87-Fernanda-Cid.Pdf

UNIVERSIDAD DE LA FRONTERA Facultad de Ingeniería y Ciencias Doctorado en Ciencias de Recursos Naturales

Characterization of bacterial communities from Deschampsia antarctica phyllosphere and genome sequencing of culturable bacteria with ice recrystallization inhibition activity

DOCTORAL THESIS IN FULFILLMENT OF THE REQUERIMENTS FOR THE DEGREE DOCTOR OF SCIENCES IN NATURAL RESOURCES

FERNANDA DEL PILAR CID ALDA

TEMUCO-CHILE

2018

“Characterization of bacterial communities from Deschampsia antarctica phyllosphere and genome sequencing of culturable bacteria with ice recrystallization inhibition activity”

Esta tesis fue realizada bajo la supervisión del Director de tesis, Dr. Milko A. Jorquera Tapia, profesor asociado del Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería y Ciencias, Universidad de La Frontera y co-dirigida por el Dr. León A. Bravo Ramírez, profesor titular del Departamento de Ciencias Agronómicas y Recursos Naturales, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera. Esta tesis ha sido además aprobada por la comisión examinadora. Fernanda del Pilar Cid Alda

……………………………………………

……………………………………. Dr. MILKO JORQUERA T.

Dr. Francisco Matus

DIRECTOR DEL PROGRAMA DE ………………………………………….

DOCTORADO EN CIENCIAS DE Dr. LEON BRAVO R. RECURSOS NATURALES

………………………………………….

Dra. MARYSOL ALVEAR Z......

Dr. Juan Carlos Parra ………………………………………… DIRECTOR DE POSTGRADO Dra. PAULINA BULL S. UNIVERSIDAD DE LA FRONTERA

…………………………………………

Dr. LUIS COLLADO G.

…………………………………………

Dra. ANA MUTIS T

Dedico esta tesis a mis padres, Lillie Alda Leiva y Fernando Cid Verdugo quienes dedicaron sus vidas a la tarea de enseñar en la Universidad de La Frontera, Temuco-Chile.

Abril, 2018

Agradecimientos /Acknowledgements

This study was supported by the following research projects: Doctoral Scholarship from

Conicyt (no. 21140534) and La Frontera University. Antarctic Chilean Institute

Scholarship (code DT_01-13) for Doctoral studies and for the permits given by them to collect plants in Antarctic protected areas n° 125, 126 and 150. International

Cooperation project Conicyt-USA code USA2013-0010 and MECESUP FRO1204 also financed this study. I would like also to acknowledge the Doctoral Program in Natural

Resources Management and for its academic and economic supports. Especially Dra.

Maria de la Luz Mora that encouraged me for doing this graduate studies. I want also to give special thanks to the administrative staff, Mariela Gonzalez and Gladys Oyarzun for their commitment and dedication at any time.

I would also like to give special thanks to Dr. Milko Jorquera Tapia for his constant support, patience, dedication and friendship in the development of this thesis in his laboratory. I want to thank also Dr. Fumito Maruyama for his friendship and voluntary support in my thesis and also for all his assistance in my scholarship in Kyoto

University-Japan. I want to refer also to Dr. Ralf Greiner for receiving me in Max

Rubner Institute in Karlsruhe-Germany, and I want also to give special thanks to Dr.

Steffen Graether for his support in my internship in Guelph University in Canada and all his support while writing all papers done in my thesis. In addition, I want also to thank all researchers who have participated as co-authors in our papers, for their collaboration and helpful comments during manuscript revisions.

I want to highlight the good vibes received always from laboratory mates; Nitza

Inostroza that helped me in the experiments done in my thesis and encouraged me to keep working despite hard situations in life. Ignacio Rilling who helped me in the

Agradecimientos /Acknowledgements

phylogenetic analyses of antifreeze protein sequences. Giovanni Larama who helped me in the bioinformatics searches and analysis of antifreeze proteins. Oscar Navarrete who helped me also with antifreeze protein sequences databases. Lorena Lagos and Patricio

Barra who supported me always with their previous experience in laboratory and Luis

Marileo for his expertise help in DGGE development. Paola Durán for teaching me at the beginning in the laboratory and also, Jacqueline Acuña who was always able to read or give her opinion to improve the work done in laboratory. I will always be grateful to all of you.

Finally, thanks to my family for their unconditional love and support. I love you, we are the best team ;).

Abbreviations

ACC deaminase = 1-aminocyclopropane-1-carboxylate deaminase

AFP = antifreeze protein

C = carbon

ColAFP = Colwellia sp. SLW05 AFP

DGGE = denaturing gradient gel electrophoresis

ERIC = enterobacterial repetitive intergenic consensus

HCN = hydrogen cyanide

IBP = ice binding protein

IAA = indole acetic acid

INP = ice nucleating/nucleation protein

IRI = ice recrystallization inhibition

InaA = gene for ice nucleation in Erwinia ananas (= Pantoea ananatis).

InaX = gene for ice nucleation in Xanthomonas campestris

-like = protein denoted as putative since their activity have not been confirmed experimentally.

MpAFP = Marinomonas primoryensis antifreeze protein

N = nitrogen

NCBI = National Center for Biotechnology Information

III

Abbreviations

P = phosphorus

PCR = polymerase chain reaction

PGP = plant growth-promoting

PV = phenotypically verified

TH = thermal hysteresis

Glossary

ACC deaminase : Is an enzyme from bacteria that converts ACC synthase, a plant precursor of ethylene, to ammonia and α-ketobutyrate compounds that are readily assimilated. Thus the reduction of ethylene levels ameliorates the effect of

“stress by ethylene” in plants.

Antifreeze protein : A protein that bind to ice crystals by adsorption causing a thermal hysteresis or ice recrystallization inhibition.

Ethylene : Hormone from plants that is produced as a response of stress, inducing processes such as senescence, chlorosis, and abscission that may lead to a significant inhibition of plant growth and survival.

Hydrogen cyanide : Is a secondary metabolite produced by prokaryotes especially within the phylum Proteobacteria. HCN is a broad-spectrum antimicrobial compound that has been found to be implicated in the suppression of different plant root diseases.

Ice binding protein : A protein found in organisms that live at subzero and near-zero temperatures, that binds to ice. Ice-binding proteins include AFPs and proteins that inhibit IRI of ice, and might include proteins that favors ice formation such as INP.

Ice nucleating protein : A protein that induces ice formation at high subzero temperatures.

Ice recrystallization inhibition : As a consequence of the adhesion of an AFP to ice, ice recrystallization inhibition prevent the generation of large ice crystals by boundary migration of smaller ice crystals.

Glossary

Identity percentage : The extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, often expressed as a percentage.

Indole acetic acid : It is an auxin, a plant phytohormone involved in plant growth regulation, specifically affects cells division, extension and differentiation. IAA synthesized by bacteria may be involved in plant growth promotion since it has been proven to increase root surface area and length, and thereby provides the plant a greater access to soil nutrients.

Median coverage : Average number of reads that include a given nucleotide in the aligned sequences.

PGP mechanisms : Have been described in bacteria among other microorganisms to facilitate nutrient uptake by plant, provide defense against pathogens and insects and/or contribute to ameliorate stress in plants, favoring plant development.

Psychrophilic bacteria : Microorganisms adapted to live in extremely cold or freezing temperatures

Similarity percentage : The extent to which nucleotide or protein sequences are related.

Thermal hysteresis : A non-colligative effect as a consequence of the adhesion of an AFP to ice, lowering the freezing point below the melting point of a solution.

Thesis summary

Thesis summary:

Frost events generate considerable damages in agriculture world-wide, thus the study of mechanisms present in Antarctic plant-associated microorganisms such as antifreeze proteins (AFP), could represent a source to search and develop natural strategies to ameliorate frost injury and economical losses in agriculture. The phyllosphere microorganisms of Antarctic vascular plants and their potential application in agriculture have not been investigated in detail so far. Thus, the objective of this thesis is to characterize the bacterial communities from Deschampsia antarctica phyllosphere and to identify putative genes encoding for AFP by determining the whole genomes of culturable bacteria showing antifreeze activity. After a general introduction, this thesis contains the information of three consecutive published manuscripts that includes; firstly a review, which was focused on properties from bacterial ice binding proteins

(IBP) that regulates ice growth, both AFP and ice nucleating proteins (INPs), and their biotechnological applications, perspectives in agriculture and shows some information regarding this protein sequences deposited in public databases. Secondly, another paper describes the bacterial community structure on the phyllosphere of Deschampsia antarctica plant and evaluate the presence of ice recrystallization inhibition (IRI) activity in these isolates. Through denaturing gradient gel electrophoresis (DGGE) analysis, it was possible to observe differences in bacterial community compositions among Antarctic plants, nevertheless the most dominant orders found were

Pseudomonadales followed by Rhizobiales. Regarding IRI activity, 21% (32 isolates) presented IRI activity in cold acclimated bacterial cultures and 3% (5 isolates) in nonacclimated cultures. And the third manuscript, contains the analysis of the draft genome sequences of bacteria isolated from the Deschamspsia antarctica phyllosphere.

Genome analyses are being used to characterize plant growth promoting (PGP) bacteria

VII

Thesis summary living in different plant compartments, thus Genomes of bacteria showing IRI activity from previous work, were sequenced and annotated according to their functional gene categories. Five strains were identified as Pseudomonas and one as Janthinobacterium and genes associated with PGP traits were found in most strains. However, no genes associated with reported AFPs were found in these genomes. Genes connected to ice nucleating proteins (InaA) were found in all Pseudomonas strains. With this thesis work we put together the state of art of IBP and their possible applications in agriculture biotechnology. Through bacterial culture dependent and independent techniques, it has been possible to study the diversity of organisms living in the phyllosphere of D. antarctica as well as to obtain candidates of AFP producing PGP bacteria to be tested.

Nevertheless, further work on characterization, purification and use of AFP or inoculation of PGP bacteria are required to be confirm their biotechnological potential in agriculture against frost damages in plants, under laboratory and field conditions.

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Thesis outline

In the world, frost events cause important economic losses in agriculture. To our knowledge, in Chilean agriculture there are no studies focused on strategies to ameliorate the damages by frost in crops, particularly in Southern Chile. Psychrophilic bacteria have developed a wide set of physiological adaptations to undergo freezing or cold temperatures. Among these, AFP are considered as adaptations to tolerate extreme cold temperatures. AFP bind to ice crystals by adsorption and can act by i) thermal hysteresis (TH) and ii) IRI. Studies have demonstrated the presence of AFP in bacteria isolated from marine polar waters, Antarctic lakes and rhizosphere from Arctic plants.

Thus, psychrophilic bacteria with the ability to produce AFP could represent an alternative to ameliorate frost injury in frost sensitive plants.

The first chapter of this thesis includes a general overview of cold temperature damages in crops and the mechanisms developed by living organisms and agriculture to counteract this damages and also includes the overall hypothesis of this work and objectives of this doctoral thesis. In the second chapter, we include a review that analyses the properties and biotechnological applications of IBP, both AFPs and INPs in various fields such as food industry and medicine. This review emphasizes on less studied fields such as agriculture and the use of INP bacteria antagonists and AFP- producing bacteria as a strategy to ameliorate frost damages in crops. This work also includes UniProt database analyses of reported IBPs in bacteria and indicate that major studies are required.

The hair grass Deschampsia antarctica is one of the two vascular plants that inhabits Antarctic continent and the symbionts on the phyllosphere, defined as the aerial part of plant leaves, have been scarcely studied. Thus, the third chapter of this thesis

Thesis outline refers to the bacterial community structure on the phyllosphere of D. antarctica plant and the diversity and representative bacterial genera associated to this plant phyllosphere. In addition, one of the mechanisms present in organisms in cold environments is IRI activity. This activity has been previously detected visually in bacteria by analyzing ice crystals growth behavior, whereas in this thesis we did a modification of this detection technique by using a spectrophotometer analysis. This technique was standardized and crude protein extracts of culturable bacteria isolated from D. antarctica phyllosphere were evaluated. In this context, genome sequencing technique has also been proven to be a valuable tool for the identification of functional genes involved in relevant processes and for the comparison of genetic features contributing to a particular lifestyle or phenotype. Therefore, fourth chapter of this thesis, presents the analysis of draft genome sequences of bacteria isolated from D. antarctica phyllosphere. Bacteria selected in previous work showing IRI, an activity related to the presence of antifreeze proteins (AFPs), were used for this study.

Finally, the fifth chapter of this thesis, gives a general discussion derived from the results obtained in this study and the sixth chapter gives the final conclusions of this thesis and the perspectives reached. And, the last chapter (7th) of this thesis, includes all the literature used in this work.

Table of contents

TABLE OF CONTENTS

Agradecimientos / acknowledgements i

Abbreviations iv

Summary vi

Thesis outline viii

Table of contents xi

Figures Index xiv

Tables Index xix

Supplementary material index xxi

Chapter 1. General introduction 1

1.1. General introduction 2

1.2. Hypothesis 6

1.3. General objective 6

1.4. Specific objectives 6

Chapter 2. “Properties and biotechnological applications of ice-binding 7

proteins in bacteria”

Abstract 8

2.1. Introduction 9

2.2. Antifreeze proteins 11

2.3. Bacterial AFPs 12

2.4. Ice-nucleating proteins 15

2.5. Bacterial INPs 15

2.6. Biotechnological applications 17

2.7. Perspectives in agriculture 19

Table of contents

2.8. IBPs in public databases 26

2.9. Conclusions and future directions 32

Acknowledgements 33

Funding 33

CHAPTER 3. “Bacterial community structures and ice recrystallization 34 inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica”

Abstract 35

3.1. Introduction 36

3.2. Materials and methods 38

3.2.1. Plant collection 38

3.2.2. Bacterial community structure 40

3.2.3. Isolation of culturable bacteria 42

3.2.4. Selection of isolates by genotyping 44

3.2.5. IRI detection 44

3.2.6. Screening for IRI in isolates 46

3.2.7. Statistical analysis 48

3.3. Results 48

3.3.1. Bacterial community structure 48

3.3.2. Isolation and genotyping of culturable bacteria 50

3.3.3. Detection and screening for IRI in isolates 51

3.4. Discussion 54

Acknowledgements 59

CHAPTER 4. “Draft genome sequences of bacteria isolated from the 60

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Table of contents

Deschampsia antarctica phyllosphere”.

Abstract 61

4.1. Introduction 62

4.2. Materials and methods 64

4.2.1. Bacterial strains and DNA extraction 64

4.2.2. Genome sequencing, assembly and taxonomic affiliation 65

4.2.3. Genome analysis. 65

4.2.4. Ice recrystallization inhibition activity 66

4.3. Results 69

4.3.1. Genome sequencing and taxonomic assignment 69

4.3.2. Genome analyses 71

4.3.3. Genes related to PGP traits 73

4.3.4. Genes related to ice recrystallization inhibition activity 75

4.4. Discussion 80

4.5. Conclusions 88

Acknowledgements 87

Supplementary figures 89

CHAPTER 5. General discussion 91

5. General discussion 92

CHAPTER 6. General conclusions and perspectives 101

6. General conclusions and perspectives 102

Chapter 7. References 104

7. References 105

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Tables index

Figures index:

Chapter 2. Properties and biotechnological applications of ice-binding proteins in bacteria

Figure 2.1. Structure of MpAFP from Marinomonas primoyensis based on Garnham et al.

(2011) model. A cartoon representation of this Antarctic bacterial AFP (PDB id 3P4G) is shown with coil structure colored white, α-helix colored orange and β-strands colored blue. The figure is oriented with the N-terminus at the top of the page and the ice-binding face towards the viewer. Page 14

Figure 2.2. Phylogenetic analysis of phenotypically verified AFPs (red) and putative AFPs

(black). Amino acid sequences were taken from the UniProt database (http://www.uniprot.org/) and aligned using Clustal Omega (http://www.clustal.org/omega/). In brackets, protein accession numbers were included. The neighborjoining phylogenetic tree was built using the

PHYLIP package (http://evolution.genetics.washington.edu/phylip.html), and interpreted using

FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Marinomonas primoryensis

(A1Y1Y2), Colwellia sp. SLW05 (A5XB26), Flavobacterium frigoris PS1 (H7FWB6),

Flavobacteriaceae bacterium 3519-10 (B3GGB1), Pseudomonas putida GR12-2 (Q68VA9), P. syringae SM (S3MTC9), Pseudomonas sp. UW4 (K9NL46), Pseudomonas sp. Chol1

(K5YJR0), Serratia fonticola AU-AP2C (U2NA57), S. fonticola AU-P3 (U2LCA8),

Xanthomonas campestris pv. campestris (A0A0C7DZ94), X. campestris pv. campestris

ATCC33913 (Q8P6Q0), X. campestris pv. raphani 756C (G0CBK7), Rhizobium fredii HH103

(G9A7B7), Bradyrhizobium diazoefficiens SEMIA 5080 (A0A099IPF9), Ensifer adhaerens

(Sinorhizobium adhaerens) OV14 (W8HVP5), Herbaspirillum frisingense GSF30 (R0G033), H. seropedicae SmR1 (D8IRJ0), Herbaspirillum sp. Gw103 (I3CTQ5), Bacillus sp. OxB-1

(A0A0A8JKC3), Paenibacillus alvei DSM29 (K4ZQG5), P. alvei TS-15 (S9SIL7),

Paenibacillus sp. JDR-2 (C6D7M4), Paenibacillus sp. R7-277 (W4DV37). Page 24

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Figure 2.3. Phylogenetic analysis of phenotypically verified INPs (red) and putative INPs

PHYLIP package (http://evolution.genetics.washington.edu/phylip.html) and interpreted using

FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Pseudomonas syringae (K4WYP7),

P. savastanoi pv. savastanoi (G7ZJR9), P. syringae SM (S3MD81), P. syringae (D2U789), P. mandelii JR-1 (A0A024E7B8), Pseudomonas sp. GM30 (W6VKH0), P. syringae pv. syringae

B728a (Q4ZW16), Pantoea ananatis LMG 20103 (D4GJ13), Xanthomonas gardneri ATCC

19865 (F0C4Z6), X. campestris pv. raphani 756C (G0CIB0), X. campestris pv. campestris

ATCC 33913 (Q8PD38), Rhodobacter capsulatus ATCC BAA-309 (D5AV30), R. capsulatus

SB1003 (O68078), Bacillus thuringensis MC28 (K0FS92), P. ananas InaA (P20469), P. syringae pv. syringae InaZ (P06620), P. ananas InaU (Q47879), X. campestris InaX, (P18127),

P. fluorescens InaW (P09815), P. syringae InaK (O30611), P. syringae InaQ (B0FXJ3), P. agglomerans iceE (P16239), P. syringae InaV (O33479). Page 27

Figure 2.4. Motifs in bacterial phenotypically verified INPs as represented by the motif discovery tool MEME (http://meme.nbcr.net/meme/). Black bars indicate the octapeptide repeat sequence. The UniProt accession numbers are given in parenthesis. Page 30

Chapter 3. Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant

Deschampsia antarctica

Tables index

Figure 3.1. Map of Antarctic Peninsula. In detail, sampling sites of D. antarctica plants (red circles) on South Shetland Islands. Plants 1, 2, and 3 were taken from Gemelos Hill

(62º11’45.73’’S; 58º59’39.95’’W). Plants 4, 5, and 6 were taken from Collins Glacier

(62º10’9.00’’S; 58º51’21.06’’W). Plant 7 was taken from Ardley Peninsula (62º12’36.78’’S;

58º56’59.25’’W). Plant 8 was taken from Hanna Point (62º39’15.64’’S; 60º36’51.82’’W).

Page 39

Figure 3.2. (a) Denaturing gradient gel electrophoresis (DGGE) banding profile of bacterial communities present in eight plants of the Deschampsia antarctica phyllosphere. Arrows indicate excised and sequenced bands. Analysis of similarity percentages of dendrogram (b) and non-metric multidimensional scaling (c) of DGGE profiles (16S rRNA gene) from bacterial communities from eight plants of the Deschampsia antarctica phyllosphere. Page 43

Figure 3.3. Dendrogram of ERIC-PCR genotyping of culturable bacteria from Deschampsia antarctica phyllosphere. (a) Isolates grown in Pseudomonas agar (PA) plates and (b) grown in

NM1 culture media. Page 45

Figure 3.4. Standardization of detection of ice recrystallization inhibition (IRI) in culturable bacteria of the Deschampsia antarctica phyllosphere. (a) Background theory, (b) Absorbance at different wavelengths (from 300 to 650 nm), (c and d) Absorbance at 500 nm at different BSA and AFP protein concentrations (from 0.01 to 2.0 mg ml-1). AFP: antifreeze protein in a 30% sucrose solution; BSA: bovine serum albumin in a 30% sucrose solution; Blank: 30% sucrose solution. Page 51

Figure 3.5. Ice recrystallization inhibition in the phyllosphere (a) Screening of IRI activity in crude protein extracts (1 mg ml-1) from bacteria isolated from the Deschampsia antarctica

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Tables index phyllosphere using Pseudomonas and Actinomyces agar plates. (b) Detection of IRI activity in crude protein extracts (0.5 mg ml-1) from bacteria grown without cold temperature acclimatization. Asterisks denote significant (p≤0.05, Dunnet’s test) differences in comparison with crude protein extracts of E. coli JM109 (negative control).

Page 53

Chapter 4. Genome sequencing and search of putative antifreeze proteins in bacteria isolated from Deschampsia antarctica phyllosphere

Figure 4.1. Abundance and functional categorization of total coding-genes in draft genomes of phyllosphere Pseudomonas (a) and Janthinobacterium (b) strains as revealed by the SEED system. The reference Pseudomonas (Pf5 and L10.10) and Janthinobacterium (KBS0711 and

PAMC25724) strains are presented on the left side of each graph, followed by the sequenced strains (43NM1, 38PA, 28NM1, 20AA, 19PA and 15PA). Page 72

Figure 4.2. Abundance of encoded-genes associated with carbon (a and b), nitrogen (c and d) and phosphorus (e and f) metabolism in draft genomes of phyllosphere Pseudomonas (a, c and e) and Janthinobacterium (b, d and f) strains, as revealed by the SEED system. The reference

Pseudomonas (Pf5 and L10.10) and Janthinobacterium (KBS0711 and PAMC25724) strains are presented on the left side of each graph, followed by the sequenced strains (43NM1, 38PA,

28NM1, 20AA, 19PA and 15PA). Page 74

Figure 4.3. Abundance of encoded-genes associated with stress response in draft genomes of phyllosphere Pseudomonas (a) and Janthinobacterium (b) strains as revealed by the SEED system. The reference Pseudomonas (Pf5 and L10.10) and Janthinobacterium (KBS0711 and

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Tables index

PAMC25724) strains are presented on the left side of each graph, followed by the sequenced strains (43NM1, 38PA, 28NM1, 20AA, 19PA and 15PA). Page 76

Figure 4.4. Phylogenetic analysis of 36 known antifreeze proteins sequences present in different taxa as reported in the Uniprot database (see also Table 1). The amino acid sequences were aligned using Mega 7.0 software (http://www.megasoftware.net/) (Kumar et al. 2016) and the UPGMA tree was constructed using Geneious 8.4.1, and interpreted using FigTree software

(http://tree.bio.ed.ac.uk/software/figtree/). Domain DUF3494 detected by SMART web server is shown in the graph with asterisks (*). The colored branches indicate the corresponding taxa.

Red: fishes, blue: arthropods, green; plants, light blue: fungi, yellow: algae and purple: bacteria.

Page 84

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Tables index:

Chapter 2. Properties and biotechnological applications of ice-binding proteins in bacteria

Table 2.1. Characteristics of phenotypically verified AFPs isolated from bacteria.

Page 16

Table 2.2. Summary of bacterial ice-binding protein applications. Page 20

Table 2.3. Summary of taxonomic affiliation of the phenotypically verified ice-binding proteins

(IBPs) and putative IBPs present in the UniProt database. Page 22

Chapter 3. Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant

Deschampsia antarctica

Table 3.1. Phylogenetic assignment of representative DGGE bands. Page 47

Table 3.2. Bacterial counts (cfu g-1 of leaf), ERIC-PCR genotyping and IRI activity of culturable bacteria isolated from Deschampsia antarctica phyllosphere.

Page 49

Chapter 4. Genome sequencing and search of putative antifreeze proteins in bacteria isolated from Deschampsia antarctica phyllosphere.

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Tables index

Table 4.1. Known antifreeze proteins used in the search for potential AFP coding genes in the draft genomes of phyllospheric strains. Page 68

Table 4.2. Summary of results obtained from the genome sequencing of the phyllospheric strains. Page 70

Table 4.3. Summary of coding-genes associated with PGP mechanisms in draft genomes of phyllospheric strains. Page 82

Supplementary material index

Supplementary material index:

Chapter 4. Genome sequencing and search of putative antifreeze proteins in bacteria isolated from Deschampsia antarctica phyllosphere.

Supplementary figure 4.1. (a) Amino acid sequence of known antifreeze protein from

Pseudomonas putida GR12-2 (accession no. Q68VA9). The region that matches with scaffold number 5 (SC5-38PA) from Pseudomonas brassicacearum 38PA is highlighted in light blue.

(b) Detail of the alignment between Q68VA9 and SC5-38PA. (c) Amino acid sequence of known antifreeze protein from Marinomonas primoryensis (accession no. A1YIY3). The region that matches with scaffold number 68 (SC68-43NM1) from Pseudomonas lini 43NM1 is highlighted in light blue. Ice-binding region is highlighted in yellow (d) Details of the alignment between the A1YIY3 and SC68-43NM1. Page 78

Supplementary Table 4.1. Matches between draft genomes of Pseudomonas brassicacearum

38PA and Pseudomonas lini 43NM1) with known antifreeze proteins from Pseudomonas putida

GR12-2 (Q68VA9) and Marinomonas primoryensis (A1YIY3). Page 79

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Chapter 1

General Introduction

Chapter 1: General introduction

1.1. General introduction

Around the world, frost temperature causes important economic losses in agriculture. In

USA, citrus industry has been devastated on several occasions, resulting in fruit and tree losses and costs estimated in billion dollars (Martsolf et al., 1984). In April 2007, damages produced by frost in USA were estimated in losses of 50% and 87% in peach and blueberry crops, equivalent to USD $65 million with respect to previous years

(Chevalier et al., 2012). Thus, it has been reported that frost events generated more damages than any other weather-related phenomenon occurred in USA (Pearce, 2001;

Snyder and Melo-Abreu, 2005; Purugganan and Fuller, 2009). The negative effects of frost in agriculture not only are related to reduced economic incomes, but also to unemployment and economic-social impasses (Snyder and Melo-Abreu 2005). To our knowledge, in Chilean agriculture there are no researches focused on evaluating strategies to ameliorate the damages by frost in crops, particularly in southern Chile.

However, losses between 22-100% in crops such as blueberries are commonly reported and described as a limitation factor in regional agriculture. Therefore, studies focused on establishing efficient strategies to ameliorate frost damage in Chilean crops are highly required.

Plants are differentially affected by frosts, defined as the occurrence of an air temperature of 0°C or lower, measured at 1-2 m above soil level. Some plant species cannot bear freeze injury being seriously damaged (even lethal), whereas other plant species regulate freezing point depression by the presence of cryoprotectants (trehalose and sucrose) allowing plants to reach lower temperatures in their tissues, this characteristic is known as super-cooling (Anchordoguy et al., 1987; Pearce, 2001).

Nevertheless, at frost temperatures close to cero (between -2 and -6°C), plants can also be affected by the occurrence of bacteria able to produce INP that promote the

Chapter 1: General introduction formation of ice (Lindow et al., 1982). In freezing-sensitive plants, ice nucleation by bacterial activity produces injury in tissues, particularly in the phyllosphere (considered as leaf surface or total above-ground surfaces of plants), facilitating the development of pathogenesis (Cambours et al., 2005). Diverse studies have described frost injury produced by various INP bacterial species (Pseudomonas flourescense, Pseudomonas syringae, Xanthomonas campestris and Pantoea aglomerans) on frost sensitive crop plants such as willows (Salix viminalis), sour cherry (Prunus cerasus), snap bean

(Phaseolus vulgaris) and eucalyptus (Eucalyptus globulus) (Lindow and Brandl, 2003;

Cambours et al., 2005; Lindow, 1983; Sule and Seemuller, 1987; Hirano et al., 1999).

Current methods to prevent frost damages on plants are mostly physical e.g., heaters, wind machines, artificially generated fogs, water sprinkling directly to plants and mechanical protection (plastic thermal blankets-covered cultures). Other methods have attempted to mitigate frost effects by control of INP bacterial populations, and consequently, enhancing the natural ability of plants to supercool (Lindow, 1983).

Bactericides (such as oxytetracycline and streptomycin) have been applied as protectant to prevent the establishment of INP bacteria on plant tissues showing significant frost control on different crops such as corn, beans, potatoes, squash, tomatoes, pear, almond, citrus and avocado (Lindow, 1984). However, the rate, type, application frequency and environmental concerns of bactericides must be previously established. Another method is the use of ice nucleating inhibitors such as Na2CO3 (0,1M), Urea (0,5M) + ZnSO4,

Urea + Na2CO3 (0,1M) and hyamine, which inactivate ice nucleus without necessary bactericidal action, with significant reductions in frost injuries. However, this activity offers a “day before” type of frost prevention.

Antagonistic bacteria have also been applied to reduce the number of INP bacterial strains. Considering that the abundance ice nucleus from bacterial INP are

Chapter 1: General introduction reduced at higher threshold temperatures (-2°C), the occurrence of non-INP bacteria populations could reduce the number of INP ice nucleus. Nevertheless, competitors should also be selected considering the ability to produce antibiotics or the effective colonization of phyllosphere. In this context, Lindow (1987) reported the reduction of frost injury in plants pretreated with non-INP bacteria mutant strains, compared with non-treated plants.

Psychrophilic bacteria are microorganisms adapted to live in extremely cold or freezing temperatures and they could represent an important source to search and develop natural strategies to ameliorate frost injury in plants. Psychrophilic bacteria have developed a wide battery of physiological tools to undergo cold or freezing temperatures such as changes in membrane fluidity, protein synthesis and enzyme kinetics (Hébraud and Potier, 1999; Feller and Gerday, 2003). Psychrophilic bacteria can also avoid ice formation through accumulation of sugars, amino acids or polyhydric alcohols with the consequent depression of freezing point in cellular structures

(colligative properties), preventing protein and membrane denaturation (Kawahara,

2008). In this context, AFP have been considered as an adaptation under extreme cold conditions to tolerate ice formation (Venketesh and Dayananda 2008). AFP bind to ice crystals by adsorption and can act by i) TH and ii) IRI. The first one is a non-colligative effect defined by a difference between the freezing and melting points of a solution

(Raymond and DeVries 1977). In IRI, AFPs prevent the generation of large ice crystals by boundary migration of smaller ice crystals (Yue and Zhang 2009).

The presence of AFP in bacteria isolated from Antarctic lakes and glacial has been previously described (Raymond and Knight, 2003; Gilbert, 2004; Raymond et al.,

2007 and 2008). In addition, Sun et al., (1995), discovered that Pseudomonas putida

GR12-2 isolated from the rhizosphere of plants growing in the Canadian High Arctic,

Chapter 1: General introduction were able to promote root growth at 5°C and survival of spring and winter canola

(Brassica campestris and Brassica napus respectively) exposed to freezing temperatures

(-20 and -50°C).

By the other hand, ice nucleus could be catalyzed by different substrates such as dust particles, clay minerals and also by molecules such as sylver iodine applied in the production of snow. Nevertheless, ice nucleus of biological origin i.e. bacterial INP, are the most efficient ice nucleating agents, even favoring the development of pathogenesis.

In this context, AFPs from fish origin have been proven to inhibit ice nucleus from bacterial origin such as common epiphytic organisms found in the phyllosphere of plants (Parody-Morreale and Murphy, 1988; Du et al., 2003). Thus the inhibition of efficient ice nucleus of bacterial origin and other ice nucleating agents such as dust particles present in the phyllosphere of plants, could reduce the damages generated by frost events and concomitantly reduce the prevalence of pathogenesis related to bacterial ice nucleus.

Despite the phyllosphere of plants is an oligotrophic substrate composed of a cutin layer coating the epidermis, this structure gives to epiphytic organisms a heterogeneous habitat with different structures such as stomatas, trichomes and hidatodes facilitating leaf colonization to a great diversity of bacterial species exposed to important daily and seasonally variations in physical (temperature, light, dehydration, etc.) and nutritional conditions (Lindow and Brandl, 2003; Cambours et al., 2005;

Hirano and Christen, 2000). Considering the ubiquitous presence of INP bacteria around the world and the discovery of bacteria with AFP in different ecosystems exposed to freezing temperatures, bacteria with the ability to produce AFP could be found in the phyllosphere ecosystem. The aim of this work is to characterize the bacterial

Chapter 1: General introduction communities from Deschampsia antarctica phyllosphere and to identify genes encoding for AFP by determining the whole genomes of culturable bacteria with IRI activity.

1.2. Hypothesis

Genomes of bacteria isolated from Deschampsia antarctica phyllosphere showing IRI activity, will have genes encoding for AFP.

1.3. General objective

To characterize the bacterial communities from Deschampsia antarctica phyllosphere and to identify putative genes encoding AFP by determining the whole genomes of culturable bacteria with IRI activity.

1.4. Specific objectives

1.4.1. - To determine the diversity and representative bacterial genera associated to

Deschampsia antarctica phyllosphere.

1.4.2. - To select bacterial strains isolated from Deschampsia antarctica phyllosphere with IRI activity.

1.4.3. - To determine whole bacterial genomes of selected culturable bacteria with IRI activity.

CHAPTER 2

Properties and biotechnological applications of ice-binding proteins in bacteria

FEMS Microbiology Letters 363, 2016

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Abstract

IBPs, such as AFPs and INPs, have been described in diverse cold-adapted organisms, and their potential applications in biotechnology have been recognized in various fields.

Currently, both IBPs are being applied to biotechnological processes, primarily in medicine and the food industry. However, our knowledge regarding the diversity of bacterial IBPs is limited; few studies have purified and characterized AFPs and INPs from bacteria. PV-IBPs have been described in members belonging to

Gammaproteobacteria, Actinobacteria and Flavobacteriia classes, whereas putative IBPs have been found in Gammaproteobacteria, Alphaproteobacteria and Bacilli classes.

Thus, the main goal of this minireview is to summarize the current information on bacterial IBPs and their application in biotechnology, emphasizing the potential application in less explored fields such as agriculture. Investigations have suggested the use of INP-producing bacteria antagonists and AFP-producing bacteria (or their AFPs) as a very attractive strategy to prevent frost damages in crops. UniProt database analyses of reported PV-IBPs and putative IBPs also show the limited information available on bacterial IBPs and indicate that major studies are required.

Keywords: Antarctic bacteria; antifreeze proteins; ice-nucleating proteins; ice-binding proteins; psychrophilic bacteria.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

2.1. Introduction

Cold environments have opened a new window in bioprospecting, which is the discovery and commercialization of new products (such as enzymes and bioactive compounds) from biological resources (Vester et al. 2015). Two of the most amazing examples are psychrophilic and psychrotolerant bacteria. Psychrophilic bacteria are defined as bacteria with an optimal growth temperature at≤15°C, while psychrotolerant bacteria are defined as bacteria with optimal growth at>15°C, but that can tolerate and survive at lower temperatures (Helmke and Weyland 1995). In nature, finding bacteria that are able to survive and proliferate at low temperatures is not surprising given that

71% of the earth’s surface is covered by oceans (with an average temperature from 2°C to 4°C below 1000 m depth), of that 15% is polar regions, glaciers and alpine mountains and permafrost (Feller 2013).

Bacteria from cold environments are characterized by long term survival at low temperatures with low water and nutrient availability (Vishnivetskaya et al. 2006); these bacteria have been reported in a wide variety of habitats, such as Antarctic subglacial lakes (D’Elia et al. 2008), Antarctic glacial ices (Bidle et al. 2007) and the deep sea (Xu et al. 1998). However, bacteria that have adapted to cold environments have been scarcely explored, and their roles in nature have primarily been associated with the dissolution and oxidation of minerals in sediments beneath ice masses, and with the degradation of recalcitrant soil organic matter in permafrost (Pautler et al. 2010).

IBPs such as AFPs have been found in organisms that survive and proliferate at cold temperatures. AFPs act by binding to ice crystals causing (i) TH and (ii) IRI (Lorv et al. 2014). TH is a non-colligative effect defined by a difference between the freezing and melting points of a solution (Raymond and DeVries 1977). In IRI, AFPs prevent the

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria generation of large ice crystals by boundary migration of smaller ice crystals (Yue and

Zhang 2009). Another type of IBPs is INPs, which induces ice formation at high subzero temperatures (Lindow et al. 1982). Water freezing is a stochastic process that is determined by the temperature and orientation of water molecules. Spontaneous or homogeneous ice nucleation in pure water occurs at a temperature of−38.5°C, and bacterial INPs can act as water molecule organizers that promote ice nucleation at higher temperatures (−2°C to−10°C) (Lee et al. 1995).

IBPs are highly attractive for biotechnologists as they can be applied in various fields, mainly in food industry (Fletcher et al. 1997; Kontogiorgos et al. 2007; Yeh et al.

2009; Zhang et al. 2010) and in medicine (Koushafar and Rubinsky 1997; Muldrew et al. 2001; Amir et al. 2003; Hirano et al. 2008; Lee et al. 2012). For example, AFPs are used in ice-cream production to keep their quality (Warren et al. 1992; Feeney and Yeh

1998) and INPs are used for the production of artificial snow (Cochet and Widehem

2000). Nevertheless, few IBPs from bacteria have been described to date, and their applications have only been demonstrated at the laboratory level. In the light of these facts, a better understanding of current and future biotechnological applications of bacterial IBPs is required. Thus, this minireview summarizes the current information on bacterial IBPs (both AFPs and INPs) and their application in biotechnology, with an emphasis on the potential application in less explored fields such as agriculture.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

2.2. Antifreeze proteins

AFPs were first identified in Antarctic teleost fish (DeVries and Wohlschlag 1969).

However, AFPs are not limited to fish and have subsequently been found in a great variety of living organisms from cold environments, such as plants (Bravo and Griffith

2005; Middleton et al. 2009), insects (Graether and Sykes 2004; Kristiansen et al. 2011) and microorganisms such as fungi (Hoshino et al. 2003; Xiao et al. 2010) microalgae

(Janech et al. 2006; Raymond et al. 2009), yeast (Lee et al. 2010a) and archaea

(Saunders et al. 2003).

Based on differential ice-binding patterns, AFPs have been classified into two primary categories: moderately active and hyperactive. The difference between moderately active and hyperactive AFPs is still being actively researched. A main driving force for their difference may be in their selectivity for different faces of ice, where the hyperactive AFPs bind to the prims and planes basal of ice and block growth along its c-axis (Pertaya et al. 2008). Indirect observation in a mutational study on an insect protein (Tenebrio molitor AFP, TmAFP) supports this mechanism, where the modification of the residues from threonine to valine resulted in lower activity of AFP, possibly due to a lower binding affinity of the protein for the basal plane of ice (Liou et al. 2000).

Moderately active AFPs bind only to the prism plane, leaving the basal plane uncovered (Scotter et al. 2006). In addition, extensive ice surface coverage by hyperactive AFPs increases TH activity up to 6°C in the meal worm T. molitor at submillimolar concentrations (Graham et al. 1997). In comparison, moderately active

AFPs, commonly found in fish, are characterized by a lower TH of 0.5°C–1.0°C at millimolar protein concentrations (Hanada et al. 2014).

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

2.3. Bacterial AFPs

Studies describing the isolation, purification and characterization of AFPs produced by environmental bacteria are scarce. As shown in Table 2.1, the first study that discovered

AFPs in bacteria was performed by Duman and Olsen (1993) in Micrococcus cryophilus and Rhodococcus erythropolis. However, these AFPs remain uncharacterized. The second study, by Sun et al. (1995), presented a glycoprotein AFP from Pseudomonas putida GR12-2 isolated from the rhizosphere of plants grown in the

Canadian Arctic. These authors reported an apparent molecular weight of∼34 kDa with a low TH of∼0.1°C. Subsequently, Xu et al. (1998) characterized this AFP as an extracellular protein with an apparent molecular weight of 164 kDa. They have also described that aggregates of this AFP display ice nucleation activity, which favors extracellular ice nucleation, followed by the stabilization effect of IRI. Both activities are suggested to maintain ice crystals at a small harmless size.

A hyperactive AFP was reported in the marine bacterium Marinomonas primoryensis, (MpAFP), with a molecular weight >1000 kDa, and was shown to be a calcium-dependent protein with a TH of 2°C at a concentration of 0.5 mg mL−1 (Gilbert et al. 2005; Garnham et al. 2008) (Table 2.1). The X-ray crystallographic structure of

MpAFP is shown in Figure 2.1 (Garnham et al. 2011). The side chain atoms of threonine and asparagine residues in the AFP contribute to the binding of water molecules to the ice facets. The flatness of the ice-binding face, another likely important property in the interaction between this protein and its ligand, is reminiscent of two insect AFP structures (Graether et al. 2000; Liou et al. 2000). The MpAFP structure also revealed that the internal Ca2+ atoms are coordinated by the side chain carboxyl groups from aspartate residues and the backbone carbonyl groups from glycine residues down one side of the protein. Removal of the Ca2+ results in a loss of antifreeze protein

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria activity, showing that these cations are essential for maintaining the correct AFP structure. Recently, MpAFP was described as an outer membrane anchored protein, serving to bind bacteria to ice (Guo et al. 2012).

Raymond et al. (2007) isolated an extracellular protein from Colwellia sp.

(ColAFP), with a molecular weight of 25 kDa with a TH<0.1°C. However, studies with recombinant ColAFP characterized this protein as a member of the β-helix hyperactive

AFP family, with no repetitive sequence motif and a TH of∼4°C at a concentration of

0.14 mM (Hanada et al. 2014). The differences in TH reported in these studies could be principally attributed to a lower protein concentration that was not given by Raymond et al. (2007). Recently, ColAFP has also been shown to have a sequence similar to the

AFP (54 kDa) that is produced by a Flavobacteriaceae bacterium isolated from subglacial Vostok Lake at a 3519 m depth (Raymond et al. 2008). This finding suggests that AFP genes might be transferred between microorganisms by horizontal gene transfer events (Raymond et al. 2008; Sorhannus 2011; Raymond and Kim 2012). A lipoprotein with a similar molecular weight (52 kDa) was also discovered in Moraxella sp. by Yamashita et al. (2002). Other extracellular and intracellular AFPs that have been reported were produced by P. fluorescens and Flavobacterium xanthum, with TH values of 0.059°C and 0.04°C, respectively (Kawahara et al. 2004; Kawahara et al. 2007).

Recently, a 25.7 kDa AFP produced by F. frigoris PS1 (FfIBP), and firstly reported by

Raymond and Kim (2012), showed a TH of 2.2°C at a concentration of 0.005 mM (Do et al. 2014). Thus, the TH values given in these investigations are dependent on the concentrations and purity of recombinant protein assays and may have no physiological meaning regarding their activity in nature. At natural concentrations, bacterial IBPs have negligible TH and thus are probably not true AFPs. Their purpose may be to just modify the growth of ice to preserve water channels or liquid pockets to create a

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Figure 2.1. Structure of MpAFP from Marinomonas primoyensis based on Garnham et al.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria habitat for bacterial growth (Raymond et al. 2008; Raymond and Kim 2012; Davies

2016).

2.4. Ice-nucleating proteins

The molecules that act as ice-nucleating agents are diverse in nature (such as dust and pollen) and have temperature ranges for ice nucleus formation between −8°C and −15°C

(Margaritis and Bassi 1991). Despite that INPs have been found in insects (Duman

2001), fungi (Tsumuki et al. 1992) and plants (Brush et al. 1994) that live in cold environments. Nevertheless, most INP studies have been performed in bacteria which are active ice nuclei at temperature ranges between 0°C and −10°C (Cochet and

Widehem 2000). In this context, Schmid et al. (1997) provided a classification method used to describe subpopulations of culturable bacteria; this classification is based on the heterogeneous threshold temperatures of INP activity. The most effective bacterial INPs are termed type I, and can initiate ice formation from 0°C to −4°C. Type II INPs initiate ice formation from −5°C to −7°C, whereas type III INPs initiate ice formation from

−8°C to−10°C. The basis for these subpopulations of INPs is their monomer, trimer and oligomer aggregation states (Burke and Lindow 1990). This assumption was demonstrated through radiation inactivation of these proteins, which showed that 150 kDa is the minimum molecular mass needed to nucleate ice at temperatures in the range from −12°C to −13°C (Govindarajan and Lindow 1988).

2.5. Bacterial INPs

In bacteria, the INP phenotype (known as Ina+) is encoded by a structural gene that expresses a membrane-bound protein. This INP is located at the outer membrane of

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Table 2.1. Characteristics of PV-AFPs isolated from bacteria.

Molecular weight Reported Name (kDa) TH (°C) concentration Isolated from Reference Uncharacterized n.d. 0.35 n.d. Micrococcus cryophilus Duman and Olsen, protein 1993 Uncharacterized n.d. 0.29 n.d. Rhodococcus Duman and Olsen, protein erythropolis 1993 Glycoprotein ~34 ~0.11 n.d. Pseudomonas putida Sun et al., 1995 GR12-2 Glycoprotein 164 n.d n.d. Pseudomonas putida Xu et al., 1998 GR12-2 Lipoprotein 52 ~0.171 0.1 mg mL-1 Moraxella sp. Yamashita et al., 2002 Uncharacterized 80 0.059 n.d. Pseudomonas Kawahara et al., 2004 protein fluorescens KUAF-68 Ca2+dependent n.d. ~0.8 11 mg mL-1 Marinomonas Gilbert et al., 2005 protein (MpAFP) primoryensis Recombinant >1000 2 0.5 mg mL-1 Marinomonas Garnham et al., 2008 (MpAFP) primoryensis Adhesin ice 1500 n.d. n.d. Marinomonas Guo et al., 2012 binding activity primoryensis (MpAFP) β-helix protein ~25 <0.1 n.d. Colwellia sp. SLW05 Raymond et al., 2007 (ColAFP) Recombinant β- n.d. ~4 0.14 mM Colwellia sp. SLW05 Hanada et al., 2014 helix protein (ColAFP) Malate-dependent 59 0.04 0.7 mg mL-1 Flavobacterium Kawahara et al., 2007 protein xanthum IAM12026 Ice binding 54 n.d n.d Flavobacteriaceae Raymond et al., 2008 protein Ice binding ~26 2.2 0.005 mM Flavobacterium frigoris Do et al., 2014 proteins (FfIBP) PS1

TH: thermal hysteresis; n.d.: not described.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria bacteria and catalyzes ice formation at high subzero temperatures (Lindow 1987).

Wolber et al. (1986) cloned the InaZ gene responsible for ice nucleation in

Pseudomonas syringae S203 and described its primary structure as an octapeptide repeat sequence; the Ala-Gly-Tyr-Gly-Ser-ThrLeu-Thr motif is repeated along a 1200 amino acid sequence. Two other orders of periodicity were detected, following a 16 residue repeat and another repeat of 48 residues found in two regions of the sequence.

The regular repeating pattern present in the protein sequence is likely responsible for organizing water molecules by influencing their spatial orientation and by favoring molecule alignment for water crystallization (Gurian-Sherman and Lindow 1992;

Zachariassen and Kristiansen 2000). Xanthomonas campestris, which causes considerable damage in plants due to its ice nucleation activity (Cambours et al. 2005), secretes an INP encoded by the InaX gene and characterized by 153 octapeptide units repeated in the sequence (Zhao and Orser 1990). Erwinia uredovora carries the InaU gene in its genome, which encodes a 1034 amino acid protein with a central repetitive domain of 16 amino acids (Michigami et al. 1994). Similarly, E. herbicola (now named

Pantoea agglomerans) (Lindow et al. 1978; Gavini et al. 1989) carries the IceE gene in its genome and secretes a protein with a sequence of 1258 amino acids, with similar codon usage to that found in the InaZ gene from P. syringae S203 and the InaW gene from P. fluorescens MS1650 (Warren and Corotto 1989).

2.6. Biotechnological applications

Many industrial and biotechnological processes use cold-active biomolecules from organisms adapted to cold environments (Cavicchioli et al. 2011). IBPs, such as AFPs and their recombinants, have been primarily applied in diverse fields such as food industry and medicine (Amir et al. 2003; Kontogiorgos et al. 2007; Hirano et al. 2008)

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

(Table 2.2). Recombinant AFPs from fish are used in the industry to improve food preservation during freezing (Fletcher et al. 1997; Yeh et al. 2009). Recombinant AFPs from the yeast Leucosporidium sp. have been used for cryopreservation of red blood cells (Lee et al. 2012b). In cryosurgery, the production of needle-like ice crystals at high

AFP concentration (≥5mg ml−1) favors the ablation of cells (Koushafar and Rubinsky

1997). Only fish AFPs have been successfully used in the ablation of subcutaneous rat tumor cell lines; however, despite that those studies have been done at laboratory level, a great application potential for the medical industry is predicted (Koushafar and

Rubinsky 1997; Muldrew et al. 2001).

INPs have also been applied in various fields (Table 2.2), particularly those bacterial INPs with activity at the warmest temperature (type I) (Gurian-Sherman and

Lindow 1992). Several food technology studies have reported favorable results when using bacterial INPs to increase the ice nucleation temperature, with consequently reduced freezing times and ice-crystal size and improved frozen solid food quality

(Zhang et al. 2010b). As the consequence of ice formation at higher temperatures, a lower energy cost is required to freeze foods. Freezing food with less cooling has also been shown to be possible when using biological ice nuclei produced by bacteria (i.e. from P. syringae and E. herbicola). Widehem and Cochet (2003) demonstrated that freezing processes could be improved by the addition of lyophilized cells of P. syringae which could act as ice nuclei, leading to a significant decrease in the super-cooling point, forming a greater number of ice crystals. Notably, INP-producing bacteria have been closely related to the water cycle due to the presence of these organisms in samples collected from rainfall and snow (Morris et al. 2008). In fact, the presence of bacteria in cloud water has revealed that this mechanism of transport has made biological ice nucleation a widespread phenomenon in the atmosphere, affecting

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria meteorological processes such as rainfall and hailstorms (Christner et al. 2008). In addition, one of the most important applications in the industry is the current use of

Snomax (Telemet, Inc, New York, USA), a freeze-dried bacterial INPs for the production of artificial snow (Cochet and Widehem 2000).

Despite that engineering or genetic modifications for enhancing the function of

IBPs are scarce, improvements in recombinant protein production has been achieved by linking a determined protein function to the encoding gene. This technology has increased our ability to modify specific peptides to obtain desired properties in vitro

(Daugherty 2007). As an example, protein engineering, by modification of certain residues in isoforms of AFPs, resulted in a gain of antifreeze activity as described by

(Garnham et al. 2012).

2.7. Perspectives in agriculture

Frost damage is responsible for more economic losses in agriculture than any other weather-related phenomenon in the United States, and in many other regions across the globe (Pearce 2001; Chevalier et al. 2012). Sessile organisms such as plants have primarily adopted IRI mechanisms to tolerate cold temperatures (Thomashow 1998;

Lorv et al. 2014). Studies have revealed that freeze-tolerant plants such as winter rye

(Secale cereale) and ryegrass (Lolium perenne) secrete AFPs into intercellular spaces and the apoplast, affecting the freezing point and ice crystal growth (Griffith et al. 1992;

Middleton et al. 2012). Freeze tolerant plants (L. perenne and others) accumulate fructans as carbohydrate source that are also considered to play a relevant role in freezing-tolerance (Valluru and Van Den Ende 2008; Valluru et al. 2008). In contrast, other plant species regulate freezing point depression by the presence of cryoprotectants

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Table 2.2. Summary of bacterial ice-binding protein applications.

Organism source Level of Protein Application field (protein type) Main effect application Reference AFP Food industry Fish (Type III) Applied as a recombinant protein Industrial Fletcher et al., 1997; to improve milk fermentation to Feeney and Yeh, 1998 store frozen yogurt and ice-cream production Food industry Plant (IRI) Screened for proteolytic and Laboratory Kontogiorgos et al., lipolytic activity to obtain a heat- 2007 stable IRI protein Food industry Fish (Type I) Applied as recombinant protein to Laboratory Yeh et al., 2009 improve frozen meat and dough quality Medicine Fish (Type I and Applied to improve the Laboratory Amir et al., 2003 III) cryopreservation of rat hearts Medicine Fish (Type III) Applied as natural and Laboratory Hirano et al., 2008 recombinant protein to improve cryopreservation of mammalian cells Medicine Yeast Applied as a recombinant protein Laboratory Lee et al., 2012 to improve the cryopreservation of red blood cells Medicine Fish (Type I) Applied in cryosurgery of Laboratory Koushafar and subcutaneous rat tumor cell lines Rubinsky, 1997; Muldrew et al., 2001 Agriculture Fish (AFGP) Inhibition of ice nucleating Laboratory Parody-Morreale and activity of membrane vesicles of Murphy, 1988 Erwinia herbicola bacterium Agriculture Bacteria Applied as biofertilizer to Laboratory Sun et al., 1995; increase plant growth at cold temperature Agriculture Fish (Type III) Inhibition of ice nucleation Laboratory Du et al., 2003 process by adsorbing to ice nuclei and dust particles. Agriculture Plant Recombinant Lollium perenne Laboratory Tomalty and Walker, (Lp (AFP)) reduced ice nucleation 2014 temperature of bacterial ice nucleating proteins (INP) INP Food industry Bacteria Applied as active bacterial cells to Laboratory Zhang et al., 2010 improve frozen food quality by reducing ice crystal size. Food industry and Bacteria Freezing processes improved by Laboratory Widehem and Cochet, wastewater adding lyophilized P. syringae 2003 treatment cells, decreasing super-cooling point and higher ice crystals size Agriculture Bacteria Inoculation of non-INP bacteria Laboratory Lindow, 1984; will reduce the frequency of Lindow, 1987 biological ice nucleus and damages in plants Agriculture Bacteria Applied as active bacterial cells to Laboratory Lee et al., 2001; control insect pest in plants Castrillo et al., 2001 Environmental Bacteria Applied as active bacterial cells to Theoretical Levin et al., 1986; science promote artificial rainfall Joly et al., 2013 Sport/entertainment Bacteria Applied as active bacterial cells Industrial Cochet and Widehem, for artificial snow production 2000 (Snow-max: Genecor, Rochester, USA)

AFP: antifreeze protein; INP: ice-nucleating protein; IRI: ice recrystallization inhibition; AFGP:

antifreeze glycoprotein.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

(trehalose and sucrose) that allow the plants super-cooling, reaching lower temperatures in tissues without ice formation (Kawahara, 2008). Studies in blueberry (Vaccinium sp.) also have shown that controlled ice nucleation is a beneficial mechanism for avoiding supercooling in the protoplasm, inducing extracellular freezing and/or accommodating ice crystal in specific tissues (stem bark) (Kishimoto et al. 2014).

However, frost sensitive plants can be affected by the occurrence of INP-producing bacteria, which induce ice formation, causing damages and dehydration in plant cells

(Lindow et al. 1982; Cambours et al. 2005).

Current methods to prevent frost damage in agroecosystems are primarily physical, i.e. heaters, wind machines, artificially generated fog and water sprinkling directly on plants. However, these methods are costly. A different method to mitigate frost damage effects is the control of INP-producing bacteria populations to enhance the natural ability of plants to super-cool (Lindow 1983b). In this context, bactericides

(such as oxytetracycline and streptomycin) have been applied as protectants to prevent the establishment of INP-producing bacteria in plants (Lindow 1984). Nevertheless, the use of antibiotics is undesirable in foodstuffs given that the remaining antibiotics could affect intestinal flora, generate allergic reactions, and also facilitate bacterial resistance to antibiotics (Jing et al. 2009). Thus, the inoculation of antagonist bacteria against INP- producing bacteria has also been proposed to diminish the frequency of biological ice nucleation damage on plants as shown by Lindow (1984, 1987) (Table 2.2). Moreover, as also shown in Table 2.2, active bacterial INPs have shown to be effective at pest control by reducing the capacity of potato beetles to resist freezing temperatures

(Castrillo et al. 2001; Lee et al. 2001). In the environmental sciences, INP-producing bacteria have also been proposed as a cloud condensation inducer to produce artificial

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Table 2.3. Summary of taxonomic affiliation of the PV-IBPs and putative IBPs present in the

UniProt database.

Antifreeze proteins Ice-nucleating proteins Class Branch 1 Branch 2 Branch 3 Branch 1 Branch 2 Branch 3 Alphaproteobacteria Rhizobium Bradyrizobium Rhodobacter Ensifer

Betaproteobacteria Herbaspirillum

Gammaproteobacteria Marinomonas* Colwellia Xanthomonas Pantoea Pseudomonas Pseudomonas Pseudomonas Pseudomonas Xanthomonas Serratia

Bacilli Paenibacillus Bacillus Bacillus

Flavobacteriia Flavobacterium

∗Bold letters denote the presence of PV-IBP activity in strain within bacterial genus.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria rainfall (Levin et al. 1986; Joly et al. 2013). Clearly, major efforts will be required to refine and implement these applications before it becomes effective and economically viable for use in the field.

Another mechanism that has been scarcely explored is the use of plant growth- promoting bacteria. The only example reported in the literature is the inoculation of P. putida GR12-2 isolated from the rhizosphere of Canadian high Arctic plants, which secretes an extracellular AFP (Sun et al. 1995). The inoculation of seeds of spring and winter canola with P. Putida GR12-2 resulted in an increase in root elongation at 5°C.

Bacteria, as epiphytic organisms living on the phyllosphere of plants, have also been found to colonize intercellular and apoplast spaces in leaves (Lindow and Brandl 2003), thus the inoculation of AFP producing epiphytic bacteria could be applied to reduce frost damages in sensitive plants (Table 2.2). This possibility is supported by the finding that AFPs isolated from fish have been experimentally demonstrated to inhibit the ice- nucleation activity of an INP-producing E. herbicola, a common organism inhabiting the phyllosphere of plants (Parody-Morreale et al. 1988). Similar results were found using type III AFP, which inhibit ice nucleation process by adsorbing onto both the surface of growing ice nuclei and dust particles (Du et al. 2003). On the other hand, a recent article demonstrated that a plant AFP could also control ice growth, and act as a defensive strategy against bacterial ice nucleation (Tomalty and Walker 2014). Recent advances in metagenome analyses have also suggested that the survival of Antarctic moss is achieved by the accumulation of bacterial AFPs activity (Raymond 2015). This occurs because homologs of the same domain (DUF3494) were identified in bacterial gene sequences discovered in metagenome analysis of the moss leaves (Davies 2016).

The same domain (DUF3494) was reported to be transmitted by horizontal gene transfer between prokaryotic organisms such as ColAFP and Flavobacteriaceae bacterium

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Figure 2.2. Phylogenetic analysis of PV-AFPs (red) and putative AFPs (black). Amino acid sequences were taken from the UniProt database (http://www.uniprot.org/) and aligned using

Clustal Omega (http://www.clustal.org/omega/). In brackets, protein accession numbers were included. The neighbor joining phylogenetic tree was built using the PHYLIP package

(http://evolution.genetics.washington.edu/phylip.html), and interpreted using FigTree software

(http://tree.bio.ed.ac.uk/software/figtree/). Marinomonas primoryensis (A1Y1Y2), Colwellia sp.

SLW05 (A5XB26), Flavobacterium frigoris PS1 (H7FWB6), Flavobacteriaceae bacterium

3519-10 (B3GGB1), Pseudomonas putida GR12-2 (Q68VA9), P. syringae SM (S3MTC9),

Pseudomonas sp. UW4 (K9NL46), Pseudomonas sp. Chol1 (K5YJR0), Serratia fonticola AU-

AP2C (U2NA57), S. fonticola AU-P3 (U2LCA8), Xanthomonas campestris pv. campestris

(A0A0C7DZ94), X. campestris pv. campestris ATCC33913 (Q8P6Q0), X. campestris pv. raphani 756C (G0CBK7), Rhizobium fredii HH103 (G9A7B7), Bradyrhizobium diazoefficiens

SEMIA 5080 (A0A099IPF9), Ensifer adhaerens (Sinorhizobium adhaerens) OV14 (W8HVP5),

Herbaspirillum frisingense GSF30 (R0G033), H. seropedicae SmR1 (D8IRJ0), Herbaspirillum

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria sp. Gw103 (I3CTQ5), Bacillus sp. OxB-1 (A0A0A8JKC3), Paenibacillus alvei DSM29

(K4ZQG5), Paenibacillus alvei DTS-15 (S9SIL7), Paenibacillus sp. JDR-2 (C6D7M4),

Paenibacillus sp. R7-277 (W4DV37).

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria and eukaryotic microorganisms such as fungi and copepods (Hanada et al. 2014). The high expressions of AFP domains by eukaryotic microbial communities suggest an essential role for this protein in survival in Arctic and Antarctic sea ice (Uhlig et al.

2015).

In summary, in the context of significant economic losses produced by frost events in agriculture, the potential application of IBPs-producing bacteria or their IBPs to diminish damage by ice-crystal formation and promote growth at seasonal cold temperatures, presents an attractive strategy that should be explored in further research.

2.8. IBPs in public databases

Sequence databases are currently widely used in bioprospecting biotechnological and industrial attractive microorganisms (Lee and Lee 2013). The current UniProt database suggests that bacterial IBPs might be found in diverse bacterial groups (Table 2.3). In this context, database-based analyses can also be used to detect conserved regions in genes or proteins, which are very useful for the detection of unknown (or novel) microbial genes and proteins from environmental bacteria and predicting their function by molecular approaches. However, very limited information on IBPs is present in databases. Few PV-AFPs and INPs in bacteria have been deposited in public databases, and most of IBP-like proteins are noted as ‘putative’, but their activities have not been confirmed. Figure 2.2 shows a phylogenetic analysis based on amino acid sequences from reported bacterial PV-AFPs and representative AFP-like proteins from plant and soil associated bacteria. The phylogenetic tree suggests clustering of three major branches. Branch 1 grouped the AFPs from M. primoryensis (MpAFP; A1Y1Y2) and

P. putida GR12-2 (Q68VA9) with AFPs-like proteins from Gammaproteobacteria

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Figure 2.3. Phylogenetic analysis of representative bacterial INPs (red) and INP-like proteins

PHYLIP package (http://evolution.genetics.washington.edu/phylip.html) and interpreted using

FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Pseudomonas syringae (K4WYP7),

Pseudomonas savastanoi pv. savastanoi (G7ZJR9), Pseudomonas syringae SM (S3MD81),

Pseudomonas syringae (D2U789), Pseudomonas mandelii JR-1 (A0A024E7B8), Pseudomonas sp. GM30 (W6VKH0), Pseudomonas syringae pv. syringae B728a (Q4ZW16), Pantoea ananatis LMG 20103 (D4GJ13), Xanthomonas gardneri ATCC 19865 (F0C4Z6), Xanthomonas campestris pv. raphani 756C (G0CIB0), Xanthomonas campestris pv. campestris ATCC 33913

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

(Q8PD38), Rhodobacter capsulatus ATCC BAA-309 (D5AV30), Rhodobacter capsulatus

SB1003 (O68078), Bacillus thuringensis MC28 (K0FS92), Pantoea ananas InaA (P20469),

Pseudomonas syringae pv. syringae InaZ (P06620), Pantoea ananas InaU (Q47879),

Xanthomonas campestris InaX, (P18127), Pseudomonas fluorescens InaW (P09815),

Pseudomonas syringae InaK (O30611), Pseudomonas syringae InaQ (B0FXJ3), Pantoea agglomerans iceE (P16239), Pseudomonas syringae InaV (O33479).

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

(Xanthomonas and Pseudomonas) and Alphaproteobacteria (Ensifer and Rhizobium) classes. The second branch grouped members of the Bacilli class (Paenibacillus and

Bacillus), but no reported AFP was present in this cluster. The third branch was subdivided into two minor branches. One of the branches grouped AFPs from F. frigoris PS1 (FfIBP; H7FWB6) and Colwellia SLW05 (ColAFP; A5XB26) with AFPs- like from Alphaproteobacteria (Bradyrhizobium) and Gammaproteobacteria

(Pseudomonas), whereas the other minor branch grouped Flavobacteriaceae bacterium

3519-10 (B3GGB1) with AFPs-like proteins from Betaproteobacteria (Herbaspirillum) and Gammaproteobacteria (Serratia and Pseudomonas) classes.

Figure 2.3 shows the phylogenetic relation of reported bacterial INPs and representative INP-like proteins from plant and soil-associated bacteria. The phylogenetic tree suggests clustering of three major branches. Branch 1 was formed by a reported INP from X. campestris (InaX; P18127) and INP-like proteins from

Xanthomonas spp. The second branch was subdivided into two minor branches. One of the branches grouped INPs from Pantoea ananas (InaA [P20469] and InaU [Q47879]) and P. agglomerans (iceE; P16239) strains with INP-like proteins from Pantoea.

Another minor branch grouped the INP from P. fluorescens (InaW; P09815) with INP- like proteins from Alphaproteobacteria (Rhodobacter), Gammaproteobacteria

(Pseudomonas) and Bacilli (Bacillus) classes. Branch 3 grouped the INPs from P. syringae (InaZ [P06620]), InaK [O30611], InaQ [B0FXJ3] and InaV [O33479]) with

INP-like proteins from P. syringae. Notably, two INP-like proteins from P. savastanoi and P. syringae were not grouped with the other P. syringae sequences.

In addition, when MEME software (http://meme.nbcr.net/meme/; Bailey et al.

(2006) is used to discover novel motifs (recurring, fixed-length patterns) in amino acid

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Figure 2.4. Motifs in bacterial PV-INPs as represented by the motif discovery tool MEME

(http://meme.nbcr.net/meme/). Black bars indicate the octapeptide repeat sequence. The UniProt accession numbers are given in parentheses.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria sequences from reported bacterial INPs, the results suggest three motifs which were all based on the repetitive octapeptide region (Ala-Gly-TyrGly-Ser-Thr-Leu-Thr) described previously by (Wolber et al. 1986) (Figure 2.4). The octapeptide region also contains a

Thr-X-Thr motif (where ‘X’ is any amino acid), which has been found to be responsible for ice-binding by AFPs from insects (Graether et al. 2000). This finding has led to the suggestion that both AFPs and INPs can have similar structures yet different effects on ice (Graether and Jia 2001). It is clear that the first motif identified by the MEME tool is very similar to those previously described, while the other motifs were not particularly well conserved and have not been previously recognized in INPs. Therefore, these results must be taken with caution, because in fact represented motifs may simply be produced by tandemly-repeated amino acids. Homology models can also be generated using protein database files (i.e. Swiss-Model and PDB) and AFPredictor (Doxey et al.

2006). This analysis can identify AFPs from a large set of structures with greater accuracy than sequence alone. However, when MEME and AFPredictor were used with reported bacterial AFPs, reliable results were not obtained (data not shown). This implies that current database is too small to motifs may simply be produced by tandemly-repeated amino acids.

Homology models can also be generated using protein database files (i.e. Swiss-

Model and PDB) and AFPredictor (Doxey et al. 2006). This analysis can identify AFPs from a large set of structures with greater accuracy than sequence alone. However, when MEME and AFPredictor were used with reported bacterial AFPs, reliable results were not obtained (data not shown). This implies that current database is too small to provide adequate coverage of IBPs present in the bacterial world.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

2.9. Conclusions and future directions

The importance of ice-binding proteins as potential biotechnological tools has been recognized in various fields. However, only a limited amount of bacterial antifreeze proteins and ice-nucleating proteins have been studied and characterized.

Phenotypically verified ice-binding proteins, both antifreeze and ice-nucleating proteins, have been described in members of the taxa Gammaproteobacteria (Pseudomonas,

Pantoea, Xanthomonas, Marinomonas, Colwellia and Moraxella), Actinobacteria

(Micrococcus and Rhodococcus) and Flavobacteriia (Flavobacterium); whereas antifreeze-like proteins from plant- and soil-associated bacteria have also been reported in Gammaproteobacteria (Pseudomonas, Xanthomonas and Serratia) and other groups such as Alphaproteobacteria (Rhodobacter, Bradyrhizobium, Rhizobium and Ensifer),

Betaproteobacteria (Herbaspirillum) and Bacilli (Paenibacillus and Bacillus). However, our knowledge regarding the roles, mechanisms and factors that regulate their activity at cold temperatures in nature is very limited. In terms of biotechnological applications, both ice-binding proteins are being investigated and applied primarily in medicine and the food industry. Ice-nucleating protein producing bacteria have been associated with frost injury and pathogenicity in agriculture, with concomitant economic losses. Thus, the potential use of Ice-nucleating protein producing bacteria antagonists and antifreeze protein producing bacteria (or their antifreeze proteins) as biotechnological tools in agriculture to prevent frost damage in crops is a very attractive strategy. However, the current database is clearly limited, and major efforts are required to improve our knowledge regarding ice-binding proteins present in bacterial world and their application in biotechnology.

Chapter 2.- Properties and biotechnological applications of ice-binding proteins in bacteria

Acknowledgements:

F. P. Cid thanks the doctor scholarships of Antarctic Chilean Institute (code DT 01-13) and Conicyt (no. 21140534).

Funding:

This minireview was financed by FONDECYT project no. 1120505 and International

Cooperation project Conicyt-USA code USA2013-0010

CHAPTER 3.

“Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica”

Polar Biology 40, 2016

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica Abstract

Despite the recognized interest in Antarctic bacteria, the relationship between bacteria and Antarctic plants has scarcely been studied. Studies have demonstrated that bacteria in the phyllosphere may contribute to plant growth, but their role in native plants such as Antarctic vascular plants living in hostile environments, is still unknown. Here we explore the bacterial community structure associated with the phyllosphere of

Deschampsia antarctica, and evaluate the presence of ice recrystallization inhibition

(IRI) activity in crude protein extracts from phyllosphere culturable bacteria.

Denaturing gradient gel electrophoresis analysis (16S rRNA genes) showed significant differences in the total bacterial community of eight sampled plants; however, members of Pseudomonadales (Pseudomonas and Psychrobacter) and Rhizobiales

(Agrobacterium and Aurantimonas) orders were dominant in all of the analyzed samples. The use of ERIC-PCR technique also revealed a high (>76%) genetic diversity in 265 isolates from the phyllosphere. With respect to IRI activity, 32 isolates (21%) showed IRI activity in crude protein extracts from cold-acclimated bacterial cultures, and 5 isolates (3%) showed IRI activity in crude protein extracts from non-acclimated cultures.

Keywords: Antifreeze proteins; Antarctic bacteria; ice-binding proteins; Deschampsia antarctica; phyllosphere; ice recrystallization inhibition

The phyllosphere is defined as the aerial part of plant leaves circumscribed by the epidermal cell wall (Hirano and Christen 2000; Borges and Lopes 2008). The phyllosphere is governed by diverse biotic (plant species, plant phenological stages, etc.) and abiotic factors (UV radiation, temperature, dehydration, humidity, etc.), which can radically change within hours, days, and even seasons thus, the phyllosphere has been categorized as an extreme environment itself (Yang et al. 2001; Lindow and

Brandl 2003). The microorganisms that colonize this habitat are known as epiphytes, and bacteria are considered to be the dominant microbial group (Bulgarelli et al. 2013).

In this context, diverse approaches (phospholipid fatty acid, denaturing gradient gel electrophoresis [DGGE], community level physiological profile, etc.) have reported the occurrence of Firmicutes (Bacillus) and Proteobacteria (Pseudomonas) phyla as the dominant bacterial groups in the phyllosphere of vegetables, such as spinach, celery, rape, broccoli and cauliflower (Zhang et al. 2010a). By using high-throughput DNA sequencing (454-pyrosequencing), members of phyla Proteobacteria (Pseudomonas,

Massilia and Pantoea), Firmicutes (Bacillus), Bacteroidetes (Flavobacterium) and

Actinobacteria (Arthrobacter) were the most represented in the phyllosphere of field- grown lettuce plants (Rastogi et al. 2012). Nevertheless, our knowledge of the role and ecological relationship between epiphytic bacterial populations and their plant host is still very limited since majority of research studies are focused on the rhizosphere, followed by endosphere of plants (Berg et al. 2014). Raja et al (2008) described that an epiphyte of the Methylobacterium strain utilizes methanol produced by cell metabolism of the host plant as an energy source, whereas the plant is favored by phytohormones

(auxins) produced by the strain, which stimulate plant growth. Phyllosphere bacteria have also been found to fulfill nitrogen requirements in host plants that have lost contact

(Brighigna et al. 1992). Cyanobacteria and Gammaproteobacteria have also been suggested to be the most active diazotrophs in the phyllosphere of the tropical rainforest

(Carludovica drudei, Grias cauliflora and Costus laevis) (Fürnkranz et al. 2008).

Currently, Antarctic bacteria are of interest as producers of biotechnological bioactive compounds (enzymes, antibiotics, pigments, etc.) (Rojas et al. 2009); however, bacteria colonizing the phyllosphere of vascular Antarctic plants have not been investigated in detail so far. In this context, ice-binding proteins (IBPs) produced by bacteria living in cold environments could be potentially applied to diverse biotechnological fields, such as medicine (cryopreservation of cells and cryosurgery), food industry (improving of ice-cream and frozen food quality) and agriculture

(inhibition ice nucleation and frost damage in plants) (Cid et al. 2016b). IBPs, such as ice nucleating proteins (INPs) and antifreeze proteins (AFPs), regulate formation and growth of ice crystals (Davies 2014), and have been found in diverse microorganisms that survive and proliferate under freezing temperatures (Kawahara et al. 2004). AFPs act by binding to ice crystals to bring about thermal hysteresis (TH) and ice recrystallization inhibition (IRI). In the first case, TH is a non-colligative effect defined by a difference of the equilibrium freezing and melting points of a solution (Raymond and DeVries 1977), whereas IRI prevents the generation of large ice crystals by boundary migration of smaller ice crystals (Yu et al. 2010). Few studies have purified and characterized AFPs from bacteria. Sun et al. (1995) isolated a Pseudomonas putida

GR12-2 from the rhizosphere of Canadian high Arctic plants, which secretes an extracellular AFP. The inoculation of spring and winter canola with these AFP- producing strains resulted in an increase in root elongation length at 5°C (Sun et al.

1995). Recent studies have identified AFPs secreted by epiphytic bacteria from

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica Antarctic moss (Raymond 2015; Davies 2016). However, AFP-producing bacteria in the phyllosphere and their contribution to adaptation and/or protection of vascular plants against freezing temperatures have not been investigated so far.

In the light of these findings, the objective of this study was to explore the bacterial community structure associated with the phyllosphere of Deschampsia antarctica, and to look for IRI activity in protein crude extracts from culturable bacteria isolated from its phyllosphere.

3.2. Materials and methods

3.2.1. Plant collection

The selection of sampling sites and sample numbers were subject to i) logistics provided by Chilean Antarctic Institute (INACH), ii) abundance of plants, iii) and weather conditions. Eight whole plants were collected during February 2014 in the South

Shetland Islands as follow. The first three plants (Plants 1, 2 and 3) were taken from a rocky area in Gemelos hill (62º11’45.73”S; 58º59’39.95”W) at a height of 40 m above sea level. The plants were separated no more than 2 meters from each other. No more plants were taken from this site given the low amount of plants present in the sampling site. Plants 4, 5 and 6 were taken from Collins glacier (62º10’9.00”S; 58º51’21.06”W) in areas with different characteristics. Plant 4 was taken from a bird breeding area, 15 m above sea level, Plant 5 was taken from an area highly populated by mosses 30 m above sea level, whereas Plant 6 was taken from a rocky place, 45 m above sea level. Only one

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

Figure 3.1. Map of Antarctic Peninsula. In detail, sampling sites of D. antarctica plants (red circles) on South Shetland Islands. Plants 1, 2, and 3 were taken from Gemelos Hill

(62º11’45.73’’S; 58º59’39.95’’W). Plants 4, 5, and 6 were taken from Collins Glacier

(62º10’9.00’’S; 58º51’21.06’’W). Plant 7 was taken from Ardley Peninsula (62º12’36.78’’S;

58º56’59.25’’W). Plant 8 was taken from Hanna Point (62º39’15.64’’S; 60º36’51.82’’W).

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica plant (Plant 7) was taken from Ardley peninsula (62°12'36.78”S; 58°56'59.25”W), 10 m above sea level. No more plants were taken from this sandy area also populated by mosses, given the low amount of plants found in this site. Another plant (Plant 8) was taken from Hanna point (62°39'15.64”S; 60°36'51.82”W). This is an area highly covered by D. antarctica plants, however only one plant was taken given the bad weather conditions when samples were collected. A map showing these sampling sites, was included in Figure 3.1. We would like to emphasize that number of samples and locations were defined in the moments according to available resources (time, plant coverage, etc.) and changing climate conditions.

The plants were transported in coolers and processed at the Laboratory of

Scientific Base ‘Prof. Julio Escudero’ of the Chilean Antarctic Institute (INACH), King

George island.

3.2.2. Bacterial community structure

Bacterial community composition was assessed by DGGE as described by Kawai et al.

(2002). First, 1 g of D. antarctica leaves were cut (aerial parts) gently washed and then vortexed for 10 min in 10 mL of sterile saline solution (0.85% NaCl). Leaves were removed and the recovered liquid was centrifuged at 15,700 × g for 10 min to collect detached bacterial cells. To obtain 1 g. of leaf it was necessary to use almost all of the leaves on a single plant. Therefore, DGGE banding profile was obtained from total

DNA extract from a composited sample of several leaves of every single plant sampled.

Bacterial cells were suspended in 50 µl of sterile distilled water, and this suspension was subsequently frozen in liquid nitrogen and thawed at room temperature three times to break the cells. The samples were then centrifuged at 15,700 × g for 40 min and the supernatant (~40 µL) was used as template DNA in the PCR reaction.

PCR with primer set EUBf933–GC/EUBr1387 as described by Iwamoto et al. (2000).

The conditions for the PCR reaction were as follows: hot-start was performed at 95ºC for 10 min, the annealing was initially set at 65ºC and was then decreased by 0.5ºC every cycle and held at 55ºC for 1 min, followed by extension at 72ºC for 3 min. Ten additional annealing cycles were then carried out at 55ºC followed by denaturation at

94ºC for 1 min and an extension at 72ºC for 3 min. The final extension step was done for 7 min at 72ºC. The DGGE runs were performed using the DCodeTM Universal

Mutation Detection System (Bio-Rad Laboratories Inc., USA). Twenty microliter aliquots of the PCR product were loaded onto a 6% (w/v) polyacrylamide gel with a 40-

65% denaturing gradient (7 M urea and 40% formamide). The electrophoresis was run for 12 h at 100 V. The gel was stained with SYBR Gold (InvitrogenTM, Thermo Fisher

Scientific Inc., USA) for 30 min and photographed on a UV transilluminator (GelDoc-

It®TS2 Imager, UVP). Clustering of DGGE banding profiles using a dendrogram was carried out with Phoretix 1D Pro Gel Analysis Software (TotalLab Ltd., UK; http://totallab.com/). Based on the matrix obtained from the Phoretix 1D analysis, differences between bacterial communities were calculated by similarity profile analysis

(SIMPROF test) with Bray-Curtis similarity index, 5% significance level and <0.1 stress values (Clarke 1993; Clarke et al. 2008), and visualized by non-metric multidimensional scaling (NMDS) analysis using Primer 6 software (Primer-E Ltd.,

UK; http://www.primer-e.com/).

A total of 19 representative bands from DGGE gels were carefully excised, purified, re-amplified by touchdown PCR and run again to avoid the inclusion of more than one band to be sent to Macrogen, Inc. (Korea) for sequencing (Figure 3.2a). The sequences obtained in this study were compared with those present in the GenBank

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica database NCBI by using BLAST tools (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify bacterial groups. More bands were sequenced, however only those having higher similarity % (≥ 90) were included in Table 3.1.

3.2.3. Isolation of culturable bacteria

Isolation of bacteria from D. antarctica phyllosphere was carried out by a plating method using 4 culture media: NM-1 (0.5 g L-1 D-glucose, 0.5 g L-1 polypeptone, 0.5 g

-1 -1 -1 -1 L sodium glutamate, 0.5 g L yeast extract, 0.44 g L KH2PO4, 0.1 g L (NH4)2SO4,

-1 -1 -1 0.1 g L MgSO4·7H2O, 15 g L agar, and 1 mL vitamin solution containing 1 g L nicotinamide, 1 g L-1 thiamine hydrochloride, 0.05 g L-1 biotin, 0.5 g L-1 4- aminobenzoic acid, 0.01 g L-1 vitamin B12, 0.5 g L-1 D-pantothenic acid hemicalcium salt, 0.5 g L-1 pyridoxamine dihydrochloride, 0.5 g L-1 folic acid (Nakamura et al.

1995)), R2A (Oxoid Ltd., Thermo Fisher Scientific Inc., UK), Pseudomonads (Oxoid

Ltd.) and Actinomyces (Oxoid Ltd.) agar medium. One gram of D. antarctica leaves in

45 mL of SSS (0.85% NaCl) was vortexed for 10 min to detach the adhered bacteria.

Then, dilutions of bacterial suspensions were spread on culture media agar plates in triplicate. The plates were incubated at 13°C until colony forming units (cfu) were observed on agar. The value of cfu per cm2 was also calculated by scanning the leaf’s surface area with Image J free software (http://imagej.nih.gov/ij/). After 1 week of incubation, 267 single colonies showing diverse phenotypes (color, brightness, form, elevation and margin; (Smibert and Krieg 1994) were transferred to fresh media, purified by streaking on agar and stored in 3:7 glycerol:LB (10 g l-1 D-glucose, 5 g l-1 yeast extract, 10 g l-1 tryptone, 5 g l-1 NaCl) broth at -20°C for further analysis.

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

The ERIC-PCR technique was used to differentiate colonies with a similar phenotype but a different genotype following the protocol described by Houf et al (2002). Briefly, crude DNA extracts from samples were obtained after boiling bacterial cell suspension followed by a quick spin down to discard cell debris. Supernatant at a concentration of

50 ng DNA µL-1 was used in the PCR mixture. DNA fragments were amplified by PCR using the primer set ERIC motifs 1R (5’-ATG TAA GCT CCT GGG GAT TCA C-3’) and 2 (5’-AAG TAA GTG ACT GGG GTG AGC G-3’). The PCR conditions were as follow: hot-start at 94ºC for 5 min, followed by 40 cycles of denaturing temperature at

94°C for 1 minute, and annealing temperatures at 25°C for 1 minute, and the extension temperature set at 72°C for 2 minutes. The final extension step was set at 72ºC for 7 minutes. PCR products were then run on a 2% agarose gel at 100 V for 1 h and stained with ethidium bromide. Electrophoretic gels were photographed and the images were analyzed with Phoretix 1D Pro Gel Analysis Software. The isolates that showed different banding profiles were considered genetically different and used for further analysis.

3.2.5. IRI detection

The detection of IRI was based on the behavior of ice at subzero temperatures. Ice recrystallization and the consequent ice crystal growth is visually different with a higher transparency, whereas in IRI ice crystals remain at small size which is visually more

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

Figure 3.3. Dendrogram of ERIC-PCR genotyping of culturable bacteria from Deschampsia antarctica phyllosphere. (a) Isolates grown in Pseudomonas agar (PA) plates and (b) grown in

NM1 culture media.

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica turbid (Budke et al. 2009; Gilbert et al. 2004). Given these differences in turbidity, ice recrystallization inhibition effect was measured in this work by a spectrophotometric method. The details of IRI detection theory are given in Figure 3.4a. Absorbance differences were determined using a microtiter plate spectrophotometer (Multiskan GO,

Thermo Fisher Scientific, Inc., USA). First, the most sensitive absorbance to detect IRI under our conditions was determined by wavelength (λ) scanning from 300 to 650 nm using type III AFP (A/F Protein, Inc., USA) and bovine serum albumin (BSA) as positive and negative protein controls respectively. Detection of IRI was assayed with both pure proteins at a final concentration of 0.04 mg mL-1 in a 30% sucrose solution according to the protocol described by Gilbert et al. (2004). The microtiter plate was frozen at -80°C for 15 min and incubated for 2 d at -6°C before reading the absorbance in a spectrophotometer. The microtiter plate wells with 30% sucrose solution were used as blanks. The detection of IRI was assayed with different total protein concentrations ranging from 0.01 to 2 mg mL-1.

3.2.6. Screening for IRI in isolates

Based on our results of IRI detection with different wavelengths and protein concentrations, the screening of IRI detection by spectrophotometry was standardized at

λ of 500 nm using crude extracts with protein concentrations from 0.01 to 2 mg mL-1.

Antarctic bacteria isolates were grown in LB broth at 15°C for 1 week and then cold acclimatized at 4°C for 1 week. The cultures were then centrifuged at 5,000× g for 10 min, and soluble proteins from the pellets were extracted by using B-PER Bacterial

Protein Extraction Reagent (Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s instructions. The protein concentrations in crude extracts were determined using the Bradford Protein Assay (Bio-Rad Laboratories, Inc., USA) and

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

Table 3.1. Phylogenetic assignment of representative DGGE bands.

Band Taxonomic group a Closest relatives or cloned sequences Identity Accession (accession no.) b (%) no. 1 Proteobacteria, Pseudomonas sp. HC7-3, 16S rRNA gene, 98.8 KU645377 Gammaproteobacteria, partial sequence from an Arctic Pseudomonadales cyanobacterial mats (JF313021) 2 Proteobacteria, Pseudomonas sp. TP-Snow-C84, 16S rRNA 97.6 KU645378 Gammaproteobacteria, gene, partial sequence from a culturable Pseudomonadales bacteria from Tibetan Snowpack (KC987014) 3 Proteobacteria, Uncultured endophytic bacterium clone str. 98 KU645379 Alphaproteobacteria, CEA10_5 from Typha angustifolia roots Rhizobiales (HM142813) 4 Proteobacteria, Agrobacterium sp. BE516, partial 16S RNA 99.8 KU645380 Alphaproteobacteria, gene, isolated from the root of Miscanthus Rhizobiales sacchariflorus (JQ764998) 5 Proteobacteria, Pseudomonas migulae strain SHZ1.3, partial 97.3 KU645381 Gammaproteobacteria, 16S rRNA gene, isolated from Arctic lake Pseudomonadales sediments (KP236597) 6 Proteobacteria, Pseudomonas sp. RC7.6, partial 16S rRNA 98.8 KU645382 Gammaproteobacteria, gene, isolated from Caesalpinia spinosa Pseudomonadales rhizosphere (KP267840) 7 Proteobacteria, Pseudomonas sp. St29 DNA complete 100 KU645383 Gammaproteobacteria, genome, isolated from Solanum tuberosum Pseudomonadales rhizosphere. (AP014628) 8 Proteobacteria, Pseudomonas sp. str. UMAB-64, partial 16S 96 KU645384 Gammaproteobacteria, rRNA gene, isolated from Antarctic soil Pseudomonadales (KF263636) 9 Proteobacteria, Pseudomonas sp. HA72, partial 16S rRNA 96.1 KU645385 Gammaproteobacteria, gene, isolated from subarctic Alaska Pseudomonadales grasslands (KF011656) 10 Proteobacteria, Pseudomonas sp. 39A_2, partial 16S rRNA 97.8 KU645386 Gammaproteobacteria, gene, an Antarctic bacteria (KC912628) Pseudomonadales 11 Proteobacteria, Uncultured Pseudomonas sp. clone 91.6 KU645387 Gammaproteobacteria, BBS8w52, partial 16S rRNA gene, obtained Pseudomonadales from beech and spruce litter (AY682157) 12 Proteobacteria, Psychrobacter sp. MVS1-N6, partial 16S 97 KU645388 Gammaproteobacteria, rRNA gene, isolated from Antarctic soil Pseudomonadales (KR023910) 13 Proteobacteria, Psychrobacter sp. SCS3-N7, partial 16S 95.9 KU645389 Gammaproteobacteria, rRNA gene, isolated from Antarctic soil Pseudomonadales (KR023909) 14 Proteobacteria, Pseudomonas sp. NSJ-3, partial 16S rRNA 90.3 KU645390 Gammaproteobacteria, gene, partial sequence from a cotton Pseudomonadales endophytic bacterium (FJ941082) 15 Proteobacteria, Pseudomonas sp. B7E, partial 16S rRNA 97.7 KU645391 Gammaproteobacteria, gene. Bacteria isolated from Antarctica Pseudomonadales (KC433647) 16 Proteobacteria, Pseudomonas frederiksbergensis strain 99.8 KU645392 Gammaproteobacteria, BDR1P1B2, partial 16S rRNA gene isolated Pseudomonadales from Zingiber sp rhizosphere soil (KJ567114) 17 Proteobacteria, Pseudomonas sp. HX32, partial 16S rRNA 95.9 KU645393 Gammaproteobacteria, gene, isolated from alpine grassland soil Pseudomonadales (EF601817) 18 Proteobacteria, Pseudomonas sp. RaSa70e, partial 16S rRNA 99.8 KU645394 Gammaproteobacteria, gene, endophyte of Arabidopsis thaliana Pseudomonadales (LN830927) 19 Proteobacteria, Aurantimonas sp. str. FB13, from Arctic and 93 KU645395 Alphaproteobacteria, Antarctic soil. (AM933504) Rhizobiales

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica IRI detection was carried out as described above. Type III AFP and BSA were used in each microtiter plate reading to verify that IRI detection was working correctly. Crude protein extracts from Escherichia coli JM109 were used as a negative control for IRI screening. E. coli JM109 was chosen because its genome information in GenBank database did not include any genetic traits associated with IBPs. This was also confirmed in our lab, where fresh LB cultures that were exposed to -20°C for 24 h. before and after freezing, serial dilutions (from 10-1 to 10-7) of E. coli JM109 culture were plated on LB agar plates, did not growth. Isolates from D. antarctica phyllosphere were also included in this assay to compare strain growth after freezing exposure.

Samples with statistically higher absorbance (see below) compared to E. coli

JM109 were considered as active isolates for IRI. These IRI active isolates were repeatedly screened to confirm IRI activity, and those isolates which always showed higher absorbance compared with E. coli JM109 were selected for additional assays for

IRI detection without cold acclimation.

3.2.7. Statistical analysis

Absorbance data were analyzed by Dunnet multiple comparisons (Dunnett 1955).

Means and standard errors were calculated in quadruplicate and a difference at p≤0.05 was considered as significant. Statistical analyses were conducted using IBM SPSS 21 software.

3.3. Results

3.3.1. Bacterial community structure

The results obtained from the DGGE analysis and visualized with a dendrogram showed that the bacterial community in D. antarctica phyllosphere was mainly distributed in

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

Table 3.2. Bacterial counts (cfu g-1 of leaf), ERIC-PCR genotyping and ice recrystallization inhibition (IRI) activity of culturable bacteria isolated from Deschampsia antarctica phyllosphere.

Growth media NM1a R2A PA AA Total Colony forming units (cfu×106 6.54±0.75b 6.65±0.77 4.70±0.63 3.57±0.33 g-1 of leaf) Total number of isolates 89 81 49 46 265 Isolates with different genotypes 227 78 (88%c) 74 (91%) 37 (76%) 38 (83%) by ERIC-PCR (85%) IRIc positives after first 23 (29%) n.d. 8 (22%) 1 (3%) 32 (21%) screening IRI positives without cold 2 (3%) n.d. 2 (5%) 1 (3%) 5 (3%) acclimatization

a Culture media: NM1 agar, R2A: Reasoners 2A agar, PA: Pseudomonas agar, and AA:

Actinomyces agar b The values represent the average (n=3) and standard errors c Percentages are relative to the total number of isolates obtained in each culture media. n.d.: Not determined.

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica two clusters (Figure 3.2b). One of the cluster showed higher similarity (>57%) among plants collected in Gemelos hill (Plants nos. 1, 2 and 3) and sharing some bands with

Plant no. 6 collected in Collins glacier, whereas the second cluster with lower similarity

(>40%) was obtained from plants taken from Collins glacier (Plant no. 4), Ardley peninsula (Plant no. 7) and Hanna point (Plant no. 8). The most dissimilar bacterial community structure was observed in Plant no. 5, collected from Collins glacier.

Differences between bacterial communities were also visualized by non-metric multidimensional scaling (NMDS) analysis (Figure 3.2c) where only a 40% of similarity was found among the plants collected from different sampling sites (Plant nos. 1, 2, 3, 6 and 7) and only a 20% similarity was observed in all samples studied.

Despite that, significant differences between bacterial communities were observed among collected plants, sequencing of representative dominant bands on

DGGE gels revealed that most members belonged to the Pseudomonadales order, followed by less abundant bands represented by the Rhizobiales order (Table 3.1).

Coincidently, the closest relative sequences deposited in GenBank database are associated with bacteria from Arctic and Antarctic ecosystems while others were also reported to be related to plant rhizosphere habitats. The sequences obtained in this study were deposited in GenBank database under accession numbers from KU645377 to

KU645395.

3.3.2. Isolation and genotyping of culturable bacteria

As shown in Table 3.2, bacterial counts in the D. antarctica phyllosphere revealed bacterial loads ranging from 3.6×106 to 6.7×106 cfu g-1 of leaf, equivalent to 3.6×104 to

6.7×104 cfu cm-2. A total of 265 isolates were obtained with a high genetic variability

(from 76 to 91%) as revealed by ERIC-PCR genotyping (Table 3.2; Figure 3.3). It is

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

Figure 3.4. Standardization of detection of IRI activity in culturable bacteria of the

Deschampsia antarctica phyllosphere. (a) Background theory, (b) Absorbance at different wavelengths (from 300 to 650 nm), (c and d) Absorbance at 500 nm at different BSA and AFP protein concentrations (from 0.01 to 2.0 mg ml-1). AFP: antifreeze protein in a 30% sucrose solution; BSA: bovine serum albumin in a 30% sucrose solution; Blank: 30% sucrose solution.

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica noteworthy that the same genotypes were found in different plants; however, this occurred in bacteria isolated from plants sampled at the same site, such as Plant nos. 4,

5 and 6, all obtained from Collins glacier (Figure 3.3).

3.3.3. Detection and screening for IRI in isolates

During standardization of the protocol for the detection of IRI activity, our results showed differences in absorbance between the positive (type III AFP) and negative

(BSA) controls at the different tested wavelengths (from 300 to 650 nm) (Figure 3.4b).

These differences in absorbance between controls were observed at 500 nm using different protein concentrations (from 0.01 to 2.0 mg mL-1) (Figure 3.4c). The screening for IRI activity was adjusted at 500 nm given that this was the wavelength in the middle of plateau region as presented in Figure 3.4b.

Compared with crude protein extract from E. coli JM109, the screening for IRI activity showed significantly higher absorbance (p≤0.05) in protein extracts obtained from 32 isolates; and therefore, 21% of isolates were considered as IRI active. The screening was repeated to confirm IRI activity in positive isolates. These data are presented in Figure 3.5a and summarized in Table 3.2. However, when ice IRI activity was screened without cold acclimation, to explore if IRI is induced by low temperature or is a constitutive phenotype of selected isolates, only 5 (3%) of isolates (strains 28

NM1, 43 NM1, 19 PA, 33 PA and 20 AA) showed significantly higher absorbances

(p≤0.05) as compared with E. coli JM109 protein extracts (Figure 3.5b).

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica

Figure 3.5. Ice recrystallization inhibition in the phyllosphere (a) Screening of ice recrystallization inhibition (IRI) activity in crude protein extracts (1 mg ml-1) from bacteria isolated from the Deschampsia antarctica phyllosphere using Pseudomonas and Actinomyces agar plates. (b) Detection of IRI activity in crude protein extracts (0.5 mg ml-1) from bacteria grown without cold temperature acclimatization. Asterisks denote significant (p≤0.05, Dunnet’s test) differences in comparison with crude protein extracts of E. coli JM109 (negative control).

The profiles obtained by DGGE (16S rRNA gene) analysis revealed differences in bacterial communities on D. antarctica phyllosphere, which varied among the different plants and collection sites. Variations of bacterial communities in the phyllosphere between different vegetables (spinach, celery, rape, broccoli and cauliflower) have previously been described by Yang et al (2001). A high variability was found in bacterial community composition in forest, among pinus trees, as described by pyrosequencing (16S rRNA gene) (Redford et al. 2010). A high diversity of bacterial composition was also detected in the phyllosphere of the leaf canopy of a tropical

Atlantic forest (Lambais et al. 2006). One of the more intriguing findings of this study was that the microbial communities varied substantially between different locations, but were more similar between plants at Gemelos hill location, sharing higher similitudes with Plant no. 6 sampled in a rocky place from Collins glacier. Some theories postulate that environment selects their colonizers, this means the local conditions regulate the abundance, composition and activity of their inhabitants (Perfumo and Marchant 2010).

In a controlled greenhouse experiment, spatial distribution of Arabidopsis thaliana plants, resulted to affect the dispersion of bacteria on plants showing similar bacterial community structure in closer plants (Maignien et al. 2014). Our results agree with this statement; however, more studies are required for confirmation. In this sense, considering that the majority of Antarctic continent is covered by ice and less than 1% of land, mostly in the Antarctic Peninsula and coastal areas, are available for plant colonization (Alberdi et al. 2002). Oceanic winds, rain, hailstones and snow could be the most important mechanism for dispersion of microorganism in this area. For example, Santl-Temkiv et al (2013) demonstrated that hailstone samples were dominated mostly by plant-surface bacteria thus, these authors hypothesize that

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica adaptations to life in the phyllosphere favors survival of airborne bacteria, affecting long distance transport and spatial distribution of bacteria on earth. In other ecosystems such as desert, dispersion of bacteria is a natural phenomenon conducted by bacteria attached to dust soil particles, contributing to the diversity of downwind ecosystem

(Yamaguchi et al. 2012). Another mechanisms that act for bacterial transport is the local fauna such as skuas and gulls to which this plant has been usually found to be attached in nests (Gerighausen et al. 2003). In addition, differences in bacterial communities in the phyllosphere could also be dependent on the location and morphology of the leaves, phenological status of plants and the amount of carbon-containing exudates released by leaf plants used by bacteria as a nutrient source (Wilson and Lindow 1994; Yang et al.

2001).

Despite differences in bacterial community composition in the phyllosphere, the sequencing of dominant DGGE bands revealed the presence of members of

Pseudomonadales (Pseudomonas and Psychrobacter) and Rhizobiales (Agrobacterium and Aurantimonas) orders. Pseudomonadales is a large taxonomic bacterial group that is metabolically versatile and collectively exhibits a high diversity of activities such as nutrient cycling, degradation of xenobiotic organic compounds, growth promotion and pathogen protection of plants (Redford et al. 2010; Loeschcke and Thies 2015). By using molecular approaches (DGGE and 454-pyrosequencing), Pseudomonadales have been reported as a dominant inhabitant in the phyllosphere of different plants such as spinach, celery, rape, broccoli, cauliflower, and Arabidopsis thaliana (Bodenhausen et al. 2013). Pseudomonadales are widely described as a common inhabitant of soil; therefore, we cannot discard the soil as a source of phyllosphere colonizers (mentioned above). Rhizobiales is also a metabolically versatile soil bacterial group characterized by the conversion of atmospheric nitrogen into ammonia to the benefit of the host plant.

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica Rhizobiales are commonly found in the phyllosphere of rice (Oryza spp.) and perennial herbaceous plants (Typha angustifolia) by metaproteogenomic analysis and 16S rDNA gene sequencing (Li et al. 2011; Knief et al. 2012; Banik et al. 2015). Both bacterial groups are also frequently isolated from Antarctic and Arctic soils (Goh and Tan 2012;

Park and Kim 2015). It is noteworthy that the sequencing of dominant DGGE bands also revealed the presence of Psychrobacter. Psychrobacter arcticus is a cold-adapted bacteria used as model for psychrophilic “cold shock” proteins which has been proposed as a key protein for life at subzero temperatures (Kuhn 2012).

In samples from D. antarctica phyllosphere, our counts ranged from 3.6×106 to

6.7×106 cfu g-1 of leaf, equivalent to 3.6×104 to 6.7×104 cfu cm-2. The bacterial loads found in D. antarctica phyllosphere seem to be similar to some plants from other climate zones. Variable bacterial loads have been reported for different plant species, such as olive plants (from 1.7×104 to 3.4×105 cfu cm-2; (Ercolani 1991), Mediterranean perennial species (from 1.3×104 to 1.4×107 cfu g-1; (Yadav et al. 2004)), evergreen subtropical forest trees (from 7.41×102 to 3.02×105 cfu g−1; (Yadav et al. 2013), and common beans (from 106 to 107 cfu cm−2; (Hirano and Upper 1989). And other authors have suggested that differences in bacterial counts could be explained mostly by the distance of leaves to the soil, as a direct source of epiphytic bacteria, and environmental factors, such as relative humidity, especially in water-saturated soil conditions (Kinkel et al. 2000).

Similar to DGGE analysis, ERIC-PCR genotyping also suggests a high genetic variability (from 76 to 91%) among the culturable bacteria from D. antarctica. In addition, this result helped us to differentiate colonies with a similar phenotype but a different genotype. Interestingly, the same genotypes determined by ERIC-PCR were found in plants sampled in the same location, but not among plants from different

Chapter 3.- Bacterial community structures and ice recrystallization inhibition activity of bacteria isolated from the phyllosphere of the Antarctic vascular plant Deschampsia antarctica sampling sites. The later result could be explained by the fact that vegetative reproduction is the most frequent and reliable means of dispersion in D. antarctica plants (Smith 2003). Other authors have postulated that the occurrence of bacteria with the same genotype in different plants could be attributed to migration of bacteria from neighboring plants and/or plant debris transported by wind, insects and other animal sources (Whipps et al. 2008; Vokou et al. 2012). In addition, numerous phyllosphere bacteria have been associated with seeds with the ability to grow on seedlings and leaves as the plant growths (Hirano and Christen 2000), thus seeds could also be considered vectors for specific bacterial genotype transference in this type of ecosystem; however this statement has not been evaluated in extreme environments such as the Antarctica.

With regards to screening for IRI activity, a total of 32 (21%) crude protein extracts from phyllosphere isolates showed a significantly higher absorbance compared with protein extract from E. coli JM109. Ice recrystallization inhibition has been shown to be a functional and cost-effective mechanism for preliminary detection of AFP activity within a solution (Capicciotti et al. 2013). Moreover, the percentage obtained of active IRI isolates (21%) was higher than that previously described by Gilbert et al.

(2004) (10%) for putative AFP-producing isolates from an Antarctic lake. However, the presence of IRI activity in culturable bacteria do not means that those are the dominant group in the bacterial community structures on the phyllosphere of D. antarctica, as well as the activity does not assure that bacterial IRI activity is expressed on leaf surfaces. In addition, as reported in other studies, the IRI effect has been mostly related to freezing tolerance as it maintains ice crystals at a small, likely harmless size (Do et al.

2014). A different theory points out that proteins secreted into the surrounding environment have an IRI activity that favors the formation of water channels (water

Raymond and Kim 2012). In this context, it has been proposed that the repeated occurrence of DUF3494 sequence (coding a protein of unknown function) in the genome of psychrophilic organisms (bacteria, archaea and eukaryotes) is correlated with

IRI activity, and that it can be transferred between prokaryotes and eukaryote by horizontal gene transfer (Raymond 2014). The discovery of epiphytic bacteria coding

DUF3494 domain in metagenomic analysis conducted in Antarctic non-vascular plants

(mosses) suggests that this activity might favor a commensal relationship between the moss and epiphytic bacteria, where the latter receives sustenance and the former freezing protection, nevertheless this statement has not been empirically proven

(Raymond 2015).

Detection of IRI activity in crude protein extracts from bacteria grown without cold acclimatization was also observed (Figure 3.5b). The constitutive expression of

AFP by bacteria could provide an immediate benefit during sudden harsh local conditions, such as freezing; however, constitutive protein expression represents a permanent metabolic cost for cells as discussed by Geisel (2011).

Finally, further studies are required to elucidate factors influencing the diversity and abundance of bacteria in the phyllosphere of Antarctic vascular plants and their activity under freezing temperature, such as the production of ice-binding proteins. This information could be pivotal for our understanding on Antarctic ecosystems and the physiological response of Antarctic vascular plants to tolerate freezing temperature and climate change (Cavieres et al. 2016).

We thank the contribution of reviewers that improved the quality of this work. This study was financed by International Cooperation project Conicyt-USA code USA2013-

0010. F.P. Cid thanks the Antarctic Chilean Institute for a Doctor Scholarship (code

DT_01-13) and for the permits given by them to collect plants in Antarctic protected areas n° 125, 126 and 150, Conicyt for a Doctor Scholarship (no. 21140534) and La

Frontera University for a Scholarship.

CHAPTER 4

“Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere”

Extremophiles 22, 2018

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere Abstract

Genome analyses are being used to characterize PGP bacteria living in different plant compartiments. In this context, we have recently isolated bacteria from the phyllosphere of an Antarctic plant (Deschampsia antarctica) showing IRI, an activity related to the presence of AFPs. In this study, the draft genomes of six phyllospheric bacteria showing

IRI activity were sequenced and annotated according to their functional gene categories.

Genome sizes ranged from 5.6 to 6.3 Mbp, and based on sequence analysis of the 16S rRNA genes, five strains were identified as Pseudomonas and one as

Janthinobacterium. Interestingly, most strains showed genes associated with PGP traits, such as nutrient uptake (ammonia assimilation, nitrogen fixing, phosphatases, and organic acid production), bioactive metabolites (indole acetic acid and 1- aminocyclopropane-1-carboxylate deaminase), and antimicrobial compounds (hydrogen cyanide and pyoverdine). In relation with IRI activity, a search of putative AFPs using current bioinformatics tools was also carried out. Despite that genes associated with reported AFPs were not found in these genomes, genes connected to ice-nucleation proteins (InaA) were found in all Pseudomonas strains, but not in the

Janthinobacterium strain.

Keywords: Antarctic bacteria; Antifreeze proteins; Ice recrystallization inhibition;

Deschampsia antarctica; Phyllosphere; Plant growth-promoting bacteria.

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere 4.1. Introduction

The plant microbiome is composed of innumerable bacterial taxa; it has been reported that some genera contribute to the stress tolerance and growth of plants, suppress diseases by phytopathogens, or degrade xenobiotic compounds in the soil (Berg et al.

2014; El Amrani et al. 2015). Bacteria are able to colonize different plant compartments, such as the rhizosphere (the soil portion influenced by plant roots), endosphere (the plant inner tissues), and phyllosphere [total above-ground portions of plants that can be further subdivided into the caulosphere (stems), phylloplane (leaves), anthosphere (flowers), and carposphere (fruits)] (Bringel and Couée 2015). In this context, studies have suggested that phyllospheric bacteria may play a pivotal role in protecting plants against diseases by conferring benefits to their plant hosts (Berlec

2012; Rastogi et al. 2013). In the phyllosphere, Methylobacterium use plant methanol (a by-product of cell wall metabolism) as a carbon source, giving back beneficial auxin- like phytohormone to the plant host (Raja et al. 2008; Kwak et al. 2014). Leaf epiphytes, such as cyanobacteria and Gammaproteobacteria, provide N to the host trees in rainforest ecosystems (Fürnkranz et al. 2008). Similarly, in plants that have lost contact with the ground, such as the perennial flowering plant Tillandsia

(Bromeliaceae), phyllospheric bacteria have also been found to fulfill N requirements of their host (Brighigna et al. 1992). However, our insights into the diversity and functionality of phyllospheric bacteria have been very limited, particularly in natural vegetation.

The Antarctic continent is a pristine and extreme ecosystem where organisms have adapted to colonize, survive, and proliferate under locally harsh conditions such as high dehydration rates and low temperatures. Despite this, Antarctic bacteria are of great interest as a source of biotechnological and bioactive compounds (e.g., enzymes,

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere antibiotics, and pigments), yet few studies have focused on unravelling the microbiome of Antarctic plants. Two vascular plants are found in Chilean Antarctic territories,

Deschampsia antarctica and Colobanthus quitensis. The 16S ribosomal RNA (rRNA) gene-based molecular approaches have revealed the presence of Proteobacteria (e.g.,

Pseudomonadales, Rhizobiales, Xanthomonadales, and Campylobacterales orders),

Actinobacteria (e.g., Micrococcales, Actinomycetales, and Bifidobacteriales orders),

Bacteroidetes (e.g., Sphingobacteriales), and Firmicutes (e.g., Clostridiales and Bacilli) phyla as predominant taxa in their rhizosphere soils (Jorquera et al. 2016; Teixeira et al.

2010; Verma et al. 2016). A recent study from our group reported the presence of

Pseudomonadales and Rhizobiales orders in the phyllosphere of D. antarctica (Cid et al.

2016a). However, the functional contribution of bacteria to the adaptation or survival of

Antarctic vascular plants remains unknown. In this sense, an AFP producing strain

(Pseudomonas putida GR12-2, isolated from the rhizosphere of Canadian high Arctic plants), has been shown to promote root elongation of both spring and winter canola grown at 5 °C (Sun et al. 1995). A recent study has suggested that bacterial genes encoding for the domain DUF3494 are homologs of AFP, and, therefore, may be involved in the survival of Antarctic mosses at freezing temperatures (Raymond 2015;

Davies 2016). Study on psychrotolerant bacteria and their contribution to promote the growth, adaptation or fitness of plants are very scarce, particularly in the phyllosphere.

The phyllosphere of plants is an intrinsically extreme environment with long UV radiation exposure times, rapid changes in temperature among short time periods (day and night), and limited nutrient sources for epiphytes (Lindow and Brandl 2003). These harsh conditions have an even more dramatic effect on plants growing in extreme environments such as the Antarctica. We have recently isolated culturable bacterial strains from D. antarctica phyllosphere that show antifreeze activity as measured by the

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere ice recrystallization inhibition (IRI) assay (Cid et al. 2016a). Bacterial IBP, both AFPs and INPs, are of great interest in various biotechnological fields (mainly medicine and food technology), making AFPs a very attractive strategy to prevent frost damage in crops (Cid et al. 2016b), but our insight into the diversity of bacterial AFPs is very limited. AFPs have been found in a variety of organisms that live in cold environments

(fishes, plants, insects, fungi, diatoms, bacteria, among others) (Cid et al. 2016b; Lee et al. 2010b). Some AFPs can be classified as families due to sequence conservation; however, no consensus sequences, motifs, or structures have been found that covers all

AFP families, and clearly greater efforts are needed to unravel the diversity of AFPs in nature. The main goals of this study were to analyze the information contained in the genomes of the selected bacterial strains isolated from D. antarctica phyllosphere.

4.2. Materials and methods

4.2.1. Bacterial strains and DNA extraction

Culturable bacteria showing IRI activity (strains 15PA, 19PA, 20AA, 38PA, 28NM1, and 43NM1) isolated from D. antarctica phyllosphere by Cid et al. (2016a) were used in this study. Bacterial cells were routinely grown in Lysogeny Broth (LB) or NM1 culture media (Nakamura et al. 1995) for 48 h at 20 °C with gently shaking (100 rpm), and then collected by centrifugation at 2300×g for 5 min. Genomic DNA was extracted using the UltraClean Microbial DNA Isolation Kit (Mo-Bio Laboratories, Inc., USA) in accordance with the manufacturer’s instructions. Quantities of extracted DNA were measured with a standard DNA quantification curve using the Quant-iT™ PicoGreen dsDNA Assay kit (Thermo Fisher Scientific, Inc., USA), and the quality was assessed by measuring the A260/A280 absorbance ratio in a microtiter plate spectrophotometer

(Multiskan GO UV/Vis, Thermo Fisher Scientific, Inc., USA).

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere 4.2.2. Genome sequencing, assembly, and taxonomic affiliation

Genome sequencing was performed using Nextera XT DNA Library Preparation Kit and Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) with paired-end strategy 2 × 300-bp reads as described by Ohnishi et al. (2014). The quality of the reads was checked and trimmed (Phred quality score > 15) with Trimmomatic version 0.3.2

(Bolger et al. 2014) and FASTQ (Andrews 2010) tools. A de novo assembly was carried out using A5-MiSeq pipeline (Coil et al. 2015). In this pipeline, BAsic

Ribosomal RNA Predictor (available at https ://githu b.com/tseem ann/barrn ap) and

HMMER (Wheeler and Eddy 2013) tools were used to predict the location of ribosomal

RNA genes and protein sequence homologs in genomes, respectively. In relation with the taxonomic assignment, identified 16S rRNA genes were compared with those deposited in the Ribosomal Database Project (https ://rdp.cme.msu.edu/) and by the

Basic Local Alignment Search Tool (BLAST; https ://blast .ncbi. nlm.nih.gov/Blast

.cgi) from the NCBI. All sequences were deposited in the Genbank/DDBJ/EMBL database under accession numbers from SRX2422488 to SRX2422493.

4.2.3. Genome analysis

The draft genome of the phyllospheric strains was annotated using the myRAST annotation server toolkit (http://rast.nmpdr .org/) (Brettin et al. 2015) and coding sequences were categorized using the SEED system (http://www.theseed.org), which organizes genes into functional categories (subsystems) based on their taxonomic assignment (Aziz et al. 2008). For comparative analysis, the genome sequences of two mesophilic and two psychrotolerant plant–soil associated strains were downloaded from

NCBI databases and used as reference genomes: (1) Pseudomonas protegens Pf5

(accession no. NC_004129), a well-known commensal bacterium that inhabits the

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere rhizosphere of plants (Paulsen et al. 2005); (2) Pseudomonas sp. L10.10 (accession no.

NZ_CP012676), an Antarctic soil bacterium with a possible PGP role (See-Too et al.

2016); (3) Janthinobacterium sp. KBS0711 (accession no. NZ_LBCO01000000), a bacterium isolated from agricultural soil (Shoemaker et al. 2015); and (4)

Janthinobacterium sp. PAMC 25724 (accession no. CYSS01000000), a bacterium isolated from Alps mountain permafrost (Kim et al. 2012). The selection of reference genomes was mainly based on the relevance of strains as PGP bacteria. In addition, annotated functions associated with some gene involved in representative PGP traits, such as nutrient uptake (fixation and assimilation of N, organic P-hydrolyzing enzymes and K/P-solubilizing by organic acids), phytostimulator (production of phytohormones and ethylene control) and biocontrol [production of siderophores and hydrogen cyanide

(HCN)], were also analyzed with the sequenced genomes.

4.2.4. Ice recrystallization inhibition activity

A search for putative AFPs was carried out to find genes responsible for the IRI activity in the isolated strains. The putative AFP coding genes in the draft genomes were first searched using the TBLASTN tool (https ://blast .ncbi.nlm.nih.gov/Blast .cgi). A total of 36 experimentally verified AFP sequences from different organisms (fishes, arthropods, plants, fungi, algae, and bacteria) were downloaded from the Uniprot database (http://www.unipr ot.org/) (Table 1) and used as the query sequences. All

TBLASTN searches were run with stringent thresholds (E values from 0.1 to 0.0001).

Three CDSs (SC1-38PA, SC5-38PA, and SC68-43NM1) that showed partial alignments with known AFP sequences were considered as potential AFP fragments for further analysis. The nucleotide sequences were translated into amino acid sequences using the

ExPASy Translate tool (https ://www.expas y.org/). The local alignment algorithm tool

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere LALIGN (Huang and Miller 1991; Altschul et al. 1997) from ExPASy

(http://embnet.vital -it.ch/softw are/LALIG N_form.html) was used to obtain the best alignment between the potential AFPs and known AFPs. The amino acid sequences of the potential AFPs were then subjected to a Protein BLAST search (BLASTP; https://blast .ncbi.nlm.nih.gov/Blast .cgi) using the non-redundant protein sequences

(nr) database from December 2016. In addition, a direct search of putative AFP coding genes in draft genomes was also done using the Web CD-Search Tool (https

://www.ncbi.nlm.nih.gov/Struc ture/cdd/wrpsb .cgi) and iAFP-Ense (http://www.jci- bioin fo.cn/iAFP-Ense), which is a recently released, sequence-based ensemble classifier for identifying AFP using pseudo-amino acid compositions (Xiao et al. 2016).

It is noteworthy to mention that iAFP-Ense predictor uses the same database from

Kandaswamy et al. (2011) which is based on 481 putative AFPs, including 221 nonredundant AFP sequences taken from Pfam database (Sonnhammer et al. 1997),

AFP-like proteins obtained through PSI-BLAST search (Altschul et al. 1997), and AFP- like proteins inferred from homology. This database includes non-AFPs with sequence similarity to AFPs, resulting in a decrease in the AFP prediction accuracy and overestimating AFP matches in our data. Therefore, we used our own database based on sequence found in Table 1. The presence of the DUF3494 domain in draft genomes of phyllospheric strains was also analyzed. First, the HMM (Hidden Markov Model)

(Johnson et al. 2010) profile of DUF3494 domain was downloaded from Pfam database

(http://pfam.xfam.org/) and HMMER version 3.1b2 software was used to search for sequence homologues in sequence databases with an E value of 10. In addition, a direct search for the DUF3494 domain in draft genomes was also done using Web CD-Search tool as mentioned above.

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Table 4.1. Known antifreeze proteins used in the search for potential AFP coding genes in the draft genomes of phyllospheric strains

Organism/Order Species name Accession Termed as ‡ Reference Fishes Perciformes Myoxocephalus Scorpius P04367 AFP Type I Kwan et al. 2005 Myoxocephalus aenaeus P20421 AFP Type I Chakrabartty et al. 1988 Hemitripterus americanus P05140 AFP Type II Gronwald et al. 1998 Zoarces americanus P19614 AFP Type III Hew et al. 1988 Zoarces americanus P07457 AFP Type III Li et al. 1985 Lycodichthys dearborni P35753 AFP Type III Li et al. 1985 Pachycara brachycephalum P12100 AFP Type III Cheng and DeVries 1989 Anarhichas lupus P12416 AFP Type III Scott et al. 1988 Lycodes polaris P24028 AFP Type III Sönnichsen et al. 1996 Paralichthys olivaceus Q8JI37 AFP Type IV Lee and Kim 2016 Myoxocephalus P80961 AFP Type IV Zhao et al. 1998 Pleuronectiformes Pleuronectes americanus B1P0S1 AFP Type I Marshall et al. 2004 Pleuronectes americanus P04002 AFP Type I Hew et al. 1986 Limanda ferruginea P09031 AFP Type I Scott et al. 1987 Osmeriformes Osmerus mordax Q01758 AFP Type II Ewart et al. 1992 Arthropods Coleoptera Tenebrio molitor O16119 THP/AFP Graham et al. 1997 Dendroides canadensis Q9NCR2 AFP Andorfer and Duman Rhagium inquisitor E5LR38 AFP Hakim et al. 2013 Collembola Hypogastrura harveyi Q38PT6 6.5 kDa AFP Graham and Davies 2005 Hypogastrura harveyi D7PBP2 15.7 kDa AFP Mok et al. 2010 Lepidoptera Choristoneura fumiferana Q9BJW7 AFP Qin et al. 2007 Calanoida Stephos longipes B8Y5Y6 AFPa Kiko 2010 Stephos longipes B8Y5Y7 AFPb Kiko 2010 Plants Apiales Daucus carota O82438 AFP Meyer et al. 1999 Poales Secale cereale A0A182BS IRI Pihakaski-Maunsbach et Lolium perenne B5T007R3 IRI Middleton et al. 2012 Fungi Leucosporidiales Leucosporidium sp. AY30 C7F6X3 AFP Lee et al. 2012a Thelephorales Typhula ishikariensis Q76CE6 AFP Hoshino et al. 2003 Kriegeriales Glaciozyma antarctica D0EKL2 AFP Hashim et al. 2013 Algae Naviculales Navicula glaciei Q108N3 IBP Janech et al. 2006 Chaetocerotales Chaetoceros neogracile D2DLE1 AFP Gwak et al. 2010 Bacteria Pseudomonadales Pseudomonas putida Q68VA9 AFP Muryoi et al. 2004 Alteromonadales Colwellia sp. SLW0 A5XB26 IBP Raymond et al. 2007 Oceanospirillales Marinomonas primoryensis A1YIY3 AFP Garnham et al. 2011 Flavobacteriales Flavobacteriaceae bacterium B3GGB1 IBP Raymond et al. 2008 Flavobacterium frigoris PS1 H7FWB6 AFP Do et al. 2014 AFP antifreeze protein, THP thermal hysteresis protein, IRIP ice recrystallization inhibition protein, IBP ice-binding protein, a Accession number in UniProt database (http://www.unipr ot.org/).

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere A search for genes connected to INPs was also conducted based on the sequences analyzed by Cid et al. (2016b). This search was carried out by checking the annotated genes downloaded from the myRAST server.

4.3. Results

4.3.1. Genome sequencing and taxonomic assignment

The summary of the genome sequencing and taxonomic assignments is listed in Table

2. The general genome stats suggest that genome sizes range from 5.6 to 6.3 Mbp with

58–63% guanine–cytosine (G + C) content. With respect to the taxonomic affiliation analysis, 5 of 6 strains showed a high sequence identity (99%) to members belonging to the Pseudomonas genus (Pseudomonas frederiksbergensis 19PA, Pseudomonas poae

20AA, Pseudomonas brassicacearum 38PA, Pseudomonas mandelii 28NM1, and

Pseudomonas lini 43NM1), mostly of strains isolated from bulk and rhizosphere soils. It is noteworthy that the strains P. poae 20AA and P. brassicacearum 38PA are closely related to strains described as disease-suppressive and PGP bacteria, respectively. On the other hand, only one strain showed a high sequence identity (99%) with a soil bacterium belonging to the Janthinobacterium genus (Janthinobacterium lividum

15PA).

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere 4.3.2. Genome analyses

The functional categorization and abundance of 27 subsystem-coding genes from draft genomes of the phyllospheric Pseudomonas and Janthinobacterium strains are shown in

Figure 4.1. As revealed by the SEED system, all strains showed similar distribution patterns of genes involved in the different subsystems; however, differences in the abundance of genes were observed. The mesophilic reference strain P. protegens Pf5 presented a higher number of categorized genes in comparison with sequenced phyllospheric strains (Figure 4.1a). In contrast, three sequenced phyllospheric strains

(P. lini 43NM1, P. mandelii 28NM1, and P. poae 20AA) showed a higher number of categorized genes (3883, 4087, and 4210 genes, respectively) in comparison with psychrotolerant Pseudomonas sp. L10.10 (3708 genes). Similarly, the analysis in J. lividum 15PA (~ 3282 genes) revealed a lower number of categorized genes compared with the mesophilic reference strain Janthinobacterium sp. KBS0711 (3467 genes), but higher than the psychrotolerant strain Janthinobacterium sp. PAMC 25724 (2971 genes)

(Figure 4.1b).

In terms of macronutrients metabolism, no striking differences among C metabolism gene abundance were found (Figure 4.2a, b). It is worth noting that, however, the remarkable differences in gene abundance related to N metabolism (Figure

4.2c, d). The lowest number of genes connected to N metabolism was in P. frederiksbergensis 19PA, which were only related to the ammonia assimilation subsystem. Interestingly, four of five phyllospheric strains showed the presence of genes associated with assimilation of nitrate and nitrite. These genes were absent in both reference Pseudomonas strains. In addition, only P. poae 20AA showed genes involved in denitrification. In the Janthinobacterium strains, genes associated with

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Figure 4.1. Abundance and functional categorization of total coding-genes in the draft genomes of phyllospheric Pseudomonas (a) and Janthinobacterium (b) strains as revealed by the SEED system. The reference Pseudomonas (Pf5 and L10.10) and Janthinobacterium (KBS0711and

PAMC25724) strains are shown on the left side of each graph, followed by the sequenced strains (43NM1, 38PA, 28NM1, 20AA, 19PA and 15PA).

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere denitrification were present in reference strains but not in J. lividum 15PA. In relation with phosphorous (P) metabolism, greater differences were observed between the analyzed strains (Figure 4.2e, f). The lowest number of genes related to P metabolism was found in the strains P. frederiksbergensis 19PA and P. brassicacearum 38PA (54 and 51 genes), mostly due to the lack of subsystems connected with phosphonate metabolism. Instead, a higher abundance of genes related to alkylphosphonate utilization was found.

In relation with genes involved in stress response, all strains showed a similar distribution pattern of genes in the different subcategories among all strains (Figure

4.3). In Pseudomonas strains, all sequenced phyllospheric strains showed a higher abundance of genes compared with the Antarctic reference strain Pseudomonas sp.

L10.10 (Figure 4.3a), mostly due to a higher abundance of subsystems involved in oxidative stress and osmotic stress (Figure. 4.3a). Similarly, J. lividum 15PA showed a higher abundance of genes, mostly due to a high abundance of genes connected to oxidative stress, compared with the two Janthinobacterium reference strains (Figure

4.3b). Genes related to the cold shock protein subsystem were found in both sequenced and reference strains, and a slightly higher number of genes were observed in

Pseudomonas compared to the Janthinobacterium strains.

4.3.3. Genes related to PGP traits

The presence of coding genes associated with PGP traits in the draft genomes of phyllospheric strains is shown in Table 3. In all analyzed strains, genes associated with nutrient uptake (N and P) and synthesis of indole acetic acid phytohormones were found. Genes associated with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and pyoverdine production were found in all sequenced Pseudomonas strains,

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Figure 4.2. Abundance of coding-genes associated with carbon (a and b), nitrogen (c and d) and phosphorus (e and f) metabolism in the draft genomes of phyllospheric Pseudomonas (a, c and e) and Janthinobacterium (b, d and f) strains, as revealed by the SEED system. The reference Pseudomonas (Pf5 and L10.10) and Janthinobacterium (KBS0711 and PAMC25724) strains are presented on the left side of each graph, followed by the sequenced strains (43NM1,

38PA, 28NM1, 20AA, 19PA and 15PA).

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere but not in the sequenced Janthinobacterium strain. Similarly, genes involved in the production of malic and gluconic acids were also found in all sequenced Pseudomonas strains but not in the sequenced Janthinobacterium strain. N2 fixation structural gene

(NifA) was only found in P. mandelii 28NM1. It is noteworthy that P. poae 20AA and

P. frederiksbergensis 19PA contained the most PGP traits compared to the references strains P. protegens Pf5 and Pseudomonas sp. L10.10, which have been used as a system to study PGP bacteria that prevent plant diseases and improve plant growth, respectively.

4.3.4. Genes related to ice recrystallization inhibition activity

Analysis of the genome sequences using known AFPs in a TBLASTN search resulted in

14 matches. All of these matches are listed in Supplementary Table 4.1. The matches numbered ‘1-2’ and ‘3-6’ occurred between the known AFP from Pseudomonas putida

GR12-2 (accession no. Q68VA9) and the sequences from P. brassicacearum 38PA in scaffold number 1 (SC1-38PA) and scaffold number 5 (SC5-38PA), respectively

(Supplementary Figure 4.1a and 4.1b). The matches numbered 7-14 occurred between the AFP from Marinomonas primoryensis (accession no. A1YIY3) and sequences from

P. lini 43NM1 in scaffold number 68 (SC68-43NM1) (Supplementary Figure 4.1c and

4.1d). It is noteworthy that the matches are located away from the ice-binding region of the M. primoryensis AFP (Supplementary Figure 4.1c).

The scaffolds SC1-38PA, SC5-38PA, and SC68-43NM1 were selected and used for further AFP analysis. The corresponding sequence was extracted from SC1-38PA, and translating it into their amino acid sequences, BLASTP analysis showed a significant sequence identity (77% identity with 99% coverage) with gene encoding 5′ nucleotidase in Pseudomonas syringae (accession no. WP_032630596). The myRAST

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Figure 4.3. Abundance of encoded-genes associated with stress response in draft genomes of phyllospheric Pseudomonas (a) and Janthinobacterium (b) strains as revealed by the SEED system. The reference Pseudomonas (Pf5 and L10.10) and Janthinobacterium (KBS0711 and

PAMC25724) strains are presented on the left side of each graph, followed by the sequenced strains (43NM1, 38PA, 28NM1, 20AA, 19PA and 15PA).

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere annotation assigned the sequence from SC1-38PA as an alkaline phosphatase (EC

3.1.3.1). With respect to SC5-38PA, BLASTP analysis of the translated amino acid sequence showed similarity (81% identity with 94% coverage) with a hypothetical protein from P. syringae (accession no. WP_050507307) and a lectin from

Pseudomonas asturiensis (67% identity with 94% coverage) accession no.

WP_073171779). Through myRAST annotation, no functional assignment was suggested. The BLASTP results of the amino acid sequence from SC68-43NM1 showed similarity (95% identity with 100% coverage) with a type I secretion system protein (C- terminal target domain) from Pseudomonas prosekii (accession no. SDS25709). The same function was found using myRAST annotation, which assigned this sequence as a type I secretion system (T1SS secreted agglutinin RTX). However, when the three scaffolds selected as putative AFPs were directly analyzed by the iAFP-Ense, these fragments were positively predicted to be AFPs. Finally, none of the draft genomes of phyllosphere strains analyzed had a match with the DUF3494 domain using two different search strategies (HMMER and Web CD-Search tool).

In relation with genes associated with INPs, it is important to mention that all sequenced Pseudomonas strains presented InaA genes. Genes connected to INPs were not found in the Janthinobacterium strain.

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere 4.4. Discussion

The genome sequence analysis revealed the presence of Pseudomonas and

Janthinobacterium strains with genome sizes ranging from 5.6 to 6.3 Mbp and 58–63%

G + C contents. The sizes and G + C contents are similar to those previously reported for Antarctic Pseudomonas and Janthinobacterium strains, such as Pseudomonas sp. strain KG01 (6.3 Mbp; 60.1% G + C) (Pavlov et al. 2015), P. putida strain ATH-43 (5.8

Mbp; 61.5% G + C) (Rodríguez-Rojas et al. 2016), and Janthinobacterium sp. Ant5-2-1

(6.1 Mbp; 62.5% G + C) (Koo et al. 2016). Pseudomonas and Janthinobacterium, among other genera (Agrobacterium, Burkholderia, Clavibacter, Leifsonia, Pantoea,

Xanthomonas, Sphingomonas, Methylobacterium, Bacillus, Massilia, and

Arthrobacter), have been described as typical inhabitants of plant phyllospheres

(Vorholt 2012; Rastogi et al. 2013; Jackson et al. 2013; Perazzolli et al. 2014). In

Antarctic plants, metagenomic studies based on 16S rRNA genes have shown that

Burkholderia (Proteobacteria), Bifidobacterium (Actinobacteria), Arcobacter

(Proteobacteria), and Faecalibacterium (Firmicutes) as dominant genera in the rhizospheres of D. antarctica and C. quitensis (Jorquera et al. 2016). However,

Pseudomonas was the dominant group in the phyllosphere of D. antarctica as revealed by DGGE technique, using also 16S rRNA genes as the target (Cid et al. 2016a). The taxonomic assignment showed that the strains 20AA and 38PA were close relative to the PGP bacteria Pseudomonas poae RE*1-1-14 (Mota et al. 2017) and Pseudomonas brassicacearum NFM421 (Ortet et al. 2011). In addition, putative PGP bacteria associated with the roots of native plants grown in extreme ecosystems have previously been reported by Acuña et al. (2016) and Jorquera et al. (2014), suggesting possibility of their contribution to survival and adaptation of plants under harsh conditions. In relation with the functional subsystems (Figure 4.1a, b), the

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere lower abundance of predicted/assigned genes in Antarctic phyllospheric isolates (4.9–

6.3 Mbp) compared to mesophilic P. protegens Pf5 could be explained by; (1) fewer annotations of genomes from psychrophilic or psychrotolerant microorganisms in the current databases and (2) the genome sizes, where P. protegens Pf5 has a bigger genome (7 Mbp) and was completely determined and annotated (Paulsen et al. 2005).

As for the genes related to macronutrient metabolism (C, N, and P), the differences in the categorized genes would be due to the local environmental conditions (Figure 4.2).

For example, the reference strain P. protegens Pf5 (isolated from the rhizosphere) harbored more genes related to C than the phyllospheric strains (Figure 4.2a, b). The rhizosphere is the plant compartment with the highest microbial activity, because root exudates represent a rapid and easy source of C and energy for the microorganisms

(Bertin et al. 2003; Lagos et al. 2014). In contrast, the phyllosphere is an oligotrophic environment with some “oases” of relatively abundant nutrient contents, such as glandular trichomes, plant cell junctions, etc. (Vorholt 2012; Bringel and Couée 2015).

Intriguingly, the higher abundance of genes associated with N metabolism was found in phyllospheric strains (Figure 4.2c, d). Phyllospheric bacteria have been suggested to be active diazotrophs and fulfill nitrogen requirements for their host (Fürnkranz et al.

2008). Interestingly, genes associated with nitrate/nitrite ammonification and denitrification were found in Pseudomonas strains used in this study. Denitrification and respiratory ammonification are two competing pathways used by bacteria (e.g.,

Shewanella loihica) under low and high carbon-to-nitrogen (C/N) ratios, respectively

(Yoon et al. 2015). Genes connected to ammonia assimilation suggest the ability of bacteria to assimilate N at a lower ammonia concentration (Burkovski 2003). In relation with P metabolism, a higher abundance of genes connected to alkylphosphonate utilization was found. Alkylphosphonates are organophosphorus compounds nowadays

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

Table 4.3. Summary of coding-genes associated with PGP mechanisms in draft genomes of phyllospheric strains.

Strains Pseudomonas Janthinobacterium Pf5† L10.10† 43NM1 28NM1 38PA 20AA 19PA KBS0711† PAMC25724 15PA † Uptake and fixation of Nitrogen Ammonia + + + + + + + + + + assimilation‡ NifA gene ‒ ‒ ‒ + ‒ ‒ ‒ + ‒ ‒

Ethylene control ACC deaminase + ‒ + + + + + ‒ ‒ ‒ activity

Synthesis of IAA phytohormone Indole-3-glycerol + + + + + + + + + + phosphate synthase Anthranilate + + + + + + + + + + phosphoribosyltransfer ase

Biocontrol Production of HCN + + + ‒ ‒ ‒ ‒ ‒ ‒ ‒ Production of + ‒ ‒ ‒ ‒ + ‒ ‒ ‒ ‒ Pyochelin Production of + + + + + + + ‒ ‒ ‒ Pyoverdine

Organic acids Oxalic acid ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ Citric acid ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ Malic acid + + + + + + + + + + Succinic acid ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ ‒ Gluconic acid + + + + + + + + ‒ ‒

Uptake of organic phosphorus Phytase + + ‒ ‒ ‒ + + + ‒ + Acid phosphatase + + ‒ + + + + + + + Alkaline phosphatase + + + + + + + + + + Glucose-1-phosphatase ‒ + ‒ ‒ ‒ + ‒ ‒ ‒ ‒ Phosphoprotein + ‒ ‒ ‒ ‒ + + ‒ ‒ ‒ phosphatase Fructose-1,6- + + + + + + + + + + bisphosphatase †Reference strains.

‡Glutamate dehydrogenase, Glutamate synthase and Glutamine synthetase.

ACC: 1-aminocyclopropane-1-carboxilate; HCN: hydrogen cyanide; IAA: indole acetic acid; +: present: ‒: absent.

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere widely employed in many industrial applications and household products as flame retardant, and also for pest control (Wang et al. 2016; Daughton et al. 1979). Recent studies have pointed to the global occurrence of organophosphorus compound even in remote regions of the world, which is linked to anthropogenic sources associated from the long-range transport of aerosols (Castro Jimenez et al. 2014). Genes connected with stress response showed similar abundance in mesophilic reference strains and the sequenced Antarctic strains (Figure 4.3). However, it is noteworthy that a major abundance of genes was connected to the oxidative stress response. Stress response is essential at low temperatures, since the solubility of gases increases, resulting in a higher concentration of reactive oxygen species and the potential for oxidative damage

(De Maayer et al. 2014). The second most abundant group of genes corresponded to osmotic stress. Solutes concentrations increase outside the cell as the result of ice formation, leading to an osmotic gradient across the cell membrane (Mindock et al.

2001). This effect is counteracted by the synthesis of compatible solutes (Cowan et al.

2014). However, it is necessary to mention that our functional analysis compared few relative reference strains and draft genomes, which might have been lost genes during their sequencing and analysis; therefore, future comparative genomic studies should consider a higher number of reference strains, including other taxa and revised genome drafts. Bacteria harboring PGP traits in plant roots growing in extreme ecosystems have previously been reported, suggesting their possible contribution to survival and adaptation of plants to harsh conditions (Suyal et al. 2014), and their possible application in agriculture as bioinoculant to promote plant growth has also been addressed (Banerjee et al. 2017; Jorquera et al. 2014; Viscardi et al. 2016). In this context, Sun et al. (1995) isolated an AFP-producing strain (P. putida GR12-2) from the root of plants grown in the high Arctic, and assays demonstrated its capability to

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere

(http://tree.bio.ed.ac.uk/software/figtree/). Domain DUF3494 detected by SMART web server is shown in the graph with asterisks (*). The colored branches indicate the corresponding taxa.

Red: fishes, blue: arthropods, green; plants, light blue: fungi, yellow: algae and purple: bacteria.

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere to promote the root elongation of canola plants grown at a low temperature (5 °C).

Recently, an Arthrobacter agilis strain L77, isolated from subglacial lake in Himalayas, exhibit PGP attributes as well as production of exopolysaccharides and antifreeze compounds (Yadav et al. 2015). The genome analysis of this strain revealed its metabolic versatility with candidate genes coding for hydrolytic enzymes and cold shock proteins (Singh et al. 2016). However, the possible contribution of phyllospheric bacteria to improving the growth and/ or mitigating the stress of plants has not been thoroughly studied. Our results showed the presence of genes encoding several PGP traits in the genomes of sequenced strains (Table 4.3). The ACC deaminase, for example, is an enzyme widely studied as way to increase stress tolerance in plants

(Martínez-Viveros et al. 2010; Glick 2015; Hassan et al. 2015). Bacteria with the ability to degrade ACC have been isolated from diverse plants in extreme environments, and their inoculation has resulted in a higher growth of plants exposed to abiotic stress

(Inostroza et al. 2016; Jorquera et al. 2012; Mayak et al. 2014a and b). Genes connected to indole acetic acid (IAA) phytohormone such as indole- 3-glycerol phosphate synthase and anthranilate phosphoribosyl transferase were also found in all sequenced strains

(Table 4.3). Auxins, such as IAA, are by far the most common and best studied phytohormones, and have been shown to increase the surface area and length of roots, thereby providing plants greater access to soil nutrients (Glick 2012; See-Too et al.

2016). The production by bacteria of low-molecular-weight organic acids, such as gluconic and malic acids, has also been widely studied. Their benefit in nutrient uptake by plants from P- and K-insoluble forms has been proven in several studies (Jones

1998; Vyas and Gulati 2009). In addition, genes associated with the production of hydrogen cyanide (HCN) were only found in P. lini 43NM1. The production of HCN has been described in Proteobacteria, and mostly involves the suppression of fungal and

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere bacterial soil borne pathogens (Blumer and Haas 2000). However, variable results with in vivo and in vitro assays have led to contradictory conclusions regarding the effective role of HCN as a plant–pathogen biocontrol (Michelsen and Stougaard 2012; Reetha et al. 2014). Similarly, the presence of genes connected to the production of siderophores in sequenced strains was also observed. Siderophores are low-molecular weight compounds that are produced under iron-limiting conditions to chelate ferric iron

(Fe+3), and serve as vehicles for the transport of this nutrient into bacterial cells

(Buysens et al. 1996). Siderophores have also been associated with the plant–pathogen biocontrol by bacteria in the rhizosphere through iron competition (Haas and Défago

2005; Paulsen et al. 2005). Therefore, the beneficial effect of phyllospheric bacteria on the growth and stress tolerance of Antarctic plants should be considered in future studies as well as their biotechnological application in agriculture and other fields, as was suggested by Cid et al. (2016b). The presence of AFP genes in the sequenced genomes could not be confirmed, despite the presence of IRI activity. However, three partial alignments with known bacterial AFPs were found. The scaffolds SC1-38PA and

SC5-38PA contained sequences that aligned with the known AFP from P. putida GR12-

2 (Supplementary Table 4.1 and Supplementary Figure 4.1), an extracellular protein rich in glycine and alanine containing carbohydrate and lipid moieties, with a molecular mass of 164 ± 15 kDa and an isoelectric point of 5.3 (Xu et al. 1998). Sequences analysis showed also that a hemolysin-like, calcium-binding secretion domain is one of the possible secretion mechanisms for this protein. The scaffold SC68-43NM1 contained sequences that aligned with a known AFP from M. primoryensis, but the region of similarity that was detected is located away from the ice-binding region

(Supplementary Table 4.1 and Supplementary Figure 4.1c). This AFP from M. primoryensis is an exceptionally large multi-domain ice adhesin protein (1.5 MDa) that

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere contains an ice-binding site in a 322-amino acids segment with 13 tandem 19-amino acids repeats (Garnham et al. 2011). When the amino acid sequence of the three selected scalfolds (SC1-38PA, SC5-38PA, and SC68-43NM1) were analyzed by BLASTP and

MyRAST, no direct link with any known AFP was found. In contrast, iAFP-Ense suggested that the amino acid sequence in the selected scaffolds can be classified as part of an antifreeze protein. However, this analysis is only theoretical and highly dependent on the AFP sequence database used. Experimental proof (a recombinant AFP assay) is required to confirm that the IRI activity of our isolates is due to the putative AFP genes we identified. In addition, as shown in Figure 4.4, the existence of few and diverse AFP sequences makes it difficult to search and predict for AFPs through gene analysis.

Therefore, our inability to detect AFPs with high similarity with known AFPs suggests that the current database does not provide adequate coverage of bacterial AFPs, particularly in isolates from pristine environments such as the Antarctic. It is important to highlight that sequences containing the domain DUF3494, present in 12 of the 36 known AFP sequences in this tree, were distributed together in one cluster (shown with asterisks in Figure 4.4). This domain has recently been associated with epiphytic bacteria of an Antarctic moss with antifreeze activity (Raymond 2015; Davies 2016).

The two isoforms (a and b) of the AFP described in the arthropod Stephos longipes were found together in this branch close to AFP sequences from bacteria such as Colwellia sp. These sequences were also closely distributed with AFP from algae such as

Navicula glaciei and Chaetoceros neogracile, and fungi such as Typhula ishikariensis.

The ice-binding site from Typhula ishikariensis AFP (TisAFP), for example, has been proposed to be the flattest portion of an irregular β-helix that lies alongside a α-helix

(Kondo et al. 2012). A similar sequence has been found in Leucosporidinium sp., which also contains the DUF3494 domain (Kondo et al. 2012; Lee et al. 2012a and b). The

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere distribution of DUF3494 in different taxa has been suggested to have occurred by horizontal gene transfer (Kiko 2010). In this sense, it is important to mention that genomes of organisms from less explored habitats such as the phyllosphere of Antarctic plants could contain novel protein sequences, which have not been described and deposited in databases so far (Yang et al. 2001; Martínez-Rosales et al. 2012). With regard to INPs, only sequences related to InaA genes were found in all sequenced

Pseudomonas strains. Intriguingly, InaA genes have only been previously described in

Erwinia ananas, responsible for frost injury in tea plants (Abe et al. 1989), maize

(Miller et al. 2016), and fruits such as melon (Bruton et al. 1991; Kido et al. 2008). The work from Abe et al. (1989) describes InaA proteins as similar but not identical to InaW and InaZ genes reported in Pseudomonas syringae and P. fluorescens, respectively.

However, INP-related genes in Janthinobacterium have not been described so far.

4.5. Conclusions

The sequencing and analysis of genomes provide valuable insights into the ecological role and biotechnological potential of bacteria. In this study, genome sequencing of phyllospheric bacteria with ice recrystallization inhibition activity isolated from vascular Antarctic plant (Deschampsia antarctica) revealed the presence of five

Pseudomonas and one Janthinobacterium strains. The sequencing analysis also revealed the presence of diverse plant growth-promoting traits (nutrient uptake, phytohormone production and pathogen biocontrol) in most of the sequenced strains. With respect to ice recrystallization inhibition activity, despite the detection of partial alignments in the genome of two sequenced strains with known bacterial antifreeze proteins, proof of encoded genes of antifreeze proteins in the genomes has not yet been demonstrated. Our inability to detect antifreeze proteins in sequenced genomes can be attributed to few and

Chapter 4.- Draft genome sequences of bacteria isolated from the Deschampsia antarctica phyllosphere unknown antifreeze proteins in the current public database. Therefore, greater efforts are required to experimentally confirm and characterize the presence of gene involved in ice recrystallization inhibition activity in the genome of phyllospheric bacteria from

D. antarctica. In the same sense, the role of phyllospheric bacteria on the growth, adaptation, and survival of Antarctic plants also needs to be addressed and confirmed in future investigations.

Acknowledgements:

This study was financed with funds from Chilean Antarctic Institute-INACH (code

RT_02_16), The National Fund for Scientific and Technological Development-

FONDECYT (No. 1160302) and The National Commission for Scientific and

Technological Research-CONICYT (Code USA2013-0010 and MEC no. 80140015).

This work was supported by the Ministry of Education, Culture, Sports, Science and

Technology in Japan or Japan Society for the Promotion of Science under Grants-in-Aid for Scientific Research (KAKENHI) (Grant Numbers

16H05830/16H05501/16H01782/16H02767); Kurita Water and Environment

Foundation Grant; Ichiro Kanehara Foundation Scholarship Grant for Research in Basic

Medical Sciences and Medical Care; Senri Life Science Foundation Kishimoto Grant; and Japan Agency for Medical Research and Development (AMED), and also by

National Science and Engineering Research Council of Canada (NSERC). F. Cid thanks to the doctor scholarships awarded by INACH (Code DT_01-13), CONICYT (No.

21140534), MECESUP FRO1204 and La Frontera University.

Chapter 5

General discussion

Chapter 5.- General discussion

5. General discussion

IBPs such as AFPs and INPs have been found in organisms that live at subzero and near-zero temperatures (Davies 2014). AFPs act by binding to ice crystals causing

TH and IRI (Lorv, Rose and Glick2014). TH is a non-colligative effect defined by a difference between the freezing and melting points of a solution (Raymond and

DeVries1977). In IRI, AFPs prevent the generation of large ice crystals by boundary migration of smaller ice crystals (Yuet al.2010). INPs on the contrary, induces ice formation at high subzero temperatures (Lindow, Arny and Upper1982; Lee, Warren and Gusta1995).

As revealed in our review, both IBPs are being applied in biotechnological processes such as medicine and the food industry. For example, recombinant AFPs from fish are used in the industry to improve food preservation during freezing (Fletcher et al.

1997; Yeh, Kao and Peng 2009). Recombinant AFPs from the yeast Leucosporidium sp. have been applied in laboratory level to improve the cryopreservation of red blood cells

(Lee et al. 2012). The production of needle-like ice crystals at high AFP concentration

(≥5mg ml−1), has been also assayed in cryosurgery to favor the ablation of undesirable cells (Koushafar and Rubinsky1997). Only fish AFPs however, have been successfully used in the ablation of subcutaneous rat tumor cell lines at laboratory level (Muldrew et al. 2001). In the industry INPs are used for the production of artificial snow (Cochet and

Widehem2000). In agriculture however, frost sensitive plants can be affected by the occurrence of INP-producing bacteria, which induce ice formation, causing damages and dehydration in plant cells (Lindow, Arny and Upper1982; Cambours et al.2005).

Among the applications in agriculture, the use of INP-producing bacteria antagonists and AFPs-producing bacteria (or their AFPs) have been suggested as a

Chapter 5.- General discussion strategy to prevent frost damages in crops (Cid et al. 2016b). The use of PGP bacteria however, is also addressed here as the most promising mechanism. In experimental assays, the inoculation of seeds with P. putida GR12-2 isolated from the rhizosphere of

Canadian high Arctic plants, resulted in an increase in root elongation in spring and winter canola plantlets grown at 5°C (Sun et al. 1995). In addition, epiphytic bacteria living on the phyllosphere of plants, have also been found to colonize intercellular and apoplast spaces in leaves (Lindow and Brandl 2003), thus the inoculation of AFP- producing epiphytic bacteria could be applied to reduce frost damages in sensitive plants. This possibility is supported by the finding that AFPs isolated from fish have been experimentally demonstrated to inhibit the ice-nucleation activity of an INP- producing E. herbicola, a common organism inhabiting the phyllosphere of plants

(Parody-Morreale et al. 1988). Similar results were found using type III AFP, which inhibited ice nucleation process by adsorbing onto both the surface of growing ice nuclei, and dust particles (Du et al. 2003).

It is widely known that plant-associated microbial communities, in this case inhabitants from the phyllosphere, play different roles related to nutrient cycling, plant health and productivity. Thus, microbial communities, as plant holobionts, have important effects in their host ecology and evolution (Laforest-Lapointe et al. 2017). In this context, the bacterial communities associated with the phyllosphere of

Deschampsia antarctica plant has to be considered a source of novel bacterial IBPs or

PGP bacteria. Bacterial community composition was assessed by DGGE (16S rRNA gene) and the profiles obtained revealed differences in bacterial communities in the D. antarctica phyllosphere among the different plants and collection sites. Variations of bacterial communities in the phyllosphere between different vegetables (spinach, celery, rape, broccoli, and cauliflower) have previously been described by Yang et al. (2001).

Chapter 5.- General discussion

High variability was found in bacterial community composition among Pinus trees, as described by pyrosequencing (16S rRNA gene) (Redford et al.2010). High diversity of bacterial composition was also detected in the phyllosphere of the leaf canopy of a tropical Atlantic forest (Lambais et al.2006). One of the more intriguing findings of this study is that the microbial communities varied substantially between different Antarctic locations. Some theories postulate that environments select their colonizers, meaning that local conditions regulate the abundance, composition, and activity of their inhabitants (Perfumo and Marchant 2010). In a controlled greenhouse experiment, the spatial distribution of Arabidopsis thaliana showed similar bacterial community structure in closer plants (Maignien et al. 2014). Our results agree with this finding; however, more studies are required for confirmation, concluding that oceanic winds, rain, hailstones, and snow could be the most important mechanisms for dispersion of microorganisms in this area.

Detection of IRI by a spectrophotometric method showed 32 isolates (21% of isolates assayed) as positive. IRI activity has been shown to be a functional and cost effective mechanism for the preliminary detection of AFP activity within a solution

(Capicciotti et al. 2013). Moreover, the percentage (21 %) obtained of active IRI isolates in this study was higher than that previously described by Gilbert et al. (2004)

(10 %) for putative AFP-producing isolates from an Antarctic lake. The presence of IRI activity in this bacteria however, does not mean that these are the dominant group in the bacterial community on the phyllosphere of D. antarctica and it does not ensure that bacterial IRI activity is expressed on leaf surfaces. The IRI effect has mostly been related to freezing tolerance, as it maintains ice crystals at a small, likely harmless size

(Do et al. 2014). A different theory points out that proteins secreted into the surrounding environment having IRI activity, favors formation of water channels (water pockets)

Chapter 5.- General discussion through which bacteria could obtain nutrients and divide (Raymond 2011; Raymond and Kim2012). In this thesis work, crude protein extract corresponded to intracellular proteins and proteins bound to cell membrane. This analysis allowed us to conclude also, that this technique could be applied as a rapid and cheap method for testing IRI activity.

After the two complete bacterial genome sequences were published in 1995, the study of bacteria has dramatically changed and the use of third-generation DNA sequencing has made possible to completely sequence a bacterial genome in a few hours

(Land et al. 2015). Sequencing of bacterial genome is currently a standard procedure, and the information from tens of thousands of bacterial genomes and the ability to identify, characterize and compare genome sequences has had a major impact on our views of the bacterial world. Thus, this thesis also focused on the genome sequencing of bacteria showing experimentally IRI activity (Cid et al. (2016a) and isolated from

Deschampsia antarctica phyllosphere, including the search for putative AFP sequences and PGP mechanisms by bioinformatic tools.

The draft genome sequencing data revealed the presence of five Pseudomonas

(strains 19PA, 20AA, 38PA, 28NM1, and 43NM1) and one Janthinobacterium strain

(15PA). Similarities and differences between the occurrence and abundance of genes associated with nutrient metabolisms, stress response, and PGP mechanisms were found by comparing genome sequences with the genome sequences downloaded from NCBI databases of two mesophilic and two psychrotolerant plant-soil associated strains. Some of the most remarkable differences were for example, the lower abundance of genes connected C metabolism in the phyllosphere strains. This difference has been attributed in this work to phyllosphere as an oligotrophic environment with some “oases” of relatively abundant nutrient contents (such as glandular trichomes, plant cell junctions,

Chapter 5.- General discussion etc.) (Vorholt 2012; Bringel and Couée 2015). In the same sense, genes connected to ammonia assimilation, present in high abundance in all phyllosphere strains, suggest the ability of these bacterium to assimilate N at a lower ammonia concentration (Burkovski

2003). Respect to P metabolism, a higher abundance of genes connected to alkylphosphonate utilization were found, which suggests that these bacteria have the ability to obtain P from alternative sources. Alkylphosphonates are organophosphorus compounds nowadays widely employed in many industrial applications and household products as flame retardant and also used for pest control (Wang et al. 2016; Daughton et al. 1979).

All strains also showed encoded-genes associated with PGP mechanisms in their draft genomes (Cid et al. 2018). PGP mechanisms have been described in bacteria to facilitate nutrient acquisition and uptake by plant, provide defense against pathogens and insects and/or modulate plant hormone levels and contribute to ameliorate stress in plants (Glick 2012; Glick 2015). In this sense, all phyllosphere Pseudomonas strains contained genes for ACC deaminase activity. Bacteria with the ability to degrade ACC have been isolated from diverse plants in extreme environments, and their inoculation have resulted in a higher growth of plants exposed to abiotic stresses (Jorquera et al.

2012; Jorquera et al. 2014; Mayak et al. 2014a; Mayak et al. 2014b). All phyllosphere strains also contained genes for the synthesis of phytohormones such as indole acetic acid (IAA) shown experimentally to increase the surface area and length of roots, thereby providing plants a greater access to soil nutrients (Glick 2012; See-Too et al.

2016). Despite the presence of AFP genes could not be confirmed, three partial alignments with known bacterial AFPs were found. These sequences aligned with the

AFP from P. putida GR12-2 Q68VA9 which is a protein secreted to the growth media, rich in glycine and alanine containing carbohydrate and lipid moieties (Xu et al. 1998).

Chapter 5.- General discussion

P. putida GR12-2 was isolated from the rhizosphere of Canadian high Arctic plants and has been shown experimentally to promote root elongation of both spring and winter canola grown at 5°C (Sun et al. 1995).

It is noteworthy that after checking the information on IBPs in databases, it was possible to observe that few phenotypically verified IBP have been deposited in public databases and most of IBP-like proteins are noted as ‘putative’, but their activities have not been confirmed so far. In the review of this thesis, one of conclusions was focused on the scarce information related to IBPs in nature. Therefore, our inability to detect

IBPs in genome sequences of phyllosphere was clearly limited by scarce information of bacterial IBPs in public databases. Thus, major efforts are required to improve our knowledge regarding IBPs present in bacterial world and their application in biotechnology.

Chapter 6

General conclusions and perspectives

Chapter 6.- General conclusions and perspectives

6. General conclusions and perspectives

The potential use of ice nucleation proteins (INPs)-producing bacterial antagonists and antifreezing proteins (AFPs)-producing bacteria (or their AFPs) in different fields was revised, revealing to be a very attractive strategy as biotechnological tools in agriculture to prevent frost damage in crops.

Analysis of the bacterial communites in the phyllosphere of Deschampsia antarctica, showed differences in bacterial communities between Antartic locations, where members of Pseudomonadales and Rhizobiales order were dominant. Ice recrystallization inhibition (IRI) activity detection was standardized and achieved through a spectrophotometer analysis in which a total of 32 (21 %) crude protein extracts from phyllosphere isolates showed significantly higher absorbance compared with the control protein extract from E. coli JM109.

Genome sequencing of culturable bacteria isolated from D. antarctica phyllosphere showing IRI activity revealed the presence of five Pseudomonas and one

Janthinobacterium strain. Functional analysis of genome sequencing also revealed the presence of genes connected to nutrient metabolisms, stress response and plant-growth promoting mechanisms. These genetic traits could be relevant for adaptation and survival of Antarctic plants and also could be exploited for the development of new strategies to promote plant growth and favor plant tolerance against frost events in agriculture.

The low number and high diversity of known AFPs, as well as our inability to detect AFPs in genome sequences from phyllosphere isolates, using modern bioinformatic tools, revealed that the current database is too small to provide adequate coverage of bacterial AFPs, particularly in isolates from pristine environments such as

Chapter 6.- General conclusions and perspectives the Antarctica. Further analysis is required to confirm and characterize the presence of

AFPs encoded in the genome of phyllosphere bacteria showing IRI activity from D. antarctica.

This thesis work is the first approach conducted to analyze the bacterial community that inhabit the phyllosphere of a plant living in an extreme environment such as the Antarctica. And it is also the first attempt to search for putative AFP coding- genes in bacteria isolated from the phyllosphere of D. antarctica plant, as alternative to prevent frost damage in agriculture.

Chapter 7

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