 0,042 0,082 0,140 0,446 17-21 0,054 0,062 0,044 0,315 21-24 0,034 0,016 0,042 0,152 24-29 0,0008 -0,011 0,035 0,058 29-31 -0,007 0,046 -0,030 0,007 31-33 - - - -0,009 Lag time (µ = 0)(days) 0 0 0 8** Exponential phase (µ>0)(days) 29 24 29 29

Maximum growth rate µmax 0,956 0,686 0,406* 0,446* (∆OD750*Day-1) ±0,149 ±0,235 ±0,106 ± 0,116

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Table VI-S 2: Genes differentially expressed Arthrospira sp. PCC 8005 during the early emergency reponse after irradiation

Genes belong to clusters 3, 5, 4 and 6. Clustering was done on genes having an absolute Log2 Fold change higher than 1 and a p-value corrected for multiple testing lower than 0.05, in either one of the 9 conditions.

Emergency Gene 800 800 800 1600 1600 1600 3200 3200 3200 Protein function response name T0H T2H T5H T0H T2H T5H T0H T2H T5H Antioxidants

ARTHROv5_30341 putative peroxiredoxin 2,10 1,35 0,23 1,73 1,15 0,28 1,20 1,06 0,41 Glutamate synthesis ARTHROv5_10456 hyuA 5-oxoprolinase 3,79 -1,34 0,94 3,89 -0,10 0,00 3,89 1,80 0,45 ARTHROv5_41057 proA1 Gamma-glutamyl phosphate reductase 2,32 1,28 1,44 2,69 1,44 1,32 2,87 2,05 1,35 proline dehydrogenase and 1-pyrroline-5 ARTHROv5_30794 putA 0,98 0,17 0,24 1,26 0,21 0,26 1,54 0,48 0,16 carboxylate dehydrogenase ARTHROv5_10357 pep prolyl endopeptidase (PE) 2,03 1,18 1,05 2,32 1,24 0,93 2,52 1,78 1,03 ARTHROv5_30084 aat1 Aspartate aminotransferase 2,35 0,96 1,16 2,97 1,19 1,16 3,29 1,51 1,14 Redox Metal homeostasis ARTHROv5_10584 copA1 copper transport -0,39 -0,41 -0,04 0,13 0,02 0,07 1,40 0,03 -0,17 ARTHROv5_40724 cutA copper transport 0,64 -0,04 0,50 1,06 0,14 0,38 1,67 0,50 0,29 ARTHROv5_60812 corA magnesium/nickel/cobalt transport 0,67 0,39 0,46 0,82 0,54 0,35 2,17 1,17 0,26 ARTHROv5_41061 mtgC magnesium transport -0,16 -0,16 -0,34 0,69 -0,17 -0,60 1,55 0,12 -0,74 ARTHROv5_11739 znuA zinc transport 1,25 0,06 0,16 1,10 0,19 0,20 1,00 0,60 -0,18 ARTHROv5_61253 cobB cobyrinic acid -0,28 -0,14 -0,45 -0,23 -0,07 -0,36 1,11 0,01 -0,66 ARTHROv5_30441 cbiO1 cobalt transport 0,87 -0,21 0,06 1,25 -0,16 0,12 1,36 0,08 -0,01 ARTHROv5_10935 cbiQ2 cobalt transport 2,20 1,32 1,17 2,39 1,28 1,11 2,70 1,99 1,21 ARTHROv5_61130 potassium transport 1,95 0,51 0,61 1,83 0,46 0,63 2,06 0,81 0,76 ARTHROv5_40647 feoA ferrous iron transport 1,76 -0,25 0,01 0,81 0,21 -0,3 -0,95 0,65 -1,00 ARTHROv5_40648 feoB ferrous iron transport 1,41 -0,34 -0,01 0,45 0,03 -0,21 -0,59 0,32 -0,64 ARTHROv5_60903 ferric iron transport 4,59 0,74 0,51 4,14 0,61 0,22 3,38 1,63 0,07 ARTHROv5_60473 fur ferric iron uptake regulation -0,39 0,06 0,47 0,94 0,75 0,49 1,98 1,32 0,08 iron stress-induced chlorophyll-binding protein ARTHROv5_61180 isiA 4,52 2,15 1,04 3,81 2,28 0,79 3,12 2,58 0,88 (CP43') 197

Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays

ARTHROv5_30590 transcriptional regulator with CopG/Arc/MetJ 3,27 2,29 2,07 4,16 2,19 2,15 4,04 2,77 2,64 ARTHROv5_41184 ompA Outer membrane porin 1,28 -0,10 -0,21 2,66 0,18 0,29 3,31 0,24 -0,24 ARTHROv5_41185 ompA Outer membrane porin 3,25 0,55 0,17 4,24 0,79 0,82 4,22 1,02 0,51 ARTHROv5_60877 putative permease 0,70 0,16 0,03 1,20 0,08 0,14 1,47 0,47 0,00 ARTHROv5_11605 putative permease 1,99 0,90 1,26 2,44 1,01 1,18 2,84 1,60 1,36 Protein damage clean up ARTHROv5_10934 putative metallopeptidase 0,92 0,73 0,36 1,22 0,54 0,57 2,06 0,86 0,3 ARTHROv5_40599 patG Subtilisin-like protease 2,30 1,12 1,22 2,19 1,19 1,20 1,93 1,04 1,04 ARTHROv5_30014 dnaK1 Chaperone protein 1,84 0,76 1,01 2,56 0,95 0,88 3,2 1,59 1,02 ARTHROv5_61125 hspA heat shock protein A 1,56 0,29 0,27 1,43 0,42 0,45 1,40 0,70 0,46 ARTHROv5_11999 dnaK2 Hsp70, co-chaperone with DnaJ 1,04 -0,20 -0,37 0,65 -0,42 -0,12 0,10 -0,45 -0,19 ARTHROv5_61127 cbpA curved DNA-binding protein, DnaJ homologue 1,71 0,25 0,25 1,58 0,33 0,31 1,68 0,59 0,49 ARTHROv5_41259 clpB2 protein disaggregation chaperone 1,67 -0,26 -0,09 1,51 -0,13 0,07 0,94 -0,12 0,03 iron-sulphur cluster biosynthesis ARTHROv5_11765 sufR 1,25 0,07 0,29 1,22 0,12 0,41 1,32 0,33 0,64 transcriptional regulator SufR Nitrogen metabolism ARTHROv5_61139 lysE LysE/RhtB family amino acid efflux pump 3,84 1,58 1,78 3,62 1,75 1,80 3,57 2,28 2,09 ARTHROv5_30069 ureA Urease subunit gamma 1,04 0,27 0,30 1,51 0,26 0,25 1,57 0,30 0,23 ARTHROv5_30068 ureB Urease subunit beta 0,94 0,28 0,28 1,48 0,17 0,22 1,42 0,32 0,29 ARTHROv5_30067 ureC Urease subunit alpha 1,11 0,28 0,30 1,68 0,30 0,25 1,78 0,46 0,33 ARTHROv5_30070 ureD Urease accessory protein ureD 1,84 0,64 0,80 2,64 0,70 0,82 3,02 1,02 1,14 ARTHROv5_60622 nifU Nitrogen-fixation 2,18 0,64 1,47 2,87 1,23 1,54 3,60 1,79 1,61 ARTHROv5_40220 devA putative Heterocyst specific transporter 2,53 0,97 1,50 3,24 1,16 1,45 3,31 1,84 1,47 ARTHROv5_40318 rbsK Ribokinase (PPP) 2,91 1,49 1,75 3,22 1,71 1,82 3,13 1,95 1,76 ARTHROv5_30379 potB Polyamine transport 1,06 0,60 0,42 1,54 0,92 0,58 1,98 1,14 0,61 ARTHROv5_30377 potC Polyamine transport 0,44 -0,02 -0,24 1,32 0,07 0,00 1,76 0,66 -0,16 DNA damage repair ARTHROv5_11714 putative ADP-ribose pyrophosphatase 0,34 0,26 0,36 1,93 0,45 0,37 3,59 0,82 0,85 ARTHROv5_40086 mutT NUDIX hydrolase 1,27 0,73 0,73 1,92 0,77 0,62 2,45 1,26 0,77 ARTHROv5_20108 recJ Single-strand-DNA-specific exonuclease 1,46 0,26 0,40 2,08 0,57 0,33 1,66 0,97 0,18 ARTHROv5_40732 uvrB excinulease of nucleotide excision repair, 2,02 1,01 1,32 2,60 1,19 1,24 2,73 1,84 1,17 ARTHROv5_60258 uvrC UvrABC system protein C 1,48 0,81 0,30 2,19 0,80 0,52 2,11 1,20 0,20 ARTHROv5_41027 uvrD DNA helicase, UvrD/REP 2,84 1,46 1,56 3,87 1,76 1,67 4,03 2,43 1,81 ARTHROv5_60750 helD putative DNA helicase, UvrD-family 1,13 -0,17 -0,17 1,15 -0,22 -0,10 0,92 -0,13 -0,48

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ARTHROv5_30188 Helicase-like protein 1,88 0,51 0,27 1,96 0,56 0,55 2,32 0,74 0,57 ARTHROv5_11763 ruvB ATP-dependent DNA helicase 1,12 0,08 -0,22 0,98 -0,04 -0,04 1,57 0,57 -0,18 ARTHROv5_10675 dnaG DNA primase 2,06 0,70 0,81 2,35 0,83 1,03 1,81 0,94 1,21 DNA modification and protection ARTHROv5_30008 mod Modification methylase 1,23 0,38 0,33 1,83 0,45 0,61 1,09 0,36 0,59 ARTHROv5_30623 hsdR1 Type I site-specific deoxyribonuclease, HsdR 4,19 2,50 2,58 4,47 2,84 2,61 4,63 3,53 2,85 ARTHROv5_30624 hsdR2 Type I site-specific deoxyribonuclease, HsdR 3,64 2,02 2,31 4,09 2,40 2,38 4,31 3,03 2,65 ARTHROv5_30625 hsdR3 Type I site-specific deoxyribonuclease, HsdR 2,51 0,95 0,90 3,09 1,38 0,93 3,37 2,03 0,98 ARTHROv5_60368 Type II DNA modification enzyme 1,85 0,54 0,65 2,20 0,62 0,50 1,78 1,02 0,78 asp8005 ARTHROv5_40255 ORF580 Type II DNA modification methyltransferase 1,18 0,31 0,31 1,93 0,45 0,50 1,80 0,82 0,25 0M asp8005 ARTHROv5_30352 ORF035 Type II restriction enzyme 2,68 1,06 0,80 3,09 0,57 1,25 2,21 0,42 1,59 9 ARTHROv5_50004 pvuIIR Type II restriction enzyme 2,67 0,45 0,31 2,83 0,26 0,21 3,07 0,50 0,16 DNA modification – FAX elements unknown phage of the genus Arthrospira, ARTHROv5_10168 faxB1 0,62 -0,23 -0,13 0,73 0,20 0,56 1,47 0,11 -0,10 protein B unknown phage of the genus Arthrospira, ARTHROv5_30751 faxB1 0,82 -0,05 0,27 0,93 0,40 0,74 2,06 0,46 0,29 protein B unknown phage of the genus Arthrospira, ARTHROv5_30553 faxK1f3 3,16 1,32 0,69 2,26 1,66 1,36 1,58 1,57 1,20 protein K unknown phage of the genus Arthrospira, ARTHROv5_40353 faxK4f2 3,28 1,74 1,25 2,25 1,83 1,60 1,87 1,86 1,72 protein K unknown phage of the genus Arthrospira, ARTHROv5_30731 faxO8f 2,31 0,79 0,73 2,43 1,21 1,05 2,55 1,39 1,04 protein O unknown phage of the genus Arthrospira, ARTHROv5_30730 faxP8 1,21 0,40 0,45 1,85 0,37 0,78 2,65 0,51 0,79 protein P T/TA systems ARTHROv5_11210 yefM putative antitoxin of toxin-antitoxin 2,71 1,53 1,57 3,43 1,41 1,69 3,62 1,94 1,71 ARTHROv5_11211 yoeB putative toxin of toxin-antitoxin 4,88 2,50 3,00 5,41 2,52 3,04 5,53 2,93 3,28 ARTHROv5_12008 mazF9 mRNA interferase 2,64 0,75 2,11 2,97 1,12 1,93 3,24 1,54 2,15 ARTHROv5_12009 Toxin/antitoxin 2,54 0,79 1,54 2,75 1,26 1,35 3,23 1,96 1,58 DNA modification – CRISPER elements ARTHROv5_40678 cas CRISPR-associated endonuclease Cas1 1,87 0,54 0,67 2,59 0,77 0,80 2,95 1,08 0,74

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ARTHROv5_40676 cas2 CRISPR-associated endoribonuclease Cas2 2,13 0,56 0,68 2,64 0,66 0,91 2,97 0,98 1,07 ARTHROv5_40718 CRISPR-associated RAMP protein, Csm3 2,48 1,09 1,14 2,47 1,31 1,30 1,55 1,48 1,09 ARTHROv5_40717 CRISPR-associated RAMP protein, Csm4 2,67 1,30 1,18 2,45 1,36 1,44 1,66 1,56 1,15 Storage & protection molecules ARTHROv5_41060 treS putative Trehalose synthase 1,39 0,32 0,43 1,54 0,23 0,29 1,58 0,67 0,30 ARTHROv5_11781 Transcriptional regulator degradation of sigE 1,71 0,24 0,66 1,83 0,44 0,74 1,70 0,29 0,45 glycogen ARTHROv5_10500 phaC Poly(R)-hydroxyalkanoic acid biosynthesis 2,00 0,60 1,00 2,14 0,79 1,02 2,36 0,95 1,06 ARTHROv5_10936 cbiM2 cobalamin biosynthesis protein 1,52 0,87 0,68 1,69 0,88 0,54 1,96 1,36 0,57 ARTHROv5_40927 cobW cobalamin biosynthesis protein 2,45 1,68 1,86 2,98 1,87 1,82 3,16 2,30 1,82 ARTHROv5_11097 ABC-type sugar transport 2,55 0,93 1,65 2,75 1,34 1,52 2,85 1,78 1,28 ARTHROv5_60792 ugpB ABC-type sugar transport 2,05 0,56 0,55 2,21 0,70 0,55 1,65 0,94 0,64 Lipid Degradation ARTHROv5_50008 Lipase class 3 1,22 -1,73 1,84 2,84 1,14 1,76 2,14 -0,49 1,54 ARTHROv5_10744 putative Esterase/lipase 3,05 1,52 1,91 3,31 2,02 1,82 3,58 2,61 1,78 Signal transduction ARTHROv5_10459 putative diguanylate cyclase 1,65 -0,05 0,02 1,08 0,24 -0,28 1,20 1,39 -0,44 putative diguanylate ARTHROv5_10653 0,96 0,34 0,14 1,20 0,07 0,28 1,53 0,53 0,62 cyclase/phosphodiesterase putative diguanylate ARTHROv5_10654 1,04 0,59 0,10 1,55 0,36 0,38 1,86 0,86 0,58 cyclase/phosphodiesterase putative diguanylate ARTHROv5_10656 0,86 0,41 0,27 1,95 0,30 0,56 2,70 0,83 0,80 cyclase/phosphodiesterase ARTHROv5_10963 cry Cryptochrome DASH 2,16 0,84 1,30 2,11 0,96 1,36 2,15 1,60 1,28 ARTHROv5_20097 Putative diguanylate cyclase/ (GAF) sensor 1,71 -0,11 0,51 3,13 0,83 0,73 5,42 1,83 0,30 ARTHROv5_20098 Putative diguanylate cyclase (GGDEF domain) 0,48 -0,33 0,23 1,46 0,28 0,30 4,82 1,34 0,13 putative diguanylate cyclase PleD-like ARTHROv5_30066 1,19 0,60 0,33 2,44 0,61 0,54 2,87 0,92 0,60 (fragment) GGDEF domain ARTHROv5_30396 Putative diguanylate cyclase (GGDEF domain) 1,92 0,71 0,76 1,85 0,55 0,93 2,14 1,06 0,80 putative diguanylate cyclase PleD-like ARTHROv5_30397 1,65 0,34 0,31 1,88 0,40 0,63 1,91 0,75 0,29 (fragment)GGDEF domain ARTHROv5_40303 putative Diguanylate kinase 0,57 0,11 0,08 0,99 0,33 0,20 1,82 0,56 0,21 ARTHROv5_50007 putative diguanylate cyclase 0,89 -0,04 0,42 1,36 0,46 0,43 2,09 0,92 0,14 putative Diguanylate ARTHROv5_50285 3,75 2,31 2,20 4,50 3,40 2,40 4,58 3,59 2,00 cyclase/phosphodiesterase

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Table VI-S 3: Genes silenced Arthrospira sp. PCC 8005 during the early emergency response after irradations.

Genes belongto clusters 1, 2, 7, 8 and 9. Clustering was done on genes having an absolute Log2 Fold change higher than 1 and a p- value corrected for multiple testing lower than 0.05, in either one of the 9 conditions.

Recovery period Gene name Gene function 800 800 800 1600 1600 1600 3200 3200 3200 T0H T2H T5H T0H T2H T5H T0H T2H T5H Phycobilisomes ARTHROv5_10635 apcC Phycobilisome linker -2,46 -0,46 -1,21 -3,19 -0,74 -0,82 -3,78 -1,87 -1,03 ARTHROv5_10637 apcA Allophycocyanin alpha -1,38 -0,24 -0,62 -1,64 -0,40 -0,28 -2,43 -1,21 -0,36 subunit ARTHROv5_10636 apcB Allophycocyanin beta -2,13 -0,50 -1,15 -2,49 -0,77 -0,69 -3,41 -1,98 -0,90 subunit ARTHROv5_12132 apcF allophycocyanin beta subunit -1,29 -0,61 -1,53 -2,07 -1,07 -1,28 -3,17 -1,82 -1,62 ARTHROv5_11555 cpcC1 Phycobilisome linker -0,73 -0,16 -0,11 -0,97 -0,12 -0,24 -1,80 -0,20 -0,39 polypeptide, ARTHROv5_11558 cpcE Phycocyanin alpha subunit -0,67 0,05 -0,12 -0,90 -0,00 -0,08 -1,62 -0,20 -0,04 ARTHROv5_60720 cpcT Chromophore lyase -1,16 -0,21 -0,86 -1,89 -0,50 -0,70 -1,78 -0,79 -0,98 Haem ARTHROv5_50123 hemC Porphobilinogen deaminase -0,80 -0,22 -0,60 -1,62 -0,42 -0,34 -2,46 -1,05 -0,49 ARTHROv5_60626 hemE Uroporphyrinogen -1,08 -0,18 -0,77 -1,95 -0,49 -0,53 -2,64 -1,11 -0,67 decarboxylase ARTHROv5_40397 cyoE Protoheme IX -1,22 0,54 -0,26 -1,92 0,29 0,04 -1,95 -0,46 0,04 farnesyltransferase (heme O synthase) Porphirin ARTHROv5_11943 cobA Uroporphyrinogen-III C- -0,99 -0,35 -0,16 -1,34 -1,15 -0,15 -1,71 -1,36 -0,41 methyltransferase ARTHROv5_10139 hemG Protoporphyrinogen oxidase -1,33 -0,29 -0,55 -1,93 -0,22 -0,40 -0,97 -0,60 -0,94 ARTHROv5_60173 hemN1 Oxygen-independent -2,77 -0,37 -0,97 -2,64 -0,80 -0,57 -1,89 -1,31 -0,85 coproporphyrinogen chlorophyll ARTHROv5_30766 chlG Chlorophyll a synthase ChlG -1,55 0,07 -0,33 -2,15 0,14 -0,03 -2,54 -0,60 -0,22

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ARTHROv5_11499 chlH Magnesium chelatase H -1,52 0,12 -0,41 -2,25 -0,03 -0,26 -2,90 -0,58 -0,38 subunit ARTHROv5_40023 chlP geranylgeranyl reductase -0,79 -0,07 -0,56 -1,40 -0,23 -0,45 -1,76 -0,56 -0,45 ARTHROv5_40768 bchD Mg-protoporphyrin IX -1,13 -0,37 -1,08 -2,03 -0,69 -0,69 -3,02 -1,81 -0,92 chelatase Plastoquinone ARTHROv5_10689 ndhA NADH:ubiquinone -0,90 -0,17 -0,31 -1,56 -0,30 -0,19 -2,11 -0,75 -0,29 oxidoreductase, membrane subunit H ARTHROv5_10690 ndhI NAD(P)H-quinone -1,49 -0,25 -0,62 -2,14 -0,38 -0,40 -2,70 -1,04 -0,61 oxidoreductase subunit I ARTHROv5_10691 ndhG NAD(P)H-quinone -1,02 -0,15 -0,43 -1,32 -0,27 -0,26 -1,61 -0,77 -0,29 oxidoreductase chain 6 ARTHROv5_10693 ndhE NADH:ubiquinone -1,38 0,08 -0,51 -2,03 -0,29 -0,34 -2,30 -0,79 -0,45 oxidoreductase, membrane subunit K ARTHROv5_40057 ndhH NAD(P)H-quinone -0,94 -0,02 -0,40 -1,86 -0,05 -0,16 -2,70 -0,40 -0,35 oxidoreductase chain H ARTHROv5_40541 ndhF1 NAD(P)H-quinone -1,08 0,05 -0,15 -1,61 -0,02 0,08 -2,09 -0,34 0,03 oxidoreductase chain 5 ARTHROv5_40542 ndhD1 NAD(P)H-quinone -1,36 0,03 -0,19 -1,98 -0,03 0,05 -2,23 -0,40 0,01 oxidoreductase chain 4 ARTHROv5_60547 ndhJ NAD(P)H-quinone -0,83 0,01 -0,25 -1,30 0,13 -0,16 -1,76 -0,19 -0,36 oxidoreductase subunit J ARTHROv5_60549 ndhC NAD(P)H-quinone -0,62 0,14 -0,20 -1,19 0,04 -0,12 -1,92 -0,18 -0,32 oxidoreductase subunit 3 ARTHROv5_60715 ndhD4 NAD(P)H-quinone -1,49 -0,57 -0,71 -2,40 -0,68 -0,54 -2,29 -1,46 -0,87 oxidoreductase chain 4 ARTHROv5_60716 ndhF NAD(P)H-quinone -0,92 -0,47 -0,53 -2,02 -0,43 -0,36 -2,69 -1,22 -0,71 oxidoreductase subunit 5 Cytochrome ARTHROv5_61102 cydA Cytochrome bd ubiquinol -1,42 0,25 -0,23 -1,84 0,39 -0,06 -2,33 0,34 -0,23 oxidase, subunit I ARTHROv5_61103 cydB Cytochrome bd ubiquinol -1,37 0,32 -0,21 -1,90 0,40 -0,11 -2,24 0,34 -0,22 oxidase, subunit II ARTHROv5_40400 coxA Cytochrome c oxidase -0,74 0,24 -0,06 -1,32 0,17 0,07 -1,77 -0,10 -0,04 subunit I ARTHROv5_40399 coxB Cytochrome c oxidase -0,77 0,08 -0,07 -1,14 0,10 0,08 -1,60 -0,15 -0,03 subunit II 202

Results Chapter VI: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays

ARTHROv5_40401 coxC Cytochrome c oxidase -1,35 0,21 -0,18 -2,10 0,01 -0,05 -2,58 -0,27 -0,21 subunit III ARTHROv5_40398 putative Cytochrome oxidase -0,75 0,22 -0,26 -1,99 0,23 -0,02 -2,44 -0,42 -0,19 assembly protein ATP ARTHROv5_60533 atpG2 ATP synthase B' chain -1,30 -0,74 -1,23 -2,04 -1,16 -1,11 -2,54 -1,83 -1,45 (Subunit II) ARTHROv5_60534 atpF ATP synthase B chain -1,29 -0,56 -1,22 -1,76 -0,89 -0,86 -2,31 -1,68 -1,10 (Subunit I) ARTHROv5_60535 atpH ATP synthase delta chain; -1,27 -0,45 -1,10 -1,75 -0,87 -0,88 -2,32 -1,65 -1,05 ATP synthase F1, delta subunit CO2 fixation & CBB cycle ARTHROv5_50352 cbbR RuBisCO operon -0,94 -0,16 -0,76 -1,71 -0,61 -0,47 -2,32 -1,03 -0,60 transcriptional regulator ARTHROv5_60714 CO2 hydration protein -1,98 -0,61 -0,89 -2,58 -0,82 -0,68 -3,14 -1,70 -0,93 ARTHROv5_10443 pgi Glucose-6-phosphate -0,78 0,17 -0,48 -1,47 0,06 -0,42 -1,32 -0,18 -0,70 isomerase ARTHROv5_20037 pgk phosphoglycerate kinase -0,92 -0,36 -0,53 -1,46 -0,45 -0,48 -2,49 -0,73 -0,71 ARTHROv5_41419 xfp D-xylulose 5-phosphate/D- -0,98 -0,25 -0,44 -1,84 -0,60 -0,40 -2,79 -0,71 -0,37 fructose 6-phosphate phosphoketolase Carbon metabolism ARTHROv5_30613 gap2 Glyceraldehyde-3-phosphate -0,75 -0,44 -0,71 -1,34 -0,77 -0,60 -2,61 -1,35 -0,80 dehydrogenase 2 ARTHROv5_30667 gpmB phosphoglycerate mutase -1,10 0,09 -0,93 -2,28 -0,26 -0,65 -3,31 -0,96 -0,87 ARTHROv5_30574 gpmI 2,3-bisphosphoglycerate- -1,08 -0,12 -0,69 -2,00 -0,28 -0,34 -2,41 -0,93 -0,58 independent phosphoglycerate mutase ARTHROv5_30318 pgm phosphoglucomutase -0,91 -0,35 -0,84 -1,58 -0,63 -0,75 -2,21 -1,18 -0,90 ARTHROv5_50271 glnB protein P-II -1,80 -0,28 -0,70 -2,31 -0,85 -0,43 -2,73 -1,49 -0,56 TCA cycle ARTHROv5_12017 sdhA succinate dehydrogenase -1,16 -0,53 -0,59 -1,83 -0,26 -0,42 -2,18 -0,85 -0,73 flavoprotein subunit ARTHROv5_10965 sucC Succinyl-CoA ligase -0,84 -0,24 -0,37 -1,56 -0,32 -0,21 -2,26 -0,67 -0,36 ARTHROv5_10964 sucD succinyl-CoA synthetase, -1,13 -0,10 -0,27 -1,55 -0,37 -0,15 -2,03 -0,66 -0,24

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Fatty acid biosynthesis ARTHROv5_11990 fabF2 3-oxoacyl-[acyl-carrier- -0,87 0,02 -0,05 -1,26 0,07 0,09 -1,20 -0,16 0,05 protein] synthase 2 ARTHROv5_30260 fabG1 3-oxoacyl-[acyl-carrier- -1,89 -0,37 -1,03 -2,26 -0,72 -0,81 -2,98 -1,27 -1,02 protein] reductase ARTHROv5_60058 fabG2 3-oxoacyl-[acyl-carrier- -0,76 -0,21 -0,55 -0,91 -0,40 -0,37 -2,07 -0,82 -0,44 protein] reductase ARTHROv5_30177 fabH 3-oxoacyl-[acyl-carrier- -1,46 0,11 -0,85 -2,22 -0,37 -0,75 -1,80 -1,10 -0,83 protein] synthase 3 ARTHROv5_41232 fabZ (3R)-hydroxymyristoyl- -1,81 -0,46 -1,01 -2,64 -0,82 -0,69 -2,67 -1,47 -1,02 [acyl-carrier-protein] dehydratase ARTHROv5_60707 desA Delta(12)-fatty acid -1,17 0,01 -0,58 -1,34 -0,39 -0,33 -1,94 -0,95 -0,24 desaturase ARTHROv5_40656 desD delta-6 fatty acid desaturase -0,93 0,21 -0,23 -1,49 0,21 0,08 -1,97 -0,44 -0,20 Nitrogen metabolism ARTHROv5_30825 ntcA Global nitrogen regulator -0,53 0,12 -0,20 -1,08 -0,01 0,00 -1,35 -0,34 -0,01 ARTHROv5_11376 amt1 Ammonium/methylammoniu -1,93 -1,73 -1,45 -2,21 -2,54 -1,18 -2,86 -3,33 -1,53 m permease ARTHROv5_12133 glnA glutamine synthetase -2,33 -0,80 -1,39 -2,86 -1,54 -0,89 -3,94 -2,79 -1,29 ARTHROv5_50078 glsF Ferredoxin-dependent -0,84 -0,25 -0,34 -1,50 -0,22 -0,19 -1,91 -0,61 -0,37 glutamate synthase, large subunit ARTHROv5_60175 nthA1 Nitrile hydratase alpha -2,62 -0,58 -0,83 -3,11 -0,99 -0,32 -3,65 -1,97 -0,55 subunit ARTHROv5_60176 nthB2 Nitrile hydratase beta subunit -2,53 -0,51 -0,89 -3,14 -1,19 -0,42 -3,65 -2,13 -0,65 ARTHROv5_30654 ARTHROv5_30654 Nitrilase/cyanide hydratase -1,71 -0,09 -0,56 -2,42 -0,16 -0,27 -2,44 -0,74 -0,35 ARTHROv5_40491 speB putative agmatine -0,09 1,32 1,62 -0,08 1,07 2,00 -0,30 0,62 1,92 ureohydrolase ARTHROv5_40618 nrtD ABC Nitrate transport -1,73 -0,84 -0,61 -2,01 -1,39 -0,19 -1,30 -2,14 -0,65 system ARTHROv5_40619 nrtC Nitrate transport ATP- -1,65 -0,72 -0,54 -1,89 -1,18 -0,21 -1,26 -1,89 -0,62 binding protein NrtC ARTHROv5_40620 nrtB ABC Nitrate transport -1,51 -0,87 -0,49 -2,01 -1,21 -0,09 -1,62 -2,10 -0,68 system

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ARTHROv5_40621 nrtA ABC Nitrate transport -1,49 -0,63 -0,94 -1,93 -1,31 -0,43 -2,25 -2,65 -1,01 system ARTHROv5_10485 livG leucine/isoleucine/valine -3,46 -1,20 -0,37 -3,36 -1,94 -0,04 -3,07 -2,29 -0,13 transporter subunit ARTHROv5_10486 livM leucine/isoleucine/valine -3,56 -1,73 -0,81 -4,07 -2,28 -0,07 -4,19 -2,90 -0,89 transporter subunit ARTHROv5_10487 livH leucine/isoleucine/valine -3,54 -1,31 -0,96 -4,23 -2,06 -0,36 -4,10 -2,95 -0,93 transporter subunit ARTHROv5_10488 livJ leucine/isoleucine/valine -3,11 -1,22 -1,00 -3,70 -2,16 -0,61 -4,05 -2,75 -1,02 transporter subunit ARTHROv5_40571 patA1 Putative Subtilisin-like -2,42 -0,39 -1,29 -2,46 -0,94 -0,63 -2,49 -2,44 -1,01 serine protease, PatA-like ARTHROv5_40573 patA2 Putative Subtilisin-like -2,06 -0,59 -1,66 -2,04 -0,94 -0,77 -2,64 -2,72 -1,22 serine protease, PatA-like ARTHROv5_40574 patB conserved hypothetical -2,25 -0,60 -1,52 -2,09 -1,03 -0,69 -2,26 -2,75 -1,14 protein, PatB-like ARTHROv5_40575 patC conserved hypothetical -2,57 -0,91 -1,61 -2,39 -1,27 -0,81 -2,09 -3,22 -1,39 protein, PatC-like

Table VI-S 4: Genes expressed during recovery, belonging to clusters 1, 2, 7, 8 and 9

As input the fold changes of those genes are having an absolute Log2 Fold change higher than 1 and a p-value corrected for multiple testing lower than 0.05 in either one of the 9 conditions.

Recovery period Gene name Gene function 800 800 800 1600 1600 1600 3200 3200 3200 T0H T2H T5H T0H T2H T5H T0H T2H T5H Sulphur metabolism ARTHROv5_40486 tauB ABC transport system, ATP- -0,05 1,33 1,52 -0,14 0,91 2,06 -0,42 0,31 1,72 binding component ARTHROv5_40487 tauC ABC transport system, 0,07 1,48 1,53 0,04 1,11 2,00 -0,25 0,51 1,67 permease component ARTHROv5_40488 tauA ABC transport system, 0,04 1,48 1,48 0,06 1,09 1,97 -0,20 0,54 1,68 periplasmic component

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Hydrogen production ARTHROv5_41303 hoxW Putative Ni,FE-hydrogenase 0,95 -0,15 0,52 1,24 0,08 0,73 1,47 0,07 0,62 maturation factor ARTHROv5_40489 hypB1 GTP hydrolase involved in 0,22 1,72 2,08 0,18 1,27 2,57 -0,03 0,70 2,35 nickel liganding into hydrogenases ARTHROv5_40490 hypA1 hydrogenase -0,05 1,61 1,89 -0,01 1,17 2,31 -0,15 0,64 2,20 expression/formation protein Glutathione production ARTHROv5_30647 gshB glutathione synthetase 0,94 1,71 1,78 0,76 1,46 1,79 0,95 1,65 2,04

Table VI-S 5: Genes expressed during emergency response and throughout recovery belonging to cluster 8 and 9

As input the fold changes of those genes are having an absolute Log2 Fold change higher than 1 and a p-value corrected for multiple testing lower than 0.05 in either one of the 9 conditions

Recovery Period Gene Gene function 800 800 800 1600 1600 1600 3200 3200 3200 name T0H T2H T5H T0H T2H T5H T0H T2H T5H ARTHROv5_10467 arhF conserved 3,74 4,36 4,32 2,46 4,19 4,43 3,29 4,34 4,36 hypothetical protein ARTHROv5_10468 arhE conserved 4,66 5,63 5,90 3,13 5,45 6,11 4,11 5,76 6,06 hypothetical protein ARTHROv5_10469 arhD conserved 3,81 4,28 4,02 2,77 4,14 4,25 3,21 4,25 4,23 hypothetical protein

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ARTHROv5_10471 arhB conserved 4,02 4,68 4,46 2,93 4,46 4,87 3,16 4,67 4,81 hypothetical protein ARTHROv5_10472 arhA putative ABC-type 3,43 4,57 4,27 2,39 4,33 4,46 3,38 4,71 4,76 phosphate transport

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Chapter VII Response of the spaceflight-relevant cyanobacterium Arthrospira sp. PCC 8005 to high doses of charged-particle radiation

Modified from: Hanène Badri, Marina Raguse, Ilse Coninx, Ruddy Wattiez, Ryuichi Okayasu, Ralf Moeller and Natalie Leys: Response of the spaceflight-relevant cyanobacterium Arthrospira sp. PCC 8005 to high doses of charged-particle radiation; Submitted to Astrobiology Journal

Contributions: Hanène Badri performed all experiments and analysis, with the help of lab technician Ilse Coninx. Dr. Ralf Moeller and Marina Raguse performed the irradiation experiments at HIMAC Facility. Dr. Ryuichi Okayasu was involved for logistic and technical assistance during irradiations. Prof. Ruddy Wattiez and Dr. Natalie Leys have guided Hanène Badri towards the most optimal experiment design and data interpretation.

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Abstract Arthrospira, also known as ‘spirulina’, is an edible oxygenic photosynthetic cyanobacterium that converts solar light into chemical energy to fix carbon dioxide from the environment. Its high nutritional value and chemical composition, has made it one of the most important industrially cultivated cyanobacterium on Earth. In addition, Arthrospira has also been selected for space applications, i.e. for oxygen and food production and water purification, as part of the bioregenerative life support system MELiSSA, developed by the European Space Agency (ESA) for future long-haul space missions. In this study, the effects of space radiation on Arthrospira sp. PCC 8005 were investigated by exposing active cells to high energy charged particles, i.e. helium ions (150 MeV/n) and iron ions (500 MeV/n). Physiological examination after ionizing irradiation showed successful recovery of Arthrospira sp. PCC 8005 after exposure to various final doses up to 1000 Gy and 2000 Gy of Helium and Iron ions, respectively. Photosynthesis efficiency and intracellular pigment concentrations were reduced after irradiation, but fully recovered to standard values during post-irradiation culturing in photosynthetic conditions, even after exposure to high doses to Helium and Iron particles. Hence, the obtained results demonstrate that the active photosynthetic cells of Arthrospira sp. PCC 8005 are highly resilient to cosmic radiation which is promising for its future use in space.

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VII.1 Introduction Astrobiology studies not only the origin of live, but also the limits of life, in the universe. Surviving cosmic radiation is a major challenge for any life in space. In space, an organism is exposed to different types of ionizing radiation [1]. In low Earth orbit there are three types of radiation: (i) radiation from particles trapped in the magnetic belts of the Earth, (ii) solar cosmic rays, and (iii) galactic cosmic rays [2]. Further, away from the Earth and from the sun, mainly the galactic cosmic rays (GCR) will have an impact. Galactic cosmic rays originate outside the solar system and consist of 98% of baryons and 2% electrons [3]. The baryonic components are composed of 85% Hydrogen nuclei (1H+1, protons ), 14% Helium nuclei (4He2+alpha particles ) and about 1% heavier nuclei, which are called HZE particles [4]. HZE particles are particularly dangerous since their interaction with the shielding material of a spacecraft leads to the formation of secondary radiation such as gamma rays and neutrons with various energies [5]. The kinetic energy of the particle will determine its penetration depth, its linear energy transfer (LET, in eV/µm), and its biological effects [1]. HZE and neutrons are the space radiation components with the highest relative biological effectiveness [6]. HZE particles have a much higher biological effectiveness than gamma or X- rays [7]. Gamma and X rays, which are electromagnetic radiation (i.e. photons of short wavelength emitted by radioactive isotopes) are considered as 'low LET' or 'sparsely ionizing radiation', while HZE particles are classified as 'high LET' or 'densely ionizing radiation'. Theses HZE particles produce a dense ionization that generates a high magnitude of local damage in cells, causing severe radiobiological damage to organisms [8]. As a consequence, even thought the number of HZE particles in space radiation is relatively small, they have a significant biological impact [8]. The biological impact of space radiation, and cosmic radiation or HZE particles in specific, has been studied using different animals, plants and microorganisms, via simulated irradiation experiments on ground and real space flight [9,10]. In the frame of the international consortium project STARLIFE (Intercomparison study of astrobiological model systems in their response to major components of the galactic cosmic radiation) coordinated by Dr. Ralf Moeller (DLR, Germany), the biological effect of low- and high-LET high energy charged-particle irradiation was investigated for different astrobiological model systems. Our interest was to investigate the effect of HZE on live photosynthetic microorganisms, such as the filamentous cyanobacterium Arthrospira. Despite the fact that photosynthetic organism are clearly of interest for space

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exploration, actually only very few of them have been characterised for their resistance to HZE particles in vegetative state, and for their ability to recover active photosynthesis after exposure. Arthrospira is of high interest, because of its potential use in planetary stations to support future manned space exploration. Arthrospira is a highly efficient oxygen producing edible photosynthetic bacterium which was selected to be part of the bioregenerative life support system MELiSSA, to enable future long-term human space exploration [11]. MELiSSA stands for “Micro- Ecological Life Support System Alternative” and is is aiming the complete recycling of gas, liquid and solid wastes, and the production of fresh food, during long distance space exploration missions, with the help of microorganisms and plants. The system consists of a closed loop of four interconnected bacterial bioreactors and a higher plant chamber, whith each a specific task to fulfil. th The 4 bioreactor is containing the cyanobacterium Arthrospira and has a role in CO2 removal, water purification, and O2 and food production [11]. Also the use of Arthrospira for in situ resource utilisation in space has been considered [12]. On Earth, Arthrospira has been used for human consumption since 16th century, due to its high protein content and good digestible property [13]. The last decades, Arthrospira has gained also increasing interest as health promoting food due to its anti-oxidant, anti-cholesterol, anti-inflammatory and anti-microbial properties, which might also be beneficial for future space travellers [14]. To our knowledge Arthrospira has not yet been tested in space, but several research groups, including ours, are preparing flight experiments (e.g. ArtEMISS experiment, ESA). In preparation of those space flights, this study aimed to explore in specific the impact of HZE particles as part of the space radiation, on live cells of the cyanobacterium Arthrospira sp. PCC 8005. Live planktonic cells of the cyanobacteria Arthrospira sp. PCC 8005 were irradiated with high- energy-charged Helium ions (with energy of 150 MeV/n) and Iron ions (with energy of 500 MeV/n) at the heavy ion irradiation facility (HIMAC) at the National Institute of Radiological Sciences (Chiba, Japan) and analysed for their susceptibility to recover after variable doses exposure. The analyses were based on morphology, pigment content and the PSII quantum yield.

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VII.2 Materials and methods

VII.2.1 Strain and culture conditions The strain of Arthrospira sp. PCC 8005 was obtained by us from Pasteur Culture Collection (PCC) (in 2009), and maintained as active subcultures in the lab. Three independent cultures (n=3) were used for the irradiation experiment. They were grown separately in 150 ml Zarrouk medium [15], on an orbital shaker at 120 rpm (Heidolph 2010) at 30 °C, illuminated with a photon flux density of ~ 42 µE m -2 s-1 provided by three Osram daylight tubes (PAR 400-700nm) (Binder KBW 400).

Cultures were grown to mid-exponential phase corresponding to an OD750nm ~1 (AquaMate, Unicam, Cambridge, UK). Then, only 120 ml of each culture was concentrated to 20 ml final volume (6x concentrated) using Vivaspin filters (vivaspin® Endotest VS15RXETO, Sartorius). Next, the 20 ml cultures were divided into 34 small reaction tubes of 0.5 ml, the maximum sample volume possible for the irradiation experiment, and grouped in 3 separate sets corresponding to different tested doses and different types of irradiation (Iron, Helium and X rays), including both samples for irradiation and equivalent samples for non-irradiated controls. The live cells, in the closed 0.5 ml reaction tubes, were kept in the dark and at 4 °C, and shipped from the laboratory in Belgium to the irradiation facility in Japan. The total storage period from preparation to irradiation took 15 days. Samples were irradiated and then shipped immediately from Japan back to Belgium, where they arrived 6 days after irradiation. Thus, the total storage time spanned 21 days. Additional tests were performed in parallel in order to assess the ability of Arthrospira to recover after such a long storage period. Our data showed that Arthrospira sp. PCC 8005 could be stored as concentrated biomass in dark at 4 °C up to 21 days, with only minor effects on viability (data not shown). In parallel with the particle irradiation in Japan, also a high dose X-ray irradiation was performed in Germany, on the same batch of samples, prepared, transported, and stored in the same way., The samples exposed to X-ray irradiation exceeded, however, 24 days of storage before return to the lab for analysis, due to logistic reasons, which had a significant detrimental impact on the cells masking the actual radiation effects. Therefore, those X-ray data were excluded from this paper.

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VII.2.2 Irradiation procedure The irradiation was performed in at the Heavy Ion Medical Accelerator Chiba (HIMAC) at the National Institute for Radiological Sciences (NIRS) in Chiba-Shi, Japan, in the frame of the STARLIFE project. The samples were irradiated with a Helium (4He2+ nucleus, alpha particle) and Iron (56Fe+26 nucleus) HZE particle beam, characterised by an energy of 150 MeV/u and 500 MeV/u respectively, and a Linear Energy Transfer (LET) of 2.2 KeV/µm and 200 KeV/µm respectively. The dose rate of the Helium beam was 192 Gy/h and for the Iron beam 672 Gy/h (Table VII-1: Time required for irradiation to achieve the predefined total dose of He particles or Fe particles.), and total doses of 50, 100, 250, 500, 1000 and 2000 Gy were given.

Table VII-1: Time required for irradiation to achieve the predefined total dose of He particles or Fe particles.

Helium 4He2+ Iron 56Fe+26 (energy 150 MeV/u) (energy 500 MeV/u) (LET 2.2 KeV/µm) (LET 200 KeV/µm) 192 Gy/h 672 Gy/h Doses Exposure Time Exposure Time 50 Gy 15.75 min 4.5 min 100 Gy 31 min 9. min 250 Gy 1h 18 min 22 min 500 Gy 2h 36 min 44 min 1000 Gy 5h 12 min 1h 28 min 2000 Gy - 2h 59 min

The live planktonic Arthrospira sp. PCC 8005 cells, from three biological independent cultures (n=3), were irradiated in closed 0.5 ml reaction tubes fully filled with liquid which were assembled in a 10 cm diameter petri dish, fixed in a tilted (90°) position and homogenously placed inside Bragg peak plateau of the irradiation beam. Non-irradiated samples (three independent cultures for each tested dose), were prepared and transported together with irradiated samples, but were placed out of the beam and kept in the storage condition in the laboratory during irradiation. After irradiation all samples were put back in storage conditions (dark, 4 °C) and shipped back from NIRS in Japan to SCK•CEN in Belgium for analysis. 213

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VII.2.3 Post-irradiation analysis VII.2.3.1 Culture and Cell Morphology Visible changes in culture pigmentation of the different Arthrospira sp. PCC 8005 samples after irradiation were documented with normal photography. Cell morphological examination was performed using an inverted microscope (Eclipse Ti, Nikon) at 200X magnification. The impact of particle irradiation on Arthrospira sp. PCC 8005 cells was assessed based on filament fragmentation and cell pigmentation. VII.2.3.2 Photosynthetic potential The photosynthetic activity of the Arthrospira cells sp. PCC 8005 in irradiated and non-irradiated samples was determined via fluorescence of the photosystem II (PSII) pigments, using the DUAL PAM 100 apparatus (Waltz-GmbH, Effeltrich, Germany). The 1 ml samples were dark adapted for 15 min, and then exposed to a weak modulated red light (635 nm) (ML) (3 µE m-2s-1), to determine the 'minimum fluorescence' (F0). Next, the cells were exposed to a short high energy pulse of red light called 'saturating pulse' (8000 µE m-2s-1, with duration of 0.8 s) to determine the 'maximum fluorescence' of the dark adapted cells (Fm). From these measurements, the ratio Fv/Fm, i.e. the PSII quantum yield, was calculated where the 'variable fluorescence' Fv presents the difference between 'maximum fluorescence' (Fm) and the 'minimum fluorescence' (F0). Healthy Arthrospira cells normally have a yield FV/FM of ca. 0.6 [16]. The photosynthetic potential of the Arthrospira sp. PCC 8005 cells was measured immediately after arrival of the samples in the SCK•CEN lab, 6 days after irradiation. In addition, also the recovery of the photosynthetic activity in post-irradiation cultures was measured when cultures reached exponential growth phase (OD750 ~ 0.8). VII.2.3.3 Pigment content The full absorption spectrum between 400 nm and 800 nm was recorded for each sample, using 1 ml of the cell suspension at room temperature, with a spectrophotometer (Aquamat, Unicame). More specifically, chlorophyll A pigment concentration was measured at 440 nm and 680 nm, phycocyanin antenna pigment absorption was measured at 625 nm, and carotenoid pigment concentration was measured at 496 nm. Data from irradiated samples were normalized versus their respective non-irradiated control samples and plotted as percentage versus control.

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VII.2.3.4 Recovery and proliferation In order to investigate the ability of Arthrospira sp. PCC 8005 filaments to recover and restart photosynthetic growth after irradiation, 2.5 ml aliquots of the 10x diluted irradiated and control samples were inoculated in 25 ml of fresh Zarrouk-UBP medium (inoculation of 10% v/v) and incubated in the same conditions as the cultures they were harvested from before irradiation (described above). The cell proliferation was followed via absorbance measurement at 750 nm

(optical density OD750nm) every three days using a spectrophotometer (AquaMate, Unicam, and

Cambridge, UK). The data were plotted in growth curves as OD750nm versus time. VII.2.3.5 Statistical analysis. The software Graph Pad Prism (version 5.00, GraphPad Software) was used, for preparing data graphs and for statistical analysis using One way ANOVA followed by "Dunnett Multiple Comparison Test" that compared data from irradiated versus non-irradiated samples with confidence interval 95% (p < 0.05).

VII.3 Results

VII.3.1 Recovery and Photosynthetic growth after irradiation For most types of cells the amount of survivals after irradiation is based essentially on colony forming units (CFU) determined by plate culture, where one CFU represents one survival cell. This is, however, not possible for Arthrospira, due to its multicellular filamentous shape, which means that one CFU would not represent one cell but a filament possibly containing 10-100 cells. Therefore, the effect of Helium and Iron particles on the cells was estimated based on the optical density measured at 750 nm (OD750), estimating the concentration of intact cells in the suspensions, after adding an aliquot (10% v/v) to fresh medium (day 0 of the growth curve). The irradiated samples did not show a significant decline in cell concentration after irradiation compared to the non-irradiated samples (Figure VII-1). Overall, the particle radiation had relative little effect on the intact cell concentration, which did not even decrease with 1 log, i.e. stayed well above 10%, even for the highest doses tested. Therefore, the D10 value (the dose required for killing 90% of cells and leaving only 10% survivors) for Arthrospira sp. PCC 8005 for He and Fe irradiation was well above the maximum doses tested, i.e. 1000 and 2000 Gy, respectively. Cell concentration did seem to decreases gradually with increasing doses of heavy ions showing the impact of He and Fe

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particles irradiation (Figure VII-1). But still 52% of the cells were present after exposure to the highest dose of He particles, i.e. 1000 Gy (Figure VII-1). Surprisingly the amount of intact cells after Fe particles exposure was even higher then after He exposure for all doses, i.e. still 91% of intact cells for 1000 Gy of Fe particles (Figure VII-1), although the energy deposition (LET) and dose rate of the iron particles was higher, and thus was expected to cause more cell damage.

A - He B - Fe

Figure VII-1: Percentage of Arthrospira sp. PCC 8005 cells, following exposure to different doses of (A) Helium and (B) Iron particle irradiation. The intact cell concentration was estimated based on the optical density measured at 750 nm (OD750nm). Data represent mean of three independent biological replicates (n=3), and error bars present the standard error of the mean (SEM). The data obtained for the irradiated (OD750nm) samples were normalized against control samples and are shown as percentage of their representative non-irradiated control (which was put at 100%). Statistical analysis was curated on the raw data set with One way ANOVA followed by "Dunnett Multiple Comparison Test". In accordance with this high preservation of intact cells Arthrospira sp. PCC 8005 was indeed able to resume photosynthetic regrowth after exposure to all tested doses of accelerated Helium and Iron particles (Figure VII-2). Only after exposure to 1000 Gy of Helium a significant delay in growth, i.e. 3 days of Lag time, was seen; whereas the delay was insignificant for 1000 Gy Iron exposure. The maximum growth rate was reached after 3 days for both exposed and non-exposed cells (Table VII-S 1). Surprisingly the top maximum growth rate was seen after exposure to the highest dose 1000 Gy of Helium (µmax= 0,861) (Table VII-S 1 & 2). After 26 days of post irradiation recovery, all treated samples, either with Helium or with Iron, reached the same of biomass yield as non-irradiated controls (Figure VII-2).

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A B

VII.3.2 Photosynthetic activity Oxygenic photosynthesis in cyanobacteria is driven via photosystem I and II (PSI and PSII) where the photochemical reaction takes place [17,18]. The status of PSII system in Arthrospira cells after irradiation was assessed via measurement of Chlorophyll a (Chla) fluorescence. Due to the experiment conditions including a long dark storage time during transport (21 days), there was a large variation of the Fv/Fm ratios among the different samples upon arrival in the lab for analysis (6 days after irradiation), including the non-irradiated control samples (Figure VII-3 A&B). As such, the measurement of Fv/Fm parameter immediate after reception of the samples did not reveal a clear direct effect of radiation. Therefore the PSII Quantum yield of irradiated samples was also assessed after recovery, when cultures reached exponential growth phase (OD750nm ~ 0.8) (Figure 3C&3D). Helium particles did not reveal adverse effect on the post-irradiation photosynthetic activity. Arthrospira showed 98 % of recovery following 1000 Gy helium exposure. Likewise, after exposure to 1000 Gy and 2000 Gy Iron particles, Arthrospira sp. PCC 8005 could reach 91 % till 83 % of its normal photosynthetic activity, so there was no significant effect of irradiation (p value = 0,176).

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A - He B - Fe

C - He D - Fe

Figure VII-3: Photosynthetic potential, measured as PSII quantum yield, of Arthrospira sp. PCC 8005 after exposure to different doses of Helium and Iron particle irradiation. (A) and (B) represent measurements directly upon arrival in the lab for analysis, while (C) and (D) represent present PSII quantum yield after recovery (i.e. when cultures reached exponential growth phase (OD750 ~ 0.8). Data represent mean of three independent cultures (n= 3) and error bars present the standard error of the mean (SEM).Statistical analysis was performed on raw data set using One way ANOVA followed by "Dunnett Multiple Comparison Test". VII.3.3 Pigments analysis Visual inspection of the samples immediately upon reception showed a subtle difference in Blue- Green colour between the irradiated samples and the related non-irradiated controls (Figure VII-4A), which was also obvious in morphological analysis under the microscope (Figure VII-4B).

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Controls: Non irradiated Irradiated: 1000 Gy Helium A

Figure VII-4: A: Photographs showing Arthrospira sp PCC 8005 cultures and pigmentation (A) and morphology (microscopy (200X Magnification) (B) after exposure to 1000 Gy of He particle irradiation. Therefore, a full absorption spectrum analysis was done, which revealed a clear difference in pigmentation between control and irradiated samples (Figure VII-5). Analysis of the full absorption spectrum of control and irradiated samples revealed a decrease in the 625 nm absorption region, likely indicating a reduction in the phycocyanin pigment concentration after irradiation (Figure VII-5).

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A– Control: non irradiated B – Irradiated 1000 Gy He

Phycocyanin absorption Phycocyanin absorption

Figure VII-5: Absorption spectrum of Arthrospira sp. PCC 8005 cells between 400 and 700 nm. A: Control sample; B: Irradiated with 1000 Gy He particles. The arrow indicates phycocyanin absorption region More detailed measurements of absorption at specific wavelengths, was done to assess specifically chlorophyll A pigment concentration (at 440 nm and 680 nm), the carotenoid pigment concentration (at 496 nm), and the phycocyanin pigment concentration at 625 nm (Figure VII-6, Figure VII-7). Results showed no significant changes in chlorophyll concentration after irradiation with He or Fe particles. A slight but not significant decrease in carotenoids (P value = 0,134) and phycocyanin (P value= 0,116) concentration was seen after exposure to 1000 Gy of He particles and 1000 and 2000 Gy of Fe particles (Figure VII-6).

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A– Chlorophyll a (440 nm) B – Chlorophyll a (680 nm)

C– Carotenoids (495 nm) D – Phycocyanin (625 nm)

Figure VII-6: Changes in pigmentation in Arthrospira sp. PCC 8005 cultures in function of the dose of He irradiation recieved. Three pigments were analysed: (A & B) chlorophyll measured at both 440 nm and 680 nm, (C) carotenoids measured at 495 nm, and (D) phycocyanin measured at 625 nm. Data represent mean of three independent cultures (n= 3).Statistical analysis was curated on raw data set One way ANOVA followed by "Dunnett Multiple Comparison Test".

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A– Chlorophyll a (440 nm) B – Chlorophyll a (680 nm)

C– Carotenoids (495 nm) D – Phycocyanin (625 nm)

Figure VII-7: Changes in pigmentation in Arthrospira sp. PCC 8005 cultures in function of the dose of Fe irradiation received. Three pigments were analysed: (A & B) chlorophyll measured at both, 440 nm and 680 nm, (C) carotenoids measured at 495 nm, and (D) phycocyanin measured at 625 nm. Data represent mean of three independent cultures (n= 3). Statistical analysis was curated on raw data set One way ANOVA followed by "Dunnett Multiple Comparison Test.

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VII.4 Discussion There are only a few facilities worldwide and few opportunities offering affordable access to high doses of particle irradiation for microbial experiments. These tests were part of the STARLIFE project providing access to the HIMAC facility (Japan). However, the restricted test conditions, including a limited number of pre-defined radiation doses, the very small culture volumes that had to be used for the experimental set-up and the long pre- and post-irradiation storage during transport, did limit our experimental investigations. It was impossible to assess the limit of Arthrospira sp. PCC 8005 strain to withstand higher doses of ions particles, and to investigate the cellular response to the radiation impact at the molecular level (i.e. genomics, transcriptomics, proteomics). Nevertheless, it was an exceptional opportunity to test for the first time the susceptibility of the cyanobacterium Arthrospira sp. PCC 8005 to particle irradiation, using Helium (4He2+ nucleus, alpha particle) and Iron (56Fe+26 nucleus) ions. The results showed that Arthrospira sp. PCC 8005 cells could recover photosynthetic proliferation after exposure to high acute doses of charged-particles irradiation, up-to 1000 Gy of He and 2000 Gy of Fe. Arthrospira sp. PCC 8005 cells showed a slightly higher sensitivity to helium particles comparing to iron particles. Cultures exposed to 1000 Gy of He ions, did show less intact cells and a significant delay in growth, which was not the case for cultures exposed to 1000 or 2000 Gy of Fe ion irradiation. One would expect, however, the impact of Fe ions (with 500 MeV/u and LET 200 KeV/µm) to be greater than of He ions (with 150 MeV/u and LET 2.2 KeV/µm), as the relative biological effectiveness (RBE) of a given radiation is a function of the Linear Energy Transfer (LET): as the LET increases, the RBE increases [19]. High LET radiation, deposits a highly concentrated energy in a small section [20], which enhances the number and the magnitude of local damage in cells. HZE particles produce clusters of ions and radicals throughout their passage through the cell [21,22]. Radiation can damage key molecules such lipids, proteins, or DNA, directly or indirectly by free radicals generated by radiolysis of water [1]. As the LET increases, a particle’s ability to cause direct ionization increases but the yield of the most reactive radicals (such as OH•) per unit radiation doses, i.e. the indirect effect, generally decreases [23,24]. Thus, high LET radiations are more mediators of direct damage rather than indirect damage [25]. As consequence, the probability of causing double strand breaks in DNA (DSBs), due to direct damage, is higher at dense ionizing radiation as from alpha particles (He particles with LET 2.2 keV/µm) and HZE particles (e.g., Fe

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particles with LET 200 keV/µm) [26]. IIt has been suggested that the RBE of HZE particle radiation reaches a maximum at a LET of about 100 keV/µm, which is optimal in terms of producing a biological effect [27]. At this density of ionization the average separation in ionizing events is equal to the diameter of DNA double helix, which causes significant amount of double strand breaks (DSBs) [28]. For HZE particle radiation with an LET above this 100 keV/µm, the RBE again falls to lower values [29]. Very densely ionizing radiations with a LET of 200 keV/µm or more (such as for Fe ions) easily produce double-strand breaks, but their ionizing events are too close together, resulting in a single DSB for multiple hits. The very densely ionizing radiation is just as effective per track, but less effective per unit dose, i.e. “energy is wasted” [30]. This is also called an “overkill or overshooting event” [31]. Thus one could hypothesise that this 'overkill phenomena', may have been the cause of the obtained results showing a more pronounced effect of He ion rather than Fe ions on Arthrospira sp. PCC 5008 cell survival and proliferation after irradiation with 1000 Gy. Such 'kind of overkill effect' of particles with very high LET was also observed, for dried cells of the radiation resistant bacterium Deinococcus radiodurans. Dried cells of D. radiodurans displayed resistance towards wide range of ionising particles, with very high LET, namely: 40Ar (2.8 MeV/u), 131Xe (4.4 MeV/u), 197Au (5.4 MeV/u, ~10 000 Kev/µm), 58Ni (5.9 MeV/u), 20Ne (6.5 and 11.9 MeV/u), and 12C (12.1 MeV/u), respectively [32]. Kobayashi and co-workers showed that the RBE, i.e. the D10 value (the dose required to inactivate 90% of the cells) stayed the same with increasing LET [32]. Moreover, a clear decrease in RBE (i.e. increase in D10 value) was seen for exposures to particle irradiation with very high LET [32]. After exposure to 197Au (5.4 MeV/u, ~10

000 KeV/µm), a sharp increase in D10 value was seen, i.e. very high doses of 30 KGy were needed to inactivate 90% of the cells [32]. In other tests with dehydrated Deinococcal cells, where the particle radiation had lower LET (< 200 KeV/µm), the RBE was correlated with LET, i.e. as the LET increased, the RBE increased. For example, the study of Kawaguchi and co-authors, reports that dehydrated Deinococcal cells irradiated with Helium ions (4He2+, 150 MeV/u; LET 2.2 KeV/µm) did show higher survivability than cells irradiated with Argon ions (40Ar18+, 500 MeV/u; LET 90 KeV/µm) under normal pressure [33]. Until today, only very few studies actually compared the bacterial radiation resistance to high LET and low LET particle irradiation, and to low LET electromagnetic radiation. In this study, Arthrospira sp. PCC 8005 was exposed to both low LET (He) particle radiation and high LET (Fe)

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particle radiation, in the same experimental set-up for the same dose, and as explained above the low LET (He) particle radiation seemingly had a bigger biological impact. Previously, we have tested live cells of Arthrospira sp. PCC 8005 also with acute doses of low LET electromagnetic radiation, i.e. 60Co gamma rays (photons with energies of 1.33 MeV and 1.17 MeV, and a LET of 0.2 keV/µm, given at dose rate of 527 Gyh-1) [34]. Comparing the post irradiation growth kinetics, morphology, photosynthetic activity and pigment concentration data for the same total absorbed dose (e.g 1000 Gy) of 60Co gamma rays and Helium and Iron particles, there was not so much difference in damaging effect. This was also reported for D. radiodurans, where more or less the same effects were observed after exposure to low LET gamma rays (f 0.2 KeV/µm) and high LET heavy ions, for the same absorbed dose (the exact dose was unfortunately not reported) [35]. Later, Kobayashi and Co-workers concluded that there would be in fact no difference in radiation induced DSBs at LET values ranging between 0.2-2000 KeV/µm, from the point of view of DNA repair ability of D. radiodurans [32]. D. radiodurans showed high ability to repair DNA double strand breaks induced at various LET, even after exposure to 197Au (5.4 MeV/u, ~10 000 KeV/µm). Thus, D. radiodurans can repair not only DSBs induced by low LET radiation, but also clustered DSBs damage generated by HZE radiation. In addition, a recent study reported that the order of radiation resistance for Deinococcus species to gamma rays was similar to the order in resistance against

HZE particles [33,36]. Dried D. aetherius (D10: 8000 Gy) showed higher resistance than dried D. 60 radiodurans (D10: 6700 Gy) and D. aerius (D10: 4900 Gy) for Co gamma rays (dose rates of 370- 1450 Gy/h) [36]. Likewise, D. aetherius was more resistant to Helium particles (4He2+ 150 MeV/u; LET: 2.2 KeV/µm) and Argon particals (Ar 500 Mev/u; LET: 90 KeV/µm) than D. radiodurans and D. aerius [33]. Thus it seems that when a bacterium is resistant to high doses of electromagnetic radiation, it is also resistant to particle radiation. The resistance of Arthrospira sp. PCC 8005 cells to high doses 60Co gamma rays allowed classifying this cyanobacterium as radiation resistant [37]. Arthrospira sp. PCC 8005 could withstand 6400 Gy of 60Co gamma rays, and resume normal photosynthetic growth after exposure [34]. Additional tests have shown that only a dose of 10 000 Gy of 60Co gamma rays (given at high dose rates of 20 000 Gy/h) was lethal, preventing any post-irradiation growth (Chapter VI). Sincethis irradiation campaign was limited to up to 1000 Gy for He ion and 2000 Gy for Fe ion irradiation, it was not possible to assess the limit of Arthrospira sp. PCC 8005 to withstand higher

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doses of particle irradiation, i.e., to determine the D10 value or the minimal required dose for full inactivation (MRD) at this time. Nevertheless, our results showing that Arthrospira sp. PCC 8005 can resist doses up 1000 and 2000 Gy of He ion and Fe ion irradiation are in fact quite remarkable, knowing that here active vegetative Arthrospira sp. PCC 8005 cells were exposed in liquid suspensions. In addition, Arthrospira sp. PCC 8005 cannot survive drying, lyophilisation or cryopreservation in lab conditions; and is only maintained as successive liquid cultures, at all culture collections or spirulina farms we know. Therefore, it was also prepared, transported, stored and tested here in this condition, as live vegetative cells in liquid suspension. It is known, however, that desiccation-tolerant organisms exhibit a higher resistance towards UV and ionizing radiation and that radiation resistance can be enhanced when the cells are irradiated in dried status, as reported for D. radiodurans [38] and Chrococcidiopsis [39,40]. Indeed, so far, most microbial astrobiology space experiments were done with cells in resting state, i.e. in dried or dormant state such as bacterial spores [41,42]. Irradiating the cells in dry state is expected to offer substantial protection, particular for proteins, as it would limit the indirect oxidative damage in the cell, caused by diffusion of radiogenic oxidants (mainly generated from radiolysis of water) [43]. Thus given that Arthrospira sp. PCC 8005 was here irradiated as live planktonic cells in watery suspension, we believe we can classify it also as high resistant to particle irradiation. The capability to survive the space radiation, however, is not sufficient for photosynthetic bacteria to be used in life support systems for future human space exploration. For bio-regenerative life support, active growing bacterial cells with high photosynthetic productivity are required [44]. Thus it is essential that the photosynthetic performance is not altered and can be fully recovered after exposure to acute high doses of space radiation. Assessment of photosynthesis potential of Arthrospira sp. PCC 8005 immediately after particle irradiation did not reveal a significant effect, i.e., the PSII quantum yield remained high (similar to non-irradiated controls), although the variability between biological replicates was high. The data did show a slight but not significant decrease in the carotenoid and phycocyanin pigment concentration after exposure to 1000 Gy of He, which was not the case for chlorophyll a. The highest decrease in phycocyanin pigment content was thus observed with He ions, and not Fe ions; which is consistent with the higher post-irradiation growth delay with He. This decrease in phycocyanin pigment observed for 1000 Gy of He could indicate a specific degradation of phycocyanin pigment, and the disassembly of phycobilisomes,

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the antenna assemblages of cyanobacteria that transfer the collected excitation energy from light to chlorophyll in the photosystem reaction centre [45]. It has been reported that under stress condition cyanobacteria induce proteolytic degradation of phycobilisomes antenna pigments [46], which leads to a colour change of the cells from blue-green to yellow-green, also called bleaching or chlorosis [47,48]. The cultures were indeed less dark green after particle irradiation. The same observation was made after exposure of Arthrospira cells to gamma rays. The cells had a significant decrease in phycocyanin pigment content after exposure to 3200 Gy, 5000 Gy and 6400 Gy (Badri et al., 2014). Evaluation of the PSII quantum yield of recovered samples, however, showed that Arthrospira resumed 100% of photosynthetic efficiency after exposure to the different doses of particles, including the 1000 Gy of He.

VII.5 Conclusion The edible cyanobacterium Arthrospira is a valuable candidate for various biotechnological applications in space, such as oxygen and food production in the life support system MELiSSA. The high resistant of Arthrospira to ionising radiation, is a large advantage to thrive in space environment. Here, it was shown, that live cells of the cyanobacterium Arthrospira are able to survive and fully recover their photosynthetic growth after exposure to high doses, i.e. 1000 and 2000 Gy, of He and Fe particle radiation, important components of cosmic radiation. These results urge for more and experimentation to be done with HZE particles, with experimental set-ups that allow advanced molecular and genetic analysis in order to get better insight in the protection, repair and recovery mechanisms which allow the cells to deal with such harsh radiation environment.

VII.6 Acknowledgements This work was supported through a PhD grant for Hanène Badri by SCK•CEN and ESA/Belspo via the ArtEMISS project.

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40. Billi D, Viaggiu E, Cockell CS, Rabbow E, Horneck G, et al. (2011) Damage escape and repair in dried Chroococcidiopsis spp. from hot and cold deserts exposed to simulated space and martian conditions. Astrobiology 11: 65-73. 41. Moeller R, Setlow P, Horneck G, Berger T, Reitz G, et al. (2008) Roles of the major, small, acid-soluble spore proteins and spore-specific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X rays and high-energy charged-particle bombardment. Journal of bacteriology 190: 1134-1140. 42. Moeller R, Reitz G, Li ZF, Klein S, Nicholson WL (2012) Multifactorial Resistance of Bacillus subtilis Spores to High-Energy Proton Radiation: Role of Spore Structural Components and the Homologous Recombination and Non-Homologous End Joining DNA Repair Pathways. Astrobiology 12: 1069-1077. 43. Shuryak I, Brenner D (2010) Effects of radiation quality on interactions between oxidative stress, protein and DNA damage in Deinococcus radiodurans. Radiation and environmental biophysics 49: 693- 703. 44. Hendrickx L, Mergeay M (2007) From the deep sea to the stars: human life support through minimal communities. Current opinion in microbiology 10: 231-237. 45. Grossman AR, Schaefer MR, Chiang GG, Collier JL (1993) The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiological reviews 57: 725-749. 46. Karradt A, Sobanski J, Mattow J, Lockau W, Baier K (2008) NblA, a key protein of phycobilisome degradation, interacts with ClpC, a HSP100 chaperone partner of a cyanobacterial Clp protease. The Journal of biological chemistry 283: 32394-32403. 47. Baier A, Winkler W, Korte T, Lockau W, Karradt A (2014) Degradation of phycobilisomes in Synechocystis sp. PCC6803: evidence for essential formation of an NblA1/NblA2 heterodimer and its codegradation by A Clp protease complex. The Journal of biological chemistry 289: 11755- 11766. 48. Deschoenmaeker F, Facchini R, Leroy B, Badri H, Zhang CC, et al. (2014) Proteomic and cellular views of Arthrospira sp. PCC 8005 adaptation to nitrogen depletion. Microbiology 160: 1224-1236.

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VII.8 Supplemental data

Table VII-S 1: The specific growth rate for the cultures grown after irradiation.

For each time interval between 2 time points was calculated with following formula: µ = ( ) ( ) ln OD750 at t2 −ln 푂퐷750 푎푡 푡1 for Helium particles 푡2−푡1

HE Time intervals (Days) CTR 50 Gy 100 Gy 250 Gy 500 Gy 1000 Gy (n=3) (n=3) (n=3) (n=3) (n=3) (n=3) 0-3 0,545 0,269 0,277 0,218 0,324 -0,059 3-6 0,565 0,672 0,670 0,668 0,676 0,861 6-9 0,276 0,353 0,295 0,291 0,271 0,249 9-12 0,150 0,251 0,235 0,277 0,201 0,284 12-15 0,132 0,175 0,177 0,219 0,146 0,280 15-18 0,097 0,127 0,213 0,181 0,058 0,226 18-21 0,065 0,096 0,023 0,081 0,123 0,096 21-23 0,089 0,066 0,075 0,102 0,081 0,118 23-26 0,050 0,078 0,085 0,093 0,067 0,135 26-31 0,032 0,037 0,047 0,045 0,039 0,062 31-33 0 -0,028 0 0 0 0 Lag time, where µ=0 (days) 0 0 0 0 0 3** Exponential growth phase duration where µ> 0 (days) 31 31 31 31 31 31

-1 Maximum growth rate µmax (∆OD750*Day ) 0,565 0,672 0,670 0,668 0,676 0,861* ±0,013 ±0,069 ±0,139 ±0,151 ±0,142 ±0,193

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Table VII-S 2: The specific growth rate for the cultures grown after irradiation.

For each time interval between 2 time points was calculated with following formula: µ = ( ) ( ) ln OD750 at t2 −ln 푂퐷750 푎푡 푡1 for Iron particles 푡2−푡1

FE CTR 50 Gy 100 Gy 250 Gy 500 Gy 1000 Gy 2000 Time interval (Days) (n=3) (n=3) (n=3) (n=3) (n=3) (n=3) Gy (n=3) 0-3 0,564 -0,029 0,472 0,237 0,195 0,404 -0,020 3-6 0,644 0,938 0,873 0,753 0,781 0,488 0,667 6-9 0,309 0,254 0,243 0,290 0,325 0,314 0,150 9-12 0,216 0,280 0,188 0,275 0,279 0,316 0,234 12-15 0,0697 0,248 0,159 0,247 0,207 0,151 0,390 15-18 0,107 0,228 0,092 0,227 0,164 0,226 0,354 18-21 0,093 0,056 0,107 0,038 0,077 0,094 0,130 21-23 0,086 0,117 0,099 0,160 0,091 0,118 0,202 23-26 0,056 0,094 0,066 0,067 0,060 0,109 0,180 26-31 0,025 0,059 0,038 0,032 0,037 0,011 0,015 31-33 0 0 0 0 0 0 0 Lag time, where µ=0 (days) 0 3 0 0 0 0 3 Exponential growth phase duration where µ> 0 (days) 31 31 31 31 31 31 31 Maximum growth rate µmax (∆OD750*Day-1) 0,644 0,938 0,873 0,753 0,781 0,488 0,667 ±0,063 ±0,219 ±0,254 ±0,090 ±0,253 ±0,413 ±0,224

The last row presents the maximum growth rate obtained for each radiation dose. Data represent mean of three independent cultures (n= 3). An asterisk indicates a value for the irradiated sample which is significant (p<0.05) different from the value of the corresponding non-irradiated control. Two asterisks indicate a value which is highly significant (p<0.01).

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Part IV: General Discussion, Conclusion and Perspectives

Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives

Chapter VIII General Discussion, Conclusion and Perspectives

Arthrospira belongs to the phylum of cyanobacteria, also called blue green algae; that evolved around 3.5 billion years ago and played a key role in converting the early reducing atmosphere into an oxidizing one, allowing live to further develop on Earth [1]. Electrons extracted from the water oxidation process were used to reduce CO2 to make carbohydrates with concomitant release of atmospheric oxygen. This process called photosynthesis, has contributed to half of the oxygen present in the atmosphere [1]. This primitive group of cyanobacteria are pioneering phototrophs driving the majority of the ecosystems on Earth [2]. They successfully populate freshwater and marine environments, hot springs, and cold dry valleys, coping with extremes in salinity, light quality and availability, pH, dryness, desiccation, temperature, and ionising radiation such as UVR [3]

Arthrospira has also been selected as key pioneer to create a 'habitable' environment, beyond our planet Earth, i.e. in space. For future long-duration manned space exploration, for instance to Moon or Mars, one needs to take into account the environmental and nutritional requirements of astronauts. One needs to provide an adequate atmosphere – removing carbon dioxide and producing oxygen, potable water, and food. To deal with oxygen, water and food production and waste treatment in space, the European Space Agency (ESA) is designing and testing the concept of a bio-regenerative life support system called MELiSSA, which stands for 'Micro-ecological Life Support System Alternative' (Introduction: Chapter II). The system is based on the principles of natural lake ecosystems on Earth, and consists of a closed circuit of four interconnected microbial bioreactors and a higher plant cultivation chamber. The food, water and oxygen are produced in the fourth compartment, using plants and cyanobacteria. Due to its great properties, the cyanobacterium Arthrospira was selected to be integrated in this fourth compartment of MELiSSA loop. The need to have an edible microorganism that continues to produce oxygen and conserves high nutritive value while grown in bioreactors and exposed to cosmic radiation in space, led us to study of the susceptibility of the cyanobacterium Arthrospira sp. PCC 8005 to ionising radiation.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives In previous tests at SCK•CEN, Arthrospira sp. PCC 8005 was exposed to a combination of neutron (252Californium) and gamma (137Cesium) irradiation, during three days at a low dose rate and low total cumulative dose, [4]. This set-up was chosen in order to simulate the total doses and the high and low LET radiation spectrum measured by the SCK•CEN radiation dosimetry group inside the International Space Station (ISS), orbiting the Earth at an altitude of about 400 km (called LEO – low Earth orbit) [5]. Due to technical reasons, the dose rate, however, was much higher than would be on-board ISS; the total dose expected for a stay on-board ISS for, 10 days (i.e. 200mGy) was given in 3 days. Arthrospira sp. PCC 8005 showed full recovery of active photosynthetic growth, without significant reduction in proliferation rate, after irradiation [4]. This promising result was the first to give confidence on the potential functional use of this the cyanobacterium under cosmic radiation in space. After that study, the susceptibility of Arthrospira sp. PCC 8005 to ionizing radiation was further assessed, using higher doses (3200 Gy) and higher dose rates (1075.6 Gy h- 1) of 60Cobalt gamma irradiation [4]. Arthrospira sp. PCC 8005 could tolerant to up to 1 600 Gy and 3 200 Gy of acute 60Co gamma rays without significant loss of viability tested by flow cytometry or delay in post-irradiation growth [4]. From then on, the cyanobacterium Arthrospira sp. PCC 8005 was considered as resistant to ionising radiation. Main studies on the effect of ionizing radiation on cyanobacteria, investigated only the growth or physiological impact of ionising radiation, without studying the cellular mechanisms responsible for their unique endurance to such stress [6-12]. In fact, the exact underlying molecular mechanisms behind the radiation resistance of certain microbes, and especially cyanobacteria, to ionizing radiation, are still largely unknown and subject of debate.

The aim of this PhD was to investigate in detail the capacity of the cyanobacterium Arthrospira sp. PCC 8005 to withstand various doses and types of ionizing radiation and to characterize the molecular responses and mechanisms of the cells to deal with and overcome such challenges.

VIII.1 The Development and Optimisation of Experimental set-ups and Analysis methods The impact of radiation on Arthrospira sp. PCC 8005 was studied on the physiological and biochemical level, via the analysis of the cell morphology and viability, the functionality of the Photosystem II, and the intracellular content of pigments (Phycocyanin, Allophycocyanin,

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives Chlorophyll and carotenoids), minerals and antioxidants. For many of these analyses the methodology had to be optimised for Arthrospira. Specific lab procedures were written and implemented, which are now further used by colleagues. Unravelling the cellular response mechanisms to ionizing radiation, requested also the optimisation and development of molecular tools for gene and protein expression analysis. The complete genome of Arthrospira sp. PCC 8005 had been sequenced and manually annotated by a consortium of experts (ARTAN) [4,13], offering a high value source of information and unique platform for further molecular investigations. Based on this genome sequence (version 3) (http://www.ebi.ac.uk/ena/data/view/GCA_000176895.2), a very new tiling-microarray chip for Arthrospira sp. PCC 8005 was designed by the bioinformatics experts of SCK•CEN (Dr. Pieter Monsieurs), and tested and used for the first time in this PhD project (Chapter V and VI). This is to our knowledge the first and only existing microarray chip allowing gene expression analysis for cyanobacteria of the Arthrospira genus. The gene expression analysis via microarray required however also the development of a tailor-made RNA extraction procedure, to obtain sufficient amounts of high quality RNA from Arthrospira sp. PCC 8005, including both the small regulatory RNA's (sRNA) and the larger messenger RNA's (mRNA). In general, cyanobacteria are well known to possess an extended array of secondary metabolites and exoplosaccharides that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Several optimisations were performed, to get to sufficient quantity and high quality RNA from an acceptable sample volume of Arthrospira sp. PCC 8005 (15-30ml of a culture at OD750 ~1). This development of a suitable RNA extraction method has taken a significant amount of time within the first year of the PhD project, has been further optimised throughout the following years, and was finally validated not only for microarray analysis but also for RNAseq analysis (data not shown). In addition to transcriptome analysis tools, also high quality proteomics tools, based on liquid chromatography coupled to mass spectrometry (LCMS/MS), available at UMons, were applied. This allowed to analyse quantitatively and qualitatively proteins after irradiation, and correlate the obtained data from transcriptomic analysis. Not many reports have combined and correlated transcriptomic and proteomic analysis for radiation stress responses, which makes our study rather unique. Moreover, during the course of this PhD the genome sequence of Arthrospira sp. PCC 8005 was further updated by experts from Genoscope and SCK•CEN, which led to the publication of genome version 5 in January 2014. So an additional effort was made to use this

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives improved genome version for the final interpretation of all the transcriptomic and proteomic results presented and discussed in this thesis.

Our investigations were mainly based on the transcriptomic and proteomic response of Arthrospira sp. PCC 8005 to gamma irradiation, tested at the 'RITA' and 'BRIGITTE' facilities of BR2 at SCK•CEN (Mol, Belgium). The particle irradiation experiment was performed at the Heavy Ion Medical Accelerator Chiba (HIMAC) at the National Institute for Radiological Sciences (NIRS) (Chiba-Shi, Japan). This study was limited to the physiological and morphological response of Arthrospira to heavy ions particles, due to the strict requirement of small volumes for that irradiation set-up.

Irradiation experiments need special facilities to perform such tests. As mentioned above, the Belgian Nuclear Research Centre (SCK•CEN) provides such special facilities (RITA and BRIGITTE Facility) and operators to execute and achieve a successful irradiation experiment. I was challenged, however, to deal with many safety and operational procedures (Contamination control, Dosimetry, and Access to BR2); the logistics of the transport of irradiated and control samples, immediately after each irradiation and finally the post irradiation analysis requiring immediate sample processing after irradiation. In addition, the control samples had to be kept at the same conditions as the irradiated one’s. Moreover, due to the fact that the time required for irradiation (hours, and minutes) was completely different for the two facilities due to the difference in dose rate, a different experimental planning had to be made for the different experiments. Therefore, getting an optimised irradiation set-up experiment required several repetitions. Another challenging experiment was the irradiation at HIMAC in Japan and DLR in Germany. The transport and storage effect was a major point to deal with, which finally resulted in an unsuccessful Xrays experiment.

I also contributed to the preparation and pre-flight testing of the ArtEMISS flight experiment (prepared by SCK•CEN with ESA), which aims to test the proliferation and oxygen production of Arthrospira sp. PCC 8005, under space flight conditions, inside ISS. To our knowledge Arthrospira has not yet been tested in space, thus this is also a first-off-a-kind experiment.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives VIII.2 The radiation tolerance of the cyanobacterium Arthrospira sp. PCC 8005 The first part of my Phd was devoted to the characterization the tolerance of Arthrospira cells to high doses of ionizing radiation (Chapter IV, V, and VI). Live cells of Arthrospira sp. PCC 8005 showed resistance up to 6400 Gy of 60Co gamma rays (photons with energies of 1.33 MeV and 1.17 MeV, LET 0.2 KeV/µm), at a dose rate of 527 Gy h–1, and restart normal photosynthetic growth. The exposure of live cells of the cyanobacterium Arthrospira sp. PCC 8005 to different doses of 60Co gamma rays (dose rate of 527 Gy h–1) showed clear damaging effects for doses of 3 200 Gy or higher, but still post-irradiation culture recovery even for a dose of 6400 Gy (Chapter IV). Testing the same doses with Brigitte facility (dose rate 20000 Gy h-1) resulted on similar observation with the same total absorbed dose (3200 Gy) (Chapter IV, V). Furthermore, the exposure of Arthrospira to 10000 Gy of 60Co gamma rays was lethal, killing all the cells after which no recovery was resumed (data not shown). These results confirmed the labelling of the planktonic filamentous cyanobacterium Arthrospira sp. PCC 8005 as a radiation resistant bacterium. A radiation resistant bacterium is defined as a bacterium that requires a dose of acute 60 Co gamma radiation greater than 1 000 Gy for a 90% reduction (D10) in Colony Forming Units (CFUs) [14]. High resistance to photon radiation has also been reported for other cyanobacteria, such as the planktonic filamentous cyanobacterium Anabaena which is tolerant up to 5 000 Gy of acute 60Co gamma radiation (dose rate of 6 250 Gy h–1) [9,12] and the rock-dwelling coccoidal cyanobacterium Chroococcidiopsis which is able to survive 15 000 Gy of X-ray [7]. In the seventh chapter of this thesis, we explored in specific the impact of particle radiation, as part of space radiation, on active cells of cyanobacterium Arthrospira sp. PCC 8005. The irradiation campaign of the STARLIFE project was limited to up to 1000 Gy for Helium particle and 2000 Gy for Iron particles. Consequently, it was unfeasible to assess the dose limit of Arthrospira sp. PCC 8005 to withstand higher doses of such particle radiation. Nevertheless, Arthrospira sp. PCC 8005 was capable to recover and resume normal photosynthetic growth after irradiation with a particle beam of 1 000 Gy of Helium (4He2+, 150 MeV/u, 2.2 KeV/µm) and 2 000 Gy of Iron (56Fe+26, 500 MeV/u, 200 KeV/µm), which are already relatively high doses. The comparison of the tolerance to electromagnetic and particle radiation is a well-established procedure for mammalian cells as hadron therapy (particle irradiation) is becoming an increasingly important option for radiation cancer therapy in comparison to classical X-ray therapy [15,16]. For microbes, this is only seldom studied or reported. This relation between the radiation biological

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives effectiveness (RBE) and the type of the radiation used (electromagnetic and particle radiation) [17], is typically presented in plots where RBE is plotted for a given Linear Energy Transfer (LET) of the radiation used, in comparison to the dose of X-ray needed to obtain the same effects [18]. Until today, only few papers exist, however, discussing the effect of heavy particles irradiation on bacterial cells, and they typically do not involve thorough comparison between high and low LET exposures. Comparing the post irradiation growth kinetics, morphology, photosynthetic activity and pigment concentration of Arthrospira for the same total absorbed dose (e.g 1000 Gy) of 60Co gamma rays and Helium (1000 Gy) and Iron particles (1000 Gy), no clear difference in damaging effect could be established between low LET electromagnetic radiation (Co60 gamma) and high LET particle irradiation (He and Fe), for Arthrospira sp. PCC 8005. This may seems surprising, as in general, it is believed that the damage induced by high-LET radiation (4He2+ particles with LET of 2.2 KeV/µm or 56Fe+26 particles with LET of 200 KeV/µm) is severe than by Low-LET radiation (gamma ray photons with LET of 0.2 KeV/µm) (Chapter VII). Typically, RBE increases with LET. High-LET radiations are more destructive to biological material than low-LET radiations because at the same dose, the low-LET radiations release their energy and induce the formation of lesions and radicals more sparely within a cell, whereas the high-LET radiations transfer most of their energy to a small region of the cell and cause intense clustered damage. Based on this relation, the probability of causing DSBs is lower in sparely ionizing (low LET) radiation and if DSBs are created, they are more randomly spread as an isolated damage, in comparison to densely ionizing (high LET) radiation. Some suggest that the localized DNA damage caused by dense ionizations from high-LET radiations is more difficult to repair than the diffuse DNA damage caused by the spare ionizations from low-LET radiations [15]. However, others have reported that their is no difference in radiation induced DSBs at LET values ranging between 0.2-2000 KeV/µm, at least from the point of view of DNA repair ability of Deinococcus [19]. The plotted relation between LET and RBE suggested that D. radiodurans can repair not only single DSBs but also clustered DSBs damage generated by heavy ions. Furthermore, the same degree of radiation resistance to high and Low LET radiation was indeed observed with dry cells of Deinococcus radiodurans [20]. Unexpectedly, Arthrospira cells showed a slight higher sensitivity for helium particles comparing to iron particles. Taking into account the linear energy transfer of Fe particles (LET 200 KeV/µm) which is greater than He particles (with LET 2.2 KeV/µm), we would expect more damaging effect with Iron prior to Helium particles. Based on the relation between the radiation biological

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives effectiveness (RBE) and the Linear Energy Transfer (LET) [17], RBE increases with respect to LET, but reaches a maximum at LET 100 KeV/µm [18]. At this density of ionization, the average separation in ionizing events which cause significant amount of double strand breaks (DSBs), is equal to the diameter of DNA double helix [21]. Beyond this maximum, RBE declines even if LET increases, a phenomenon that has been also called the ‘overkill’ or 'overshooting' effect. This would mean that the amount of energy deposited in a cell by one single particle traversal is already in excess regarding the amount required to kill the cell. Subsequently meaning that more particle traversals per cell or more energy deposition per transversal in the cell would not lead to more cell death [22], meaning that the ratio of the lethal effectiveness per total dose (RBE) declines. Others have suggested that saturation occurs not at the level of the biological (cell-killing) process but on the pure physical molecule-radiation interaction level [23]. An individual cell which survives irradiation with very high LET ions may either have received no lethal lesions despite having intersected by one or more tracks or it may have been missed entirely by the track [24]. The more pronounced effects observed with Helium particle rather than Iron on Arthrospira, might be explained by such '‘overkill’ or 'overshooting' effect, but would require additional investigation to confirm. Nevertheless, similar observations were reported with dry cells of Deinococcus radiodurans tested with various ionising particles [19].

VIII.3 The impact of ionising radiation on Arthrospira sp. PCC 8005

It is well known that ionizing radiation causes DNA damage, which includes double-strand breaks (DSBs), single-strand breaks (SSBs), and base damage. On average, for every 20 SSBs induced in DNA by gamma rays, there is 1 DSB. The rate of direct DSB formation is proportional to the radiation dose, i.e. radiation causes DNA double stranded breaks in a frequency of ~0.002-0.008 DSB/Gy/Mb [25] and the yield of strand breaks per unit absorbed-dose is nearly constant over a wide range of LET. The direct DSB DNA damage per dose is also constant between cell types, including eukaryotic and prokaryotic cells, but variations in radio-sensitivity is assigned to difference in repair speed and/or accuracy, which is more protected and more efficiently restored after radiation in radiation resistant cells. Moreover, radiation resistant cells can better protect the DNA from the additional indirect damages caused by ROS. Thus the final amount of DNA damage (direct and indirect) is influenced by the ability of cells to scavenge free radicals produced during ionizations prior to them causing damage, the number of ionizations that are close enough to DNA

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives to damage it, and the effectiveness of DNA repair. In D. radiodurans, for example a dose of 6000 Gy induces approximately 200 DSBs, compared to ca. 3000 SSBs [26]. We also investigated the presence of DSBs break in Arthrospira sp. PCC 8005 after exposure to gamma rays via classical constant field agarose gel electrophoresis, a method which has also been used in other studies [9]. In all the tests performed, there was no clear 'smear' of smaller DNA fragments on the gel which would indicate double strand breaks. There was nevertheless a tendency of a decrease in the total amount of high molecular intact DNA, with high doses such as 6400 and 10000 Gy. Although these observations are intriguing, they are currently still inconclusive to determine if there was or was not a significant amount of DSBs caused by gamma radiation in the DNA of Arthrospira sp. PCC 8005, and from which doses onwards. Hence, this requires additional investigation with more optimised DNA analysis techniques.

A time series experiment was carried out, allowing investigation the molecular mechanisms and related metabolic pathways involved in the response to ionizing radiation after two and five hours post irradiation recovery (Chapter VI). The results showed two major main stages: an early “emergency” response immediately upon irradiation and a “recovery" response later. The largest molecular events occurred immediately upon irradiation, during the 'emergency' response where Arthropira sp. PCC 8005 changed the expression of a high number of genes, sometimes even without correlated physiological traits. During recovery period, the cells tend to reprogram their metabolism, in a more controlled manner, in order to repair the damage and restart photosynthesis and growth.

VIII.4 The “emergency” response of Arthrospira sp. PCC 8005 immediately after irradiation Radiation causes the excessive production of ROS mainly hydroxyl radicals HO•, in the cells via the hydrolysis of water. Nevertheless, even in the absence of ROS produced radiation hydrolysis of water, photosynthetic organisms such cyanobacteria, are continuously challenged by ROS generated via photosynthetic electron transport chain and the respiratory machinery. The photosynthesis process is the main source of reactive oxygen species (ROS) including superoxide •− • anion (O2 ), hydrogen peroxide (H2O2) and the hydroxyl radical (OH ) under normal conditions. Thus, it seems logic, that in the event of high ROS production due to radiation, the cells shut down the ROS generation from photosynthesis and respiration.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives During the first hour after irradiation, several genes involved in photosynthesis and carbon fixation were indeed significantly repressed. Gene and protein expression analysis indeed confirmed an overall suppression of genes coding for the light harvesting system (phycobilisomes), the photosynthesis systems (PSII and PSI), electron transfer (plastoquinones, cytochromes, ferredoxin), carbon fixation and energy production (ATP synthase). Similar results were widely discussed in related studies on the effect of UV radiation on cyanobacteria [27]. The first study, using lower dose rates (527 Gyh-1) and thus longer irradiation times (Chapter VI), showed a clear correlation between reduced gene expression for photosynthesis, and biochemical analysis though assessment of pigments content and functionality of PSII quantum yield. The assessment of pigment content showed significant reduction of phycobiliproteins presenting the antenna pigments namely Phycocyanin and Allophycocyanin. The cells exhibited significant decrease in phycocyanin pigment content after exposure to 3200 Gy, 5000 Gy and 6400 Gy (Chapter V). This reduction of phycobiliproteins, is likely due to protein oxidation caused by the radiation-induced ROS, and potentially also the result of a controlled active phycobilisome degradation guided by the enzyme NblA [28,29]. The nblA gene was induced significantly after exposure of Arthrospira sp. PCC 8005 to high acute doses of gamma rays after exposure to 5000 Gy (Chapter V), but not significant at the doses tested 800 Gy, 1600 Gy, 3200 Gy (Chapter VI). This ordered breakdown of phycobilisomes (PBS), also called chlorosis, is reflected in a bleaching of the cells from blue green to yellow green which has been reported several times as a response to environmental stress. For instance, the expression of nblA gene was increased in the cyanobacterium Microcoleus vaginatus during desiccation [30], and in Synechosystis sp. PCC 6803 when challenged by UVB radiation [31]. It is an active response of the cell to lower the light energy harvesting, in order to minimize the effective photosynthesis and ROS production, under stress conditions. In contrast, there was no significant effect of radiation on the chlorophyll content. This finding is in agreement with other reports indicating in general a degradation of phycobiliproteins prior to chlorophyll pigment [32]. Thus it was mainly the degradation and lower proportion of phycobilisomes antenna pigments to harvest the light energy that caused the lower PSII functionality, and so reduced photosynthesis process. The assessment of the functionality of Photosystem II via fluorescence showed indeed a significant decrease after exposure high doses of gamma rays of 3200 Gy and more.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives In our second gamma irradiation study using higher dose rates (20000 Gyh-1) and thus very short irradiation times (Chapter VI), no significant variation of pigments content or PSII quantum yield, occurred. The observed reduction in gene expression was thus not yet translated to physiological change measurable immediately after irradiation. Sometimes the bleaching of the cultures did appear at later time points. This difference could be explained by the short irradiation time (minutes) compared to the first study where the exposure time was in hours. Also a lack of active phycobilisome degradation via NblA enzyme [33], that was not transcriptionally induced in this set-up could indeed explain this main difference. Consequently, the photosynthesis measurement revealed intact and functional photosystem even after exposure to the highest dose 3200 Gy in this set-up, which was not the case in previous gamma irradiation study with same dose, but lower dose rate. Also for the particle irradiation experiments, the immediate assessment of functionality of PSII via measurement of quantum yield was inconclusive towards radiation impact. Some cultures exhibited, however, clearly less green colour after particle irradiation, and there was also a slight but not significant decrease in carotenoids and phycocyanin content after 1000 Gy Helium. Hence, the observed impaired photosynthesis seems a logic response that could be explained by an effort of the cell to reduce production of oxidants, which are already enhanced by ionising radiation. In line with reduced photosynthesis, Arthrospira cells tried to reprogram the expression of genes for carbon and nitrogen assimilation during irradiation. Several genes involved in de Calvin-

Benson-Bassham-cycle (CBB-cycle), fixating CO2 in carbohydrates, and the Krebs cycle (also known as the Tricarboxylic acid or TCA cycle, or the citric acid cycle), providing the precursors for amino acids biosynthesis, were significantly repressed in early response. The cells tried to reroute their metabolic flux from glycolysis to the pentose phosphate pathway, in favour for NADPH and pentoses generation [34]. Gao and co-workers report similar observation with Synechocystis challenged with UVB radiation [35]. The reduction in TCA cycle after irradiation was also reported for Deinocccus radiodurans [36,37]. The obtained results from the two experiments (Chapter V and VI) showed an altered nitrogen uptake, assimilation and metabolism. Nitrogen is, however, an essential component for synthesis of amino acids for structural proteins and enzymes, nucleotides for DNA and RNA, and amino sugars for lipopolysaccharide and peptidoglycan in the cell envelope [38]. In general, genes for nitrogen uptake and assimilation were strongly reduced in expression after irradiation. This includes nitrate, nitrite, cyanate, and amino-acid uptake, nitrile hydrolysis and pattelamide biosynthesis. It is well known that cyanophycin (L-Arginine and L-Aspartic acid)

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives is thought to represent a dynamic reservoir of nitrogen accumulated in both non-nitrogen fixing and filamentous or unicellular N2-fixers, responding to the N regime [39]. Under nitrogen limiting conditions cyanophycin is catabolized as an internal nitrogen source, balancing the nitrogen deficiency. However, our findings did show no related genes responsible for this process namely cyanophycinase (cphA, cphB genes), confirming that the cells did not seem to experience N- limitation. At the contrary, the nitrogen metabolism seemed to be also adjusted to deal with high ammonium concentrations, possibly released from radiation damaged proteins, nucleotides and amino-sugars, which are known to be highly toxic to the cell. The ammonia produced from degradation of aminoacids, is usually quickly neutralised via urea synthesis [40]. There was after irradiation indeed also an increased transcription of genes in Arthrospira for the decomposition of urea to CO2 and ammonium. There was also a clear deactivation of genes involved in polyamine degradation, i.e. agmatine degradation to putrescine and urea, and a clear transcriptional activation of polyamines transporters (e.g spermidine and putrescine). This is possibly a mechanism to prevent even more urea and ammonium production. Polyamines are a group of nitrogen (amine) containing compounds that received recently considerable attention owing to its possible role in abiotic stress resistance [41]. Studies on polyamine transport in cyanobacteria have been scarce, but the implication of polyamine transport to protect Synechosystis against salt stress has been reported [42].

The regulation of C and N nutrient stress is typically under the control of the metabolic signal 2- oxoglutarate (in TCA cycle), which interacts with the sensor regulator PII protein (glnB), a transcriptional regulator, which in its turn induces the expression of genes involved in the signalling cascade controlling nitrogen metabolism, such as nblA. The nblA gene was induced significantly after exposure of Arthrospira sp. PCC 8005 to high acute doses of gamma rays in our previous studies (5000 Gy) (Chapter VI). The transcription of the PII protein was indeed significantly altered after radiation. The mechanism of the PII protein is well known and conserved for many organisms. It has been reported that the cellular carbon, nitrogen, energy and redox status which are sensed through ATP and 2-oxoglutarate. It is likely that the PII protein and the global transcription factor ntcA are involved in glycogen degradation [43]. The PII protein induces global transcriptional regulators such NtcA, the RNA polymerase sigma factor SigE and other response regulators such OmpR in Synechosystis sp PCC 6803 [44] and NrrA in Anabeanna sp PCC 7120 [45]

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives In the end, this significant reduction on the level of gene expression for photosynthesis and related carbon fixation and nitrogen assimilation had an impact on post irradiation cell proliferation, which was significantly delayed after exposure to damaging doses.

VIII.5 The mitigation of radiation damage in Arthrospira sp. PCC 8005: protection & detoxification In an attempt to survive, Arthrospira sp. PCC 8005 cells activated pathways for cellular detoxification and protection to fight against oxidative stress generated via ionizing radiation. In general, it is assumed that due to the hostile habitats colonized by cyanobacteria, they have developed multiple mitigation strategies to deal with such stresses (Chapter II). Here, we tried to identify the strategies that are specifically used by the cyanobacterium Arthrospira to deal with assaults of ionising radiation.

Recent studies, suggested that the mechanism of radiation resistance is mainly correlated with protection of proteins from radiation damage, to ensure the protection and repair of DNA. Protecting the activity of enzymes would enhance the efficiency of DNA repair and thereby cell recovery and cell survival from ionizing radiation [46]. Thus the capacity to prevent and tolerate protein damage (oxidation) is a major determinant of radiation resistance [47]. Cellular strategies to quickly neutralize the radiation-induced reactive oxygen species (ROS), which are causing protein oxidation, typically involve enzymatic and non-enzymatic antioxidants systems to detoxify the cell and restore its redox balance. The primary ROS scavenging is typically mediated by enzymatic antioxidants [48]. Our findings depicted, however, the lack of the catalase gene in Arthrospira sp. PCC 8005 genome, which is an exceptional trait of all Arthrospira species reported by Fujisawa and co-authors [49]. Furthermore, the results did not display radiation-induced transcription of the Fe superoxide dismutase gene (sodB). This is clearly different from what has been observed for many other radiation resistant bacteria and cyanobacteria, for which, the enzymatic antioxidant systems have been reported to play important role in combating oxidative stress. For instance, the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, induced cat and sod to cope with oxidative stress generated by UVA exposure [27]. The unicellular Synechosystis sp. PCC 6803 increased the transcription level of sod and glutathione peroxidase (gpx) upon UVB exposure [50]. In Anabeana sp. PCC 7120, the Ahp alkylhydroperoxide reductase enzyme was reported to play an important role in combating

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives various stresses, including: heat, copper, salt and ionizing radiations such as UVB [51]. Also the high radiation resistant bacterium Deinococcus radiodurans displayed induced expression of the antioxidant enzymes catalase, superoxide dismutase, thioredoxin reductase and alkylhydroperoxide reductase [52]. A slight induction of peroxiredoxine enzyme was also seen in the filamentous Anabeana sp. PCC7120 challenged by salt and UVB radiation [53]. Besides the commonly reported enzymatic antioxidants, there exists also a multitude of non- enzymatic ways to deal with oxidative stress. Several bacteria have increased radiation resistance thanks to the accumulation of small antioxidants molecules. Our findings showed that Arthrospira sp. PCC 8005 seems to rely mainly on thiol-based small antioxidant molecules, such as glutathione (GSH) but also peroxiredoxine, to cope with oxidative stress. The glutathione metabolite concentration analysis displayed a significant increase after exposure to radiation. Glutathione is a potent low molecular weight, scavenger of superoxide anion

.- • O2 , hydrogen peroxide H2O2, and the most harmful ROS hydroxyl radical OH [54,55]. The thiol group is very reactive, and quickly neutralizes radicals (Chapter V and VI). Glutathione is well known to play an important anti-oxidative role defense system for plants [56,57]. Although, Deinococcus lacks the glutathione system, very recently it was actually reported that this bacterium also relies on a thiol-based antioxidant, called Bacithiol and considered as a substitute for glutathione for its resistance to gamma rays [37]. Temporal gene expression in Arthrospira showed the coordinate activation of different genes involved in biosynthesis and regeneration of glutathione: the enzyme 5-oxoprolinase (hyuA) catalyses the generation of glutamate from 5-oxoproline, enzyme Glutamate Cysteine Ligase (gcL) converts glutamate to gamma-glutamylcystein ,and the enzyme glutathione synthetase (gshB) converts the precursor gamma-glutamylcystein to glutathione. Proline is the main precursor for glutathione synthesis and our findings suggest also a synthesis of proline in response to radiation. In addition, considered as potent antioxidant. It has been proposed that free proline can act as hydroxyl and singlet oxygen scavenger, inhibitor of lipid peroxidation and osmoprotectant [58,59]. Our results also showed the up-regulation of the putA gene contributing in glutamate synthesis from proline. Glutamate synthesis was also induced via aspartate-aminotransferase catalysing the formation of glutamate from aspartate and 2-oxoglutarate (also called α-ketoglutarate) from the TCA cycle. Glutamate can also synthesised from 2-oxoglutarate, via the GS-GOGAT pathway or the dehydrogenase (GLDH) pathway [60].

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives The GS-GOGAT pathway (also called glutamine synthetase (GS) or glutamate synthase (GOGAT) pathway) was immediately shut-down; while the glutamate dehydrogenase (GLDH) pathway was induced. Both pathways provide glutamate synthesis from NH3 and 2-oxoglutarate, but normally the GS-GOGAT pathway is used at low ammonium concentrations and when the cell is not under energy limitation (i.e. with sufficient reduced ferrodoxin or NAD(P)H), while the GLDH pathway is used when the cell is limited for energy and carbon but ammonium and phosphate are present in excess [61]. Synechocystis sp. strain PCC 6803, for example, utilizes the GS-GOGAT pathway as the primary pathway of ammonia assimilation, but the presence of GDH appears to offer a selective advantage for the cyanobacterium under non-exponential growth conditions [62]. It is known that high ammonia availability (as discussed above) leads to repression and deactivation of GS-GOGAT and induction of GDH [63]. The actual production of glutathione, which increased significantly during recovery, was confirmed via glutathione metabolite concentration analysis. Previous studies have also shown that Mn2+ cations can boosts protein protection in cells by interacting synergistically with the pool of small molecules, including orthophosphate, amino - acids, peptides, and nucleosides, generating catalytic O2 and H2O2 scavenging complexes [64-66]. It has been reported that Deinococcus in particular relies for the protection of its proteins from oxidation also strongly on the accumulation of manganese-orthophosphate (Mn-PO4) anti-oxidant complexes [67] Similar result was observed with Halobacterium salinarum [65], and the bdelloid invertebrate Adineta Vaga [66]. Cyanobacteria are well known to accumulate Mn2+ that is required specifically for PSII function. The Mn clusters in the donor side of PSII catalyses the water-splitting reaction [68]. Furthermore, also Iron (Fe) is required as a cofactor for photosynthetic electron transfer chain super-complexes. Especially the PSI system with its three 4Fe-4S clusters contains more Fe than the other super-complexes together. Total content of Manganese/Iron was assessed in Arthrospira sp. PCC 8005 cells using ICP/MS technique (Data not shown). However no clear conclusion could be established from obtained ratio, which could be linked to possible role of Manganese in proteins protection form oxidation. Our gene-expression results did show that the Arthrospira sp. PCC 8005 cells fine-tuned the active transport of some 'redox-active' metals such as Manganese, Iron, Copper, and Magnesium, in response to radiation. These metals are crucial for oxygenic photosynthesis and electron transfer, but they may also pose a serious risk to the cells via the generation of free radicals. Free transition metal species can enhance the rate of ROS generation

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives and cause extensive damage. Thus, metal transport and storage needs to be tightly regulated to ensure adequate supply and to protect against oxidative damage. [69]. Furthermore, possibly Arthrospira sp. PCC 8005 cells redirected their C-metabolism to produce polyhydroxyalkanoate (PHA), which may also have a role in cell protection. It has been reported that during desiccation Micrococcus vaginatus induces the transcription level of the PHA biosynthesis [30]. There were also some observations suggesting a putative induction of trehalose synthesis due to radation exposure. In addition to the reported role of trehalose as contributor to high salt stress tolerance [70], and desiccation tolerance [71], recent investigations suggest also a possible role of trehalose as protector of protein activity (enzymes) against IR damage, either alone or in combination with Mn2+ [72]. The addition of trehalose resulted in a significant increase in enzyme protection in vitro, up to 6000 Gy of 60Co Gamma rays [72]. Cyanobacteria have also developed a smart protection mechanism to deal specifically with the damaging effect of ionizing radiation on photosynthetic pigment proteins. While the expression of the genes coding for the main antenna pigments for the light harvesting complex of Arthrospira sp. PCC 8005 was reduced after irradiation, the cells induce in parallel the expression of the isiA gene. The isiA gene encodes the CP43’ protein, which is an auxiliary antenna complex, to compensate for the loss of phycobilisomes. In addition, this protein may also serve as a chlorophyll storage molecule contributing to the reassembly of reaction centres during recovery [73]. In general, bacteria have evolved extraordinary abilities to detect physical and chemical signals, both within their own cells and in the extracellular environment. The interaction of a signal with its receptor (usually a protein or RNA molecule) triggers a series of events that lead to reprogramming the cellular physiology, typically as a consequence of altered patterns of gene expression. In this way, the bacterial cell is able to mount appropriate and effective responses to changing physical and/or chemical environment. Molecules with PAS and GAF domains serve as specific sensors that react to oxidative stress, light, oxygen and many other signals [74,75]. Gene expression showed also the induction of the (cry dash) gene well known as photo-sensor for UV- A light induced photo-tactic movement [76]. Where a recent study reported that Syn-Cry, the Cry- DASH gene from the cyanobacterium Synechosystis sp. PCC 6803, was required for efficient restoration of photosystem activity following UV-B and PAR induced photo-damage [77]. Also the transcriptions of the genes for synthesis of the secondary messenger cyclic diguanylate (c-di- GMP) were induced. The aconitase enzyme (in TCA cycle), which showed an increased

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives transcription upon irradiation (3200 Gy) (Chapter VI), has been reported to play a role in regulation of these detoxification processes [78]. It has been proposed that aconitase, containing an iron sulphur cluster [4Fe-4S] as ROS sensor, can mediate the response to oxidative stress via transcription regulation [79].

VIII.6 The recovery of Arthrospira sp. PCC 8005 from radiation damage: repair & restart

Since irradiation causes protein oxidation, the damaged and dysfunctional proteins need also to be removed and rapidly resynthesized. Bacteria rely on different chaperones and proteases to deal with this process. Similar processes were observed in cyanobacterium Arthrospira sp. PCC 8005 after radiation exposure (Chapter V, VI). After irradiation, we observed an increase in the transcription level of heat shock proteins HSP, protease and peptidase coding genes. Such observation is likely a response to deal with the protein damage caused by the high doses of irradiation. Heat Shock Proteins, (HSP) proteins, such as the HSP70-type DnaK and GroEL/GroE are present in highly conserved forms in all bacteria, including cyanobacteria, and play crucial role in folding of newly synthesized proteins, preventing protein mis-folding or aggregation and promoting protein degradation [80]. Mis-repaired proteins are dysfunctional and need to be removed and rapidly resynthesized [81]. Requiring efficient proteolytic degradation of the damaged proteins [64]. Indeed, it has been shown for some bacteria, including Deinococcus that the level of intracellular proteolytic activity increased following radiation exposure [64,82]. Furthermore, Arthrospira sp. PCC 8005 expresses a set of protease genes, including the nblA and ftsH genes which allow specific proteolysis of key components of the photosynthesis system [83]. The FtsH protein has been shown to be involved in repair of the PSII system in Synechocystis sp. PCC 6803 [84]. Therefore it seems that the role of FtsH in PSII repair and D1 turnover might be conserved in both cyanobacteria and higher plants [85].

Alongside the protection and repair of proteins from oxidation, protection and repair of damaged DNA remains the final goal in order to survive. The gene expression profile obtained for Arthrospira sp. PCC 8005 after irradiation indeed showed the induction of genes related to SSB repair via the Nucleotide excision repair system (NER) and Mismatch repair system (MMR) (Chapter V). These were the only DNA-repair pathways that were activated in dose dependent

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives manner at early response and maintained during recovery. A high expression of mut and uvr genes during recovery, confirmed the involvement in methyl-directed mismatch repair. None of our transcriptome or proteome analyses in all of the experiments, showed strong activation of classical double strand DNA repair systems after irradiation (Chapter V and VI). Maybe the real pathway of double strand DNA repair in Arthrospira sp. PCC 8005 remains to be elucidated. Also the expression of the gene coding for the nudix hydrolase was significantly induced by irradiation. Nudix hydrolases are involved in the removal of nucleotide lesions and in the housecleaning, and they may play an important role in the fast recovery of the cells after radiation exposure, as reported for Deinococcus radiodurans [86].

It is known that in some bacteria, such as E. coli and B. subtilis, such DNA repair and clean-up is activated via an SOS response system. The inducible SOS system is regulated by two key proteins; RecA and LexA, where the RecA protease activates auto-cleavage of the LexA transcriptional repressor to induce DNA repair. It is assumed that RecA is also directly involved in the recombination repair of DSBs which entails a search for homologous DNA sites by RecA-DNA filaments. In some bacteria, however, this might function differently. In the study of D. radiodurans, however,it was demonstrate that the LexA is not required for the activation of RecA and DNA-repair following gamma radiation [87]. Despite the presence of a homolog of LexA in some cyanobacteria such as Synechocystis sp. PCC 6803 [88], this seems not to have a function in SOS response [8,89]. Our study depicted the lack of the lexA gene in the Arthrospira sp. PCC 8005 genome, which is uncommon but not exceptional. The absence of a lexA gene has also been reported for Helicobacter pylori [90]. In Arthrospira sp. PCC 8005 cells the recA gene, was not induced neither at the mRNA level nor at protein level. This might be due to a large amount of RecA protein present in the cell continuously, which in contrast to the 'inducible' nature of this gene in other bacteria such as E. coli [91]. Similar observations were reported for Helicobacter pylori, showing the lack of induced RecA protein following UV and gamma radiation [90]. This lack of induced RecA thus suggesst the existence of constitutively expressed DNA damage repair system, or an alternative regulation system to activate it, in Arthrospira sp. PCC 8005 cells. This would need to be further investigated.

Our results revealed also the presence of new set of genes and proteins highly induced, seemingly in a dose-dependent manner, following exposure to high doses of gamma rays, and highly

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives expressed throughout the full recover period (up to 5 hours) after irradiation (Chapter V, VI) The reported genes, which we called arh genes, were among the top ranked expressed genes after irradiation and maintained high expression during recovery period with respect to different doses. It is the only set of genes that displayed this kind of expression profile and that were not only found overexpressed in RNA level but three of them also at protein level for 3200 Gy and 5000 Gy. This set of genes was clustered in one genomic region, and were annotated to code for 'conserved hypothetical proteins'. The genes arhC and arhB showed some homology with the conserved domain of “chromosome segregation” (SMC) proteins. In eukaryotic cells SMC proteins function in chromosome condensation, segregation, cohesion and DNA recombination repair [92]. In addition, SMC-like proteins were found to be also present in bacteria and Archea where they have an essential function in a variety of chromosome dynamics, such as chromosome compaction, segregation, and DNA repair [93]. Some examples of SMC-like proteins in bacteria include RecN and SbcC, helping in protein assembly at replication forks. Theses Arh proteins thus may be involved in the early stages of DNA damage repair. However, these are only hypotheses, and will need to be confirming by further investigating to elucidate the real functional role of these proteins in the cell. Although no clear function could be assessed or confirmed at this moment, we can not deny their involvement in radiation resistance of Arthrospira sp. PCC 8005, and they are a highly valid target for further investigation of their role in the cell in general and their contribution to radiation resistance in particular.

During recovery, all the genes cited to be reduced in the 'emergency response' immediately after exposure, tended to increase their expression gradually, and switched back on photosynthesis, carbon fixation and nitrogen assimilation. In the end, the significant reduction of photosynthesis and related carbon fixation and investment in detoxification and repair, had a significant impact on post irradiation cell proliferation, which was significantly delayed with several hours to days after exposure to damaging doses. But for almost all irradiation types and irradiation doses tested in this study, except for 10000 Gy of gamma rays, the cultures were able to recover photosynthetic growth. This can be seen as reassuring as the true fate of Arthrospira sp. PCC 8005 for life support in space is linked to their capacity to conserve photosynthetic performance after exposure to radiation.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives VIII.7 Conclusion This PhD aimed to define the tolerance and unravel the response of the cyanobacterium Arthrospira sp. PCC 8005 to ionizing radiation. Arthrospira sp. PCC 8005 cells showed clear potential to resist high doses ionizing radiation, including electromagnetic and particle irradiation. We showed the ability of live cells of Arthrospira sp. PCC 8005 to survive and fully recover their photosynthetic growth after exposure to high doses, of 6400 Gy of gamma irradiation and 1000 and 2000 Gy, of He and Fe particle radiation. This supports the labelling of Arthrospira sp. PCC 8005 as radiation resistant cyanobacterium. Using a new specifically designed microarray chip, the molecular response of Arthrospira sp. PCC 8005 to high doses gamma rays could be further investigated. The dynamic gene expression changes of Arthrospira sp. PCC 8005 in response to ionising radiation over time, showed two main stages. During the early 'Emergency response, Arthrospira sp. PCC 8005 cells switched quickly from active growth state to survival mode. Cells shut down photosynthesis and carbon fixation and nitrogen assimilation mechanisms, and induce typically detoxification, protection and repair mechanisms. This was not only confirmed by gene and protein expression profile but also by the biochemical content and photosynthetic performance of the cell. The lack of the catA catalase gene in Arthrospira sp. PCC 8005 genome, together with non- significant expression of sodB superoxide dismutase gene, indicated that enzymatic anti-oxidants are likely not the primary detoxification and protection mechanism of Arthrospira sp. PCC 8005 against radiation. Arthrospira sp. PCC 8005 cells seem to rely mainly on a various non-enzymatic antioxidants systems, such as the glutathione system. During recovery the cells induce the cluster of arh genes with unknown function that are highly and specifically expressed in response to radiation, in a dose dependent manner. Finally, the cells expressed a restart of the vital energy and metabolic pathways, and full recover of photosynthetic proliferation could be obtained. These results, confirm that Arthrospira sp. PCC 8005 is valuable candidate for various biotechnological applications in the spaceflight environment.

Thus this PhD has led to following conclusions I. Arthrospira sp. PCC 8005 can survive high doses 60Co gamma radiation and He and Fe particle irradiation and can be classified among radiation resistant bacterium, and establish their exposure limit to high doses. II. Using the first designed tiling microarray chip, two main stages were denoted in the response of Arthrospira sp. PCC 8005 to ionising radiation:

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives i. Emergency response during which the cells suppresses photosynthesis and carbon fixation and nitrogen assimilation, going from an active growth state to a survival mode. The cells induce mechanisms for ROS detoxification and repair. ii. Recovery response during which the cells reactivate progressively all genes involved in photosynthesis, and carbon and nitrogen metabolism displayed slight increase. III. Gene expression results showed impaired photosynthesis and reduced carbon fixation and nitrogen assimilation in Arthrospira sp. PCC 8005 in response to ionising radiation IV. Arthrospira sp. PCC 8005 lacks the catalase gene in its genome, and does not induce Fe superoxide dismutase expression in response to radiation, but relies on other non- enzymatic antioxidants molecules such glutathione for cellular ROS detoxification. V. Gene expression results showed that Arthrospira sp. PCC 8005 relies on the MMR and NER pathways for radiation-induced DNA damage repair, but lacks the LexA repressor gene in its genome, and the lack of induction of RecA in response to radiation. VI. Arthrospira sp. PCC 8005 activates a set of conserved proteins of unknown function in response to ionising radiation, in dose dependent manner. VII. Live cells of the cyanobacterium Arthrospira sp. PCC 8005 can fully recover normal photosynthetic activity and cell proliferation after exposure to high doses, i.e. 6400 Gy of gamma rays or 1000 and 2000 Gy, of He and Fe particle radiation, components of cosmic radiation.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives VIII.8 Perspectives The gene and protein expression and biochemical fingerprint of the molecular response of the edible cyanobacterium Arhrospira sp. PCC 8005 to ionising radiations obtained in this PhD is a major step forward towards a better understanding of the radiation resistance of cyanobacteria, and bacteria in general. These interesting results also opened perspectives for complementary studies, which can and need to be done, to confirm or to further investigate these preliminary findings.

I. Complementary tests could be done, to further map the impact of radiation on Arthrospira sp. PCC 8005 as for instance the measurement of Lipid peroxidation and Protein Oxidation as indicators for oxidative stress damage, or of DNA damage. i. The cell membrane composed of polyunsaturated fatty acids, is a primary target for reactive oxygen attack leading to cell membrane damage and the formation lipid hydro- peroxides that could be done as reported in [94]. ii. The radiation resistance of several bacteria are linked to their power to protect their proteins from oxidation. Hence, oxidation of proteins and mainly carbonylation, is widely used as marker of protein oxidation [95]. The oxidation of protein could be assessed as reported in Krisko et al [96]. iii. During our study, it was difficult to assess the level of DNA damage following irradiation. Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines [97]. But this technique was also used with the cyanobacteria Anabaena [98]. If the FADU method is considered a sensitive technique in detecting DNA breaks induced X-rays, it would be interesting to test it with also on Arthrospira sp. PCC 8005 after radiation exposure. II. The designed array for Arthrospira sp. PCC 8005 is a tiling array that covers not only the coding sequences (5854CDS) but also inter-genic regions (3141 inter-genic regions), that can also be expressed and play a role in regulation of gene expression. Our current study was only based on exploring the coding sequences whereas the non-coding regions offers large amount of information that has to be explored in details. The microarray data obtained in this PhD project, show a distinct non-coding RNA expression profile in response to radiation, which was not yet explored. In bacteria, non-coding RNAs (ncRNAs) are a heterogeneous group of sequence-specific regulators of gene expression, normally lacking

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives a protein-coding function. They are typically smaller than 300 nt in length [99], and regulate mRNA translation or decay but sometimes also directly modulate certain protein functions. The roles of ncRNAs are diverse; they can be involved in the control of several stress responses, virulence and motility, among other functions [100]. The fact that cyanobacteria thrive in hostile habitats and are capable to adapt to vastly different environmental conditions suggests they also contain sophisticated regulatory mechanisms [101]. Therefore, various types of regulatory RNA can be expected that interplay with the different signal transduction pathways in the response to ionising radiation. III. The most novel finding was the expression of a new set of genes, the arh genes, induced in a dose-dependent manner following exposure to high doses of gamma rays. Although there are some indications that the Ahr proteins might be involved in DNA repair, no clear function could be assigned to these proteins yet. Deciphering the structure and the real function of these proteins is a new research topic that would complements our study. The additional investigations on these proteins should include: iv. The construction and study of deletion mutants for these genes. It is well established that knockout of genes offers a way to investigate the role of these proteins. Unfortunately, the genetic modification systems for Arthrospira are still limited due to the limited success to isolate viable single cells from the multicellular filaments. There is only 1 report on the genetic transformation of Arthrospira [49], but a similar approach could be tested with the set of 7 arh genes. Specifically, the search for sequence similarities did also show the conservation of the ArhB and ArhC proteins within cyanobacteria phylum. Different species of Arthrospira showed high amino acids sequence similarity: e.g Arthrospira platensis C1 (95.12%), Arthrospira maxima CS328 (94.82%) and Arthrospira platensis NIES (94.21%). high amino acids sequence similarity was seen with species from the Nostocacea family: e.g Nostoc punctiforme PCC 73102 (49.38%), Anabeana variabilis ATCC 29413 (48.46%) and Nostoc sp. PCC 7120 (47.84 %). Next to cyanobacteria, there are also species from the Deinococacea phylum that contain similar amino acids, i.e. the bacterium Deinococcus radiodurans R1 showed 40% and 31 % homology toward ArhC and ArhB respectively. One could therefore, also try to knock out the target genes in the filamentous cyanobacteria

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives Anabeana and Deinococcus radiodurans which also showed high radiation resistance in response to 60Co gamma rays. v. The determination of the proteins structure and function. As a protein structure is more conserved than its sequence, determining 3D structure of a protein of interest is an important step towards determining the for protein function. Conserved motifs in 1D sequence often corresponds to 3D structural features and an associated functional family. The conservation of theses motifs during the evolution at specific locations reflects their specific stability in protein structure. Structural similarity is a good indicator of similar function in two or more proteins. Thus, studying 3D structure of a protein can provide a hypothesis towards the role of a conserved motif. The two techniques X-raycrystallography and Nuclear magnetic resonance (NMR) offers information about the relative positions of atoms inside the molecules. The technique that has been used the most to discover the three-dimensional structure of molecules, including proteins, at atomic resolution, is x-ray crystallography. Nuclear magnetic resonance (NMR) spectroscopy has been widely used for many years to analyse the structure of small molecules, and is now increasingly applied to study small proteins or protein domains. Unlike X-ray crystallography, NMR does not depend on having a crystalline sample; it simply requires a small volume of concentrated protein solution that is placed in a strong magnetic field. The deduction of the function from the 3D protein structure can be done with programs such as Protein Data Bank (PDB) (http://www.pdb.org/such), Structural Classification Of Protein (SCOP) (http://scop.mrc-lmb.cam.ac.uk/scop/), Protein Structure Classification (CATH) (http://www.cathdb.info/). Although it is not always true that 3D structure allows determining a real function of a protein, such study nevertheless may bring some additional information about protein function. vi. Try to investigate the specificity of arh genes to irradiation or ROS. It would be of interest to assess further whether Arthrospira sp. PCC 8005 induces the set of arh genes only in response to ionising radiation, or also in response to other various ROS inducing or DNA damage conditions such: radiomimetic chemicals: Mitomycin C, Paraquat, or

a direct exposure to H2O2.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives vii. In proteins, aromatic sulfur-containing and aliphatic amino acid (AA) side chains are known to be the main targets of HO•- mediated damage. Aliphatic AAs become more reactive with an increasing number of methyl groups, but the reaction with aromatic AA residues is faster and easier for addition of HO• to double bonds, with tryptophan (W) residues being the most prone to oxidative damage by ROS [102,103]. Thus, resistance to radical-induced damage might be related to the substitution of residues highly prone to oxidative damage with residues less prone. One more target, could be to investigate this 'specialised AA usage' in arh genes to support this hypothesis.

IV. Interestingly, our results did show a high number of significantly induced Mobile genetic elements (MGEs) such Transposases and phage-like elements. These results needs to be investigated more in details, as transposable elements can lead to genetic changes and generate mutants. .

V. For its potential future use for life support in space, it is needed to further investigate if spaceflight conditions in general, including in addition to the cosmic radiation also the reduced gravity, have an effect on morphology, physiology, and metabolism of Arthrospira sp. PCC 8005. This planned within ARTeMISS project and a flight experiment to ISS.

VI. Radiation resistant Arthrospira sp. PCC 8005 holds also high potential for additional applications on Earth, which should be further explored.

i. Removal of heavy metals and radionuclides from contaminated water. Owing to the presence of exopolysaccharides EPS, cyanobacteria have been considered very promising as chelating agents for the removal of positively charged heavy metal ions from water [104]. It has been reported that spirulina contributes efficiently in the biosorption of toxic metals ions such (Cr3+, Cd2+, Cu2+) [105]. And given its high radiation resistance it could perhaps also be used as sorbent for radionuclide. The Fukushima Daiichii Nuclear Power Plant accident in March 2011, for example, released an enormously high level of radionuclides into the environment such: Cs, Sr, and I. Thus developing an effective and economical method for removing theses radionuclides from radioactive wastewater and aquatic ecosystem upon accidental releases has

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives become increasingly important. The potential use of aquatic plants, algea and cyanobacteria to eliminate theses radionuclides from the environment has been reported [106]. Comparing various taxa, cyanobacteria, green algea and ochrophytes seem to exhibit higher radionuclide uptake ability than other organisms [107]. The eukaryote green algae Coccomyxa actinabiotis, isolated from a nuclear facility, showed simultaneous extreme radio-resistance and radionuclides sequestration. This algea can withstands huge ionizing radiation doses, up to 20 000 Gy and strongly accumulates radionuclides, including 238U, 137Cs, 60Co, 54Mn, 65Zn, and 14C [108]. This could maybe give a possible use of Arthrospira as candidate for bio-decontamination in nuclear industry.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives VIII.9 References 1. Whitton BA, Potts M (2000) The Ecology of Cyanobacteria: Their Diversity in Time and Space: Springer. 2. Kamennaya N, Ajo-Franklin C, Northen T, Jansson C (2012) Cyanobacteria as Biocatalysts for Carbonate Mineralization. Minerals 2: 338-364. 3. Ashish Kumar Srivastava ANR, Brett A Neilan (2013 ) Stress Biology of Cyanobacteria: Molecular Mechanisms to Cellular Responses. 394 p. 4. Morin N (2009) Studies on the response to spaceflight related conditions in the cyanobacterium Arthrospira sp. PCC 8005 using a genomic approach: University of Liège, Belgium. 262 p. 5. Goossens O, Vanhavere F, Leys N, De Boever P, O'Sullivan D, et al. (2006) Radiation dosimetry for microbial experiments in the International Space Station using different etched track and luminescent detectors. Radiation protection dosimetry 120: 433-437. 6. Kraus MP (1969) Resistance of blue-green algae to 60Co gamma radiation. Radiation Botany 9: 481-489. 7. Billi D, Friedmann EI, Hofer KG, Caiola MG, Ocampo-Friedmann R (2000) Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. Applied and environmental microbiology 66: 1489-1492. 8. Domain F, Houot L, Chauvat F, Cassier-Chauvat C (2004) Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation. Molecular Microbiology 53: 65-80. 9. Singh H, Fernandes T, Apte SK (2010) Unusual radioresistance of nitrogen-fixing cultures of Anabaena strains. J Biosci 35: 427-434. 10. Dartnell LR, Storrie-Lombardi MC, Mullineaux CW, Ruban AV, Wright G, et al. (2011) Degradation of cyanobacterial biosignatures by ionizing radiation. Astrobiology 11: 997-1016. 11. Agarwal R, Rane SS, Sainis JK (2008) Effects of (60)Co gamma radiation on thylakoid membrane functions in Anacystis nidulans. Journal of photochemistry and photobiology B, Biology 91: 9-19. 12. Singh H, Anurag K, Apte SK (2013) High radiation and desiccation tolerance of nitrogen-fixing cultures of the cyanobacterium Anabaena sp. strain PCC 7120 emanates from genome/proteome repair capabilities. Photosynth Res 118: 71-81. 13. Janssen PJ, Morin N, Mergeay M, Leroy B, Wattiez R, et al. (2010) Genome sequence of the edible cyanobacterium Arthrospira sp. PCC 8005. Journal of bacteriology 192: 2465-2466. 14. Sghaier H, Ghedira K, Benkahla A, Barkallah I (2008) Basal DNA repair machinery is subject to positive selection in ionizing-radiation-resistant bacteria. BMC genomics 9: 297. 15. Okayasu R (2012) Repair of DNA damage induced by accelerated heavy ions--a mini review. International journal of cancer Journal international du cancer 130: 991-1000. 16. Goodhead DT (1999) Mechanisms for the biological effectiveness of high-LET radiations. J Radiat Res 40 Suppl: 1-13. 17. Hall EJ, Hei TK (2003) Genomic instability and bystander effects induced by high-LET radiation. Oncogene 22: 7034-7042. 18. IAEA (2008) Relative Biological Effectiveness in Ion Beam Therapy :Technical Reports Austria. 978– 92–0–107807–0. 19. Y Kobayashi TS, A. Tanaka G. Taucher-Scholz and H. Watanabe (1995) RBE / LET Effects of Heavy Ions on Inactivation in Dry Cells of Deinococcus radiodurans. Japan Japan Atomic Energy Research Institute JAERI. 44-46 p. 20. Kawaguchi Y, Yang Y, Kawashiri N, Shiraishi K, Takasu M, et al. (2013) The possible interplanetary transfer of microbes: assessing the viability of Deinococcus spp. under the ISS Environmental conditions for performing exposure experiments of microbes in the Tanpopo mission. Orig Life Evol Biosph 43: 411-428. 21. JAEA (2007) Dependence of Yield of DNA Damage Refractory to Enzymatic Repair on Ionization & Excitation Density of Radiation. 2188-1456.

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Discussion & Conclusion Chapter VIII: General Discussion, Conclusion and Perspectives 22. Barendsen GW, Walter HM, Fowler JF, Bewley DK (1963) Effects of different ionizing radiations on human cells in tissue culture. III. Experiments with cyclotron-accelerated alpha-particles and deuterons. Radiation research 18: 106-119. 23. Kiefer J, Rase S, Schopfer F, Schneider E, Weber K, et al. (1983) Heavy ion action on yeast cells: inhibition of ribosomal-RNA synthesis, loss of colony forming ability and induction of mutants. Advances in space research : the official journal of the Committee on Space Research 3: 115-125. 24. Parinaz MEHNATI SM, Fumio YATAGAI, Yoshiya FURUSAWA, Yasuhiko KOBAYASHI, Seiichi WADA, Tatsuaki KANAI, Fumio HANAOKA, and Hiroshi SASAKI (2005) Exploration of ‘Over Kill Effect’ of High-LET Ar- and Fe-ions by Evaluating the Fraction of Non-hit Cell and Interphase Death. J Radiat Res 46: 343-350. 25. Slade D, Radman M (2011) Oxidative stress resistance in Deinococcus radiodurans. Microbiology and molecular biology reviews : MMBR 75: 133-191. 26. Burrell AD, Feldschreiber P, Dean CJ (1971) DNA-membrane association and the repair of double breaks in x-irradiated Micrococcus radiodurans. Biochimica et biophysica acta 247: 38-53. 27. Soule T, Gao Q, Stout V, Garcia-Pichel F (2013) The global response of Nostoc punctiforme ATCC 29133 to UVA stress, assessed in a temporal DNA microarray study. Photochem Photobiol 89: 415- 423. 28. Baier K, Nicklisch S, Grundner C, Reinecke J, Lockau W (2001) Expression of two nblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp. PCC6803. FEMS Microbiol Lett 195: 35-39. 29. Aguirre von Wobeser E, Ibelings BW, Bok J, Krasikov V, Huisman J, et al. (2011) Concerted changes in gene expression and cell physiology of the cyanobacterium Synechocystis sp. strain PCC 6803 during transitions between nitrogen and light-limited growth. Plant physiology 155: 1445-1457. 30. Rajeev L, da Rocha UN, Klitgord N, Luning EG, Fortney J, et al. (2013) Dynamic cyanobacterial response to hydration and dehydration in a desert biological soil crust. Isme Journal 7: 2178-2191. 31. Hihara Y, Kamei A, Kanehisa M, Kaplan A, Ikeuchi M (2001) DNA microarray analysis of cyanobacterial gene expression during acclimation to high light. The Plant cell 13: 793-806. 32. Sauer J, Gorl M, Forchhammer K (1999) Nitrogen starvation in synechococcus PCC 7942: involvement of glutamine synthetase and NtcA in phycobiliprotein degradation and survival. Arch Microbiol 172: 247-255. 33. Karradt A, Sobanski J, Mattow J, Lockau W, Baier K (2008) NblA, a key protein of phycobilisome degradation, interacts with ClpC, a HSP100 chaperone partner of a cyanobacterial Clp protease. The Journal of biological chemistry 283: 32394-32403. 34. Ralser M, Wamelink MM, Kowald A, Gerisch B, Heeren G, et al. (2007) Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. Journal of biology 6: 10. 35. Gao Y, Xiong W, Li XB, Gao CF, Zhang YL, et al. (2009) Identification of the proteomic changes in Synechocystis sp. PCC 6803 following prolonged UV-B irradiation. Journal of experimental botany 60: 1141-1154. 36. Liu Y, Zhou J, Omelchenko MV, Beliaev AS, Venkateswaran A, et al. (2003) Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation. Proceedings of the National Academy of Sciences of the United States of America 100: 4191-4196. 37. Luan H, Meng N, Fu J, Chen X, Xu X, et al. (2014) Genome-wide transcriptome and antioxidant analyses on gamma-irradiated phases of deinococcus radiodurans R1. PloS one 9: e85649. 38. Huergo LF, Pedrosa FO, Muller-Santos M, Chubatsu LS, Monteiro RA, et al. (2012) PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity. Microbiology 158: 176-190. 39. Maheswaran M, Ziegler K, Lockau W, Hagemann M, Forchhammer K (2006) PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC 6803. Journal of bacteriology 188: 2730-2734. 40. Herrero A, Muro-Pastor AM, Flores E (2001) Nitrogen control in cyanobacteria. Journal of bacteriology 183: 411-425.

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Acknowledgments

Leaving home in 2010 and coming to Boeretang kingdom was the beginning of a new chapter in my life. Especially that I was wondering if I did the right choice to came to such place where it was completely different from my usual environment. Fortunately, the persons I used to know within these years became my colleagues, my friends and my new family.

Then my journey started in the group of the Molecular and Cellular Biology laboratory where I had the chance to know my mentor Dr Natalie Leys, the one to whom, I would express my appreciation and gratitude. She always guided and appreciated my research with providing advices and encouraging new ideas that improved the project and the quality of the work. During my whole PhD, Dr Leys was always there ready to help, kind and supportive supervisor.

In parallel, I had the honour to be promoted by Prof. Ruddy Wattiez. Despite his responsibilities and fully booked agenda, Prof Wattiez was supportive and continuously motivating me during my PhD. Furthermore, I would like to thank the team of Prof. Wattiez, namely Batiste Leroy, Catherine Shereen who were extremely professional and helpful colleagues for proteomic analysis.

I would like to thank all of the research personal in the MCB laboratory especially Microbiology group but also the Radiobiology group. I am extremely grateful for Dr. Pieter Monsieur for being available for me every time I need his help for Microarrays analysis and statistical studies. Thank you for your kindness and the discussions we had. Many thanks to Ilse Coninx for her great help in each irradiation experiment, and for staying very late with me in the evening. Many thanks also to Robin Nauts for his kind help for Glutathione measurement. I really appreciated your effort to meet my complicated experiments. I would also express my gratitude also to Dr. Felice Mastroleo, Dr. Hugo Moors, Dr. Paul Janssen, Dr. Rob Vanhoudt, Dr. Katinka Wouters, Ann Provost, and Arlette Michaux. They were always kind looking to help and to support me.

I would express my gratitude to Dr. Mohamed Islam Saiful, the first person I knew in the lab, his continuous support and help, and the invitations to his place discovering the delicious food

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prepared by his kind wife. Then, I shared the ‘aquarium’ office with Charlotte Rombouts and Kevin Tabury with whom I spent a great time.

I would like to thank all the PhD students that I was pleased to know during these years, some of them left already namely Dr Michael Bech, Dr Guiseppe Pani, Dr Kristel Mijnendoncks, Sandra Condori Catachura, Ellina Macaeva, Bo Byloos, Annelies Stussens, Marlies Gijs with whom I enjoyed discussing, laughting and working, in particular to Bjorn Baselet, the new comer to my office, thank you for kindness and your help. In addition, I was pleased to meet with Mohamed Maysara, his wife Raghda Ramadhan and their little girl Lilly. Ahmed, his wife Mercy and their little boy Zidane. It was always fun to discuss with them and to discover the Egyptien food nicely prepared by them.

I met my “New Family”

I would say that I am the luckiest women on Earth as I used to know friends of whole life, friends who take care of you, support you in gain and pain. In summary, they are and still always there for you. I will not take any order in listing them but I want to let them know that they are my new family. I would never forget the magical moments with you people. Khalil Abou-El-Ardat ‘Mr. You’, a kind-hearted and a funny brother to whom to I wish all the best in his life. Hussein Al Saghir ‘My pain supporter’, a very tender brother who was always there to support me we were so close in these last two years. Myriam Ghardi, the kindest sister, always generous (except for Dreft), understanding and always funny. I was so pleased to get to know her boyfriend Wouter Verstraate with whom I carried very funny discussions. Nada Samari, an exceptional sister with whom there is no place for routine, so reliable and supportive person I will never forget what she did in my most painful moments; without forgetting her adorable husband Marc-Antoine Hache (stop teasing me‼). Moreover, I used to know my soulmate Hanane Derradji; it was a great pleasure to share with someone the same habitudes and concerns. Her husband Yoann Descas was always kind and helpful without forgetting their awesome baby boy Ilyann who start to pronounce my name in first (Sorry Nada, I tooked your place ‘Tata N°1). I would finally list Iskander Ben Hadj, Laurianne and theirs little Yanis & Elissa, thank you for sharing the numerous moments of Happiness and your kind support.

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I will never be able to express my love, gratitude and appreciation to you Guys and I hope that we will keep always in touch.

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Curiculum Vitae

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Personal information

Name: Badri Surname: Hanène Citizenship: Tunisian Civil status: Married Adress: Boeretang 204 Bis 32 Mol 2400 Belgium Tel: (+32) 0485682556 E-mail: [email protected]

Education

January 2015: Doctor in Microbiology (SCK•CEN, Mol / UMons, Mons, Belgium) December 2007: Master Degree in Biotechnology (National Institute of Applied Sciences and Technology (INSAT), University of 7th November Carthage, Tunis – Tunisia). January 2005: Engineer in Bio-industry (National Institute of Applied Sciences and Technology (INSAT), University of Carthage – Tunisia). Honors and Awards

AWM (Aspiring Scientific Assistant) 4 years PhD grant from SCK•CEN and UMons - 2010

Research Experience

October 2010- present: “Molecular investigation of radiation resistant cyanobacterium Arthrospira sp. PCC8005”. Molecular and Cellular Biology unit, (MCB) at Belgian nuclear research center SCK•CEN and University of MONS: December 2008 – Juin 2009: “Optimization of the bioremediation potential of Cupriavidus metallidurans and Deinococcus radiodurans in radwaste and heavy metal contaminated matrices”. Laboratory of Microbiology and Molecular Biology of the National Centre for Nuclear Sciences and Technologies in collaboration with the University of Tunis ElManar September 2006 – December 2007: Training of the Master Degree in Biotechnology at Pasteur Institute of Tunis in collaboration with the National Institute of Applied Sciences and Technology (INSAT), University of 7th November Carthage, Tunis – Tunisia: “Cloning and Expression of Rieske iron sulphur gene of Leishmania Major and Leishmania Infantum in prokaryote system”. September 2004 – January 2005: Detection and confirmation of the presence of the doping agents Carvedilol and Pindolol in Urine. Engineer Diploma in Biotechnology at the National Laboratory of Drug Control of Tunis

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Training

June 2013: Bibliography training: searches in Web of Knowledge, ScienceDirect (SciVerse) and Endnote X6” June 2012: Statistics: basic training course: 3 days – 18 hours effective training, Club House, SCK•CEN. April 2011: Upgrade your written English: 3 days- 12 hours, Club House, SCK•CEN. November 2010: SCK•CEN isRP, International School for Radiological Protection, Radioprotection course English session. Club House, SCK•CEN. September 2009: Training in Molecular and Cellular Biology, (MCB) at SCK•CEN, Mol – Belgium. July 2001: Practice of ELISA test, Hematology analysis, Biochemistry analysis, Microbiology analysis. S. Meddeb Laboratoire d’Analyses Médicale (Medical Analysis Laboratory - Tunis) Scientific production

Publications with peer review

1. Hanène Badri, Pieter Monsieurs, Ilse Coninx, Ruddy Wattiez, Natalie Leys: Molecular investigation of the high radiation resistance of edible cyanobacterium Arthrospira sp. PCC 8005. In Press in MicrobiologyOpen 2. Hanène Badri, Pieter Monsieurs, Ilse Coninx, Robin Nauts, Ruddy Wattiez, Natalie Leys: Temporal gene expression of the cyanobacterium Arthrospira in response to gamma rays. Submitted to PlosOne 3. Hanène Badri, Marina Raguse, Ilse Coninx, Ruddy Wattiez, Ryuichi Okayasu, Ralf Moeller and Natalie Leys: Response of the spaceflight-relevant cyanobacterium Arthrospira sp. PCC 8005 to high doses of charged-particle radiation. Submitted to Astrobiology Journal 4. Orily Depraetere, Guillaume Pierre, Frédéric Deschoenmaeker, Hanène Badri, Imogen Foubert, Natalie Leys, Giorgos Markou, Ruddy Wattiez, Philippe Michaud, Koenraad Muylaert: Harvesting carbohydrate-rich Arthrospira platensis by spontaneous settling. Accepted for publication in Bioresource Technology. 5. Deschoenmaeker F, Facchini R, Leroy B, Badri H, Zhang CC, and Wattiez R. (2014) Proteomic and cellular views of Arthrospira sp. PCC 8005 adaptation to nitrogen depletion. Microbiology, 160, 1224-36. 6. Matallana-Surget S, Derock J, Leroy B, Badri H, Deschoenmaeker F, and Wattiez R. (2014) Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005. PLoS One, 9, e99076. Poster Presentations

1. Badri Hanène, Leys Natalie, Wattiez Ruddy: Effects of artificial light and ionizing radiation on photosynthetic cyanobacterium Arthrospira sp. PCC 8005 used for oxygen and food production in space, Event: PhD day SCK•CEN 6 october 2011. 2. Badri Hanène, Leys Natalie, Wattiez Ruddy: Effects of artificial light and ionizing radiation on photosynthetic cyanobacterium Arthrospira sp. PCC 8005 used for oxygen

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and food production in space; Conference ELGRA Biennial Symposium and General Assembly, Antwerp, Belgium: 6th - 9th September, 2011. 3. Badri H., Leys N., Wattiez R.- Effects of artificial light and ionizing radiation on photosynthetic cyanobacterium Arthrospira sp. PCC 8005 used for oxygen and food production in space.-8th European Workshop on Molecular Biology of Cyanobacteria .- Naantali , Finland, 28 August - 1 September 2011. 4. Badri H., Leys N., Wattiez R.- Molecular study of radiation resistance of cyanobacterium Arthrospira sp. PCC 8005.- Molecular Bioenergetics of Cyanobacteria From Cell to Community .- Saint Feliu de Guixol , Spain, 10-15 April 2011. 5. Badri H., Wattiez R, Leys N, . Arthrospira sp. PCC 8005 a highly radiation resistant photosynthetic cyanobacterium selected for oxygen and food production in space- Posttranscriptional regulation and epigenetics in microorganisms November 30th 2012 6. Badri H., Leys N., Wattiez R.- The edible cyanobacterium Arthrospira sp. PCC 8005, used for Life Support in space, is resistant to high doses of gamma rays.- Microbial Diversity for Science and Industry.- Brussel, Belgium, 27 November 2013.- 7. Badri H., Leys N., Wattiez R.- The edible cyanobacterium Arthrospira sp. PCC 8005, used for Life Support in space, is resistant to high doses of gamma rays.- PhD Day.- MOL, Belgium, 23 October 2013. 8. Badri H., Wattiez R, Leys N, The edible cyanobacterium Arthrospira sp. PCC 8005, used for Life Support in space, is highly tolerant to gamma rays. ESF-EMBO Symposium on Molecular Bioenergetics of Cyanobacteria, 15-20 April 2013 in Polonia Castle in Pultusk, Poland. Oral Presentations

1. Badri H., Leys N., Wattiez R.- Effects of artificial light and ionizing radiation on photosynthetic cyanobacterium Arthrospira sp. PCC 8005 used for oxygen and food production in space.- 7th International Microbial Space Workshop.- Clermont-Ferrand, France, 17-19 May 2011. 2. Mastroleo F., Condori Catachura S., Badri H., Mijnendonckx K., Wouters K., Leys N.- BD Accuri C6 applications at SCK-CEN.- BD Accuri user meeting: Micro and Marine Biology.- De Bilt, Netherlands, 22-22 May 2012. 3. Badri H, Leys N, Wattiez R: Effects of artificial light and ionizing radiation on the cyanobacterium Arthrospira sp. PCC 8005, used for oxygen and food production in space, Event: PhD day SCK•CEN 27th April 2012. 4. Janssen P., Badri H., Morin N., Barbe V., Vallenet D., Médigue C., e.a.- The Draft Genome Sequence of Arthrospira sp. PCC 8005, an Edible Cyanobacterium Tolerant to Salinity and Ionising Radiation.- CAREX Conference on Life in Extreme Environments.- Dublin, Ireland, 18-20 October 2011. 5. Badri H., Janssen P., Wattiez R., Leys N.- Photosynthetic growth of cyanobacterium Arthrospira is not effected by acute doses of gamma radiation.- Photosynthetic proteins for technological applications: biosensors and biochips (PHOTOTECH).- Antwerpen , Belgium, 10-12 April 2013. 6. Badri H., Monsieurs P., Wattiez R, Leys N,: Molecular study of Arthrospira sp. PCC 8005, a cyanobacterium highly tolerant to ionising radiation. Second PLENARY WORKSHOP PHOTOTECH meeting, on photosynthetic proteins for technological applications: Biosensors and Biochips; Istanbul, Turkey, from 9 to 11 April 2014. 7. Badri H., MonsieursP Wattiez R, Leys N: Molecular study of Arthrospira sp. PCC 8005, a cyanobacterium highly tolerant to ionising radiation. PhD Day at SCK•CEN. 24 April 2014.

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8. Badri H., MonsieursP, Wattiez R, Leys N, Bo Byloos: Molecular study of Arthrospira sp. PCC 8005, a cyanobacterium highly tolerant to ionising radiation. COSPAR Scientific Assembly, Moscow, 2–10 August 2014 Languages

 Arabic: mother tongue  English: Fluently spoken, read and written.  French: Fluently spoken, read and written.  Dutch: Basic knowledge  Italian: Basic knowledge

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