PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: PENESYAN
First name: ANAHIT Other name/s:
Abbreviation for degree as given in the University calendar: PHD
School: BIOTECHNOLOGY AND BIOIVIOLECULAR SCIENCES Faculty: SCIENCE
Title: PRODUCTION OF ANTIMICROBIAL COMPOUNDS BY MARINE EPIBIOTIC BACTERIA
Abstract
The aim of this thesis was to obtain antimicrobiai compounds from marine eukaryote-associated bacteria. The unique environment, present on the surfaces of marine eul^aryotes, creates conditions that promote and favour the production of bioactive compounds, such as antimicrobials, by giving the producers a clear advantage in the competition for nutrients.
This study has demonstrated the abundance of antimicrobial producing bacteria on two marine algae, Delisea pulchra and Ulva australis. Two antimicrobial compounds, violacein and tropodithietic acid (TDA), have been successfully purified and chemically identified from two different bacterial isolates. IVIoreover, the production of multiple bioactive compounds was observed for both these bacteria.
This study also made an attempt to understand the role of the antimicrobial compounds for the producer organisms. Consequently, the effect of violacein on biofilm formation, as well as the possible role of TDA in the defence of both the producer bacterium and the host, have been proposed.
The importance of environmental conditions for the expression of bioactive compounds has also been demonstrated, for example, by showing the necessity of high iron concentrations for the production of bioactives in isolates U156 and D245. Notably, despite the absence of a close phylogenetic relationship between these two isolates, they have shown similar trends in terms of production of bioactive compounds.
For the first time, this work also described the construction and analysis of a large insert DNA library in E. coli using genomic DNA from a collection of cultured isolates with demonstrated antimicrobial activity. This approach proved to be successful and led to a substantial increase in the positive hit rates in the functional screening of the library, and provided invaluable information concerning the genes, potentially involved not only in the biosynthesis, but also in the processes associated with the production of bioactive compounds, such as transport and resistance.
Declaration relating to disposition of project thesis/dissertation
I hereby grant to the University of New South Wales or its agents the right to archive and to mal I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ' Signature/^? Witness '' ' Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional FOR OFFICE USE ONLY Date of co mpleti on of requirements for Award: to Production of antimicrobial compounds by marine epibiotic bacteria Anahit Penesyan A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia 2010 COPYRIGHT STATEMENT 'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968.1 retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ..../.....rt^e^ Date AUTHENTICITY STATEMENT 'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.' Signed Date I^/.RBJAPIA. ORIGINALITY STATEMENT 'I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.' Anahit Penesyan Date ACKNOWLEDGEMENTS I would like to thank my supervisors, Prof. Staffan Kjelleberg and Dr. Suhelen Egan, for the opportunity to join the research team at the Centre for Marine Bio- Innovation. Dear Staffan, I would like to thank you for all the advice and support during my PhD project, and thank you for believing in me. Dear Su, I can't express all my gratitude for all your help and guidance and for the continuous support and encouragement during my PhD. It was always very comforting to know that you are there, in all ups and downs, and are always ready to help. I would also like to thank Dr Tilmann Harder for his help in the chemistry related part of my project. Dear Tilmann, thank you for enthusiasm, it was always a pleasure working with you and it was a great learning experience. I am also grateful to Dr. Torsten Thomas, Dr. Matt Lee, Dr. Carola Holmstrom, Dr. David Schleheck, Dr. Sharon Longford, Dr. Flavia Evans, Cathy Burke, Maria Yung, Jan Tebben for all their help during various stages of my project. Thank you guys, I wouldn't be able to do all I did during my project without you. Many thanks to Kirsty Collard and Adam Abdool who do a great job and ensure everything runs smoothly within the CMB. Thank you for all your help in sorting out all the various forms and documents I have encountered during my project. I am also grateful to Bill O'Sullivan for proof-reading my thesis. I would also like to thank all the people working in various labs within CMB where I also happen to work, to all the people in labs 323, 304, and 315: Anne, Sacha, Melissa, Jo, Francesco, Adrian, and everyone else whom I did not mention - thank you for all the fun and for the great working environment. At the end I would like to thank my family and my parents for all their love and support. Words will not be able to express all that I owe you; this work is dedicated to you! LIST OF PUBLICATIONS Published: •Penesvan A. Kjelleberg S and Egan S (2010). "Development of novel drugs from marine surface associated microorganisms." Marine Drugs (special issue) 8(3): 438-459. *Penesvan A. Marshall-Jones Z, Holmstrom C, Kjelleberg S and Egan S (2009). "Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs: Research article." FEMS Microbiology Ecology 69(1): 113-124. Thomas T, Evans F F, Schleheck D, Mai-Prochnow A, Burke C, Penesvan A, Dalisay D S, Stelzer-Braid S, Saunders N, Johnson J, Ferriera S, Kjelleberg S and Egan S (2008). "Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface- associated life style in the marine environment." PLoS ONE 3(9): e3252. *Matz C, Webb J S, Schupp P J, Phang S Y, Penesvan A. Egan S, Steinberg P and Kjelleberg S (2008). "Marine biofilm bacteria evade eukaryotic prédation by targeted chemical defense." PLoS ONE 3(7): e2744. Penesvan A R, Antonyan A P, Vardapetyan H R. (2004). "Interaction of purified hypericin preparations with DNA." Scientific Publications of the Yerevan State University i: 80 -85. Penesvan A R (2004). "Content of podophyllotoxin in different plant species grown in Armenia." lAELS Bulletin 9(8): 56 - 60. Penesvan A R, Vardapetyan H R, Tiratsuyan S G, Hovhannisyan A A and Kabasakalyan E E (2004). " Interactions between podophyllotoxin and DNA." lAELS Bulletin 9 (3): 168-171 Vardapetyan H R, Kirakosyan A B, Hovhannisyan A A, Penesvan A R and Alfermann W. (2003) "Effect of various elicitors on lignan biosynthesis in callus cultures of Linum austriacum. Russian Journal of Plant Physiology 50 (3): 297-300. In Preparation: *Penesvan A. Harder T, Tebben J, Lee M, Kjelleberg S and Egan S (2010). "Production of tropodithietic acid and phenol by an algal-associated alpha-proteobacterium D323 and related sponge isolates". *Penesvan A. Kjelleberg S, and Egan S (2010). "Violacein affects formation of biofilms in marine algal-associated bacterium Microbulbifer sp. D250". *Penesvan A. Novakowsky D, Thomas T, Kjelleberg S, and Egan S (2010) "Functional screening of a large DNA insert library created using the genomic DNA from a collection of antimicrobial-producing bacterial isolates, shows a substantial increase in positive hit rates and identifies genes involved in the production of bioactives". * publications relevant to this thesis CONFERENCE PRESENTATIONS * Penes van A, Harder T, Tebben J, Kjelleberg S and Egan S. "Production of tropodithietic acid (TDA) and phenol by an alga-associated alpha-proteobacterium D323". 3rd Congress of European Microbiologists (FEMS 2009), 2009; Gothenburg, Sweden *Penesvan A, Kjelleberg S and Egan S. "Diversity of cultured bacteria with antimicrobial properties associated with marine algae Delisea pulchra and Ulva australis". 12th International Symposium on Microbial Ecology (ISME-12), 2008; Cairns, Australia. Penesvan A R, Hovhannisyan A A, Kabasakalyan E Y."Ecological aspects of cultivation of Hypericum perforatum L." Conference of Youth Scientists, 2003, Yerevan, Armenia. Vardapetyan H R , Hovhannisyan A A, Penesvan A R , Kabasakalyan E.Y. and Hunanyan L. "hiteraction of hypericin with cell components." Litemational Conference on Bioantioxydants, 2002; Moscow, Russia. Vardapetyan H R., Kirakosyan A B., Hovhannisyan A A., Penesvan A R and Kabasakalyan E Y. "Accumulation of lignans under elicitors treatment in cell cultures of Linum austricum L." Symposium on Dietary Phytohemicals and Human Health, 2002; Salamanca, Spain. Vardapetyan H R , Kirakosyan A B , Hovhannisyan A A , Penesvan A R and Tiratsuyan S G. "Formation of lignans and key enzymatic activities under elicitors treatment in callus cultures of Linum austricum I." Symposium on Dietary Phytohemicals and Human Health, 2002; Salamanca, Spain. Vardapetyan H R, Hovhannisyan A A and Penessvan A R. Biosynthesis of Triterpenoids in Cell Cultures of Bryonia alba L Litemational Symposium on Flavour and Fragrance Chemistry, 2000; Compabasso, Italy. Vardapetyan H., Alfermaim W and Pencsvan A.: Induction of biosynthesis of podophyllotoxin in callus cultures of different Linum species. PSE Meeting, 2000; Lisbon, Portugal. '-presentations relevant to this thesis ABSTRACT The aim of this thesis was to obtain antimicrobial compounds from marine eukaryote-associated bacteria. The unique environment, present on the surfaces of marine eukaryotes, creates conditions that promote and favour the production of bioactive compounds, such as antimicrobials, by giving the producers a clear advantage in the competition for nutrients. This study has demonstrated the abundance of antimicrobial producing bacteria on two marine algae, Delisea pulchra and Ulva australis. Two antimicrobial compounds, violacein and tropodithietic acid (TDA), have been successfully purified and chemically identified from two different bacterial isolates. Moreover, the production of multiple bioactive compounds was observed for both these bacteria. This study also made an attempt to understand the role of the antimicrobial compounds for the producer organisms. Consequently, the effect of violacein on biofilm formation, as well as the possible role of TDA in the defence of both the producer bacterium and the host, have been proposed. The importance of environmental conditions for the expression of bioactive compounds has also been demonstrated, for example, by showing the necessity of high iron concentrations for the production of bioactives in isolates U156 and D245. Notably, despite the absence of a close phylogenetic relationship between these two isolates, they have shown similar trends in terms of production of bioactive compounds. For the first time, this work also described the construction and analysis of a large insert DNA library in E. coli using genomic DNA from a collection of cultured isolates with demonstrated antimicrobial activity. This approach proved to be successful and led to a substantial increase in the positive hit rates in the functional screening of the library, and provided invaluable information concerning the genes, potentially involved not only in the biosynthesis, but also in the processes associated with the production of bioactive compounds, such as transport and resistance. LIST OF ABBREVIATIONS degree Celsius micro (10-^) 2,2-D 2,2-dipyridyl 4,4-D 4,4-dipyridyl A ampere ABC ATP-Binding Cassette ACN acetonitrile AD adapter ADSF Automated DNA Sequencing Facility AHL 7V-acyl homoserine lactone AM antimicrobial AP adapter specific primer Ap ampicillin APCI Atmospheric Pressure Chemical Ionization ATP adenosine triphosphate BCA bicinchoninic acid BHI Brain Heart Infusion BLAST Basic Local Alignment Search Tool BOA Bioautography Overlay Assay bp base pair(s) BSA bovine serum albumin CAS chrome azurol sulphonate CBA Columbia Blood Agar CCR Carbon Catabolism Repression CEE crude ethylactetate extract (obtained at pH 2) CHP conserved hypothetical protein Cm chloramphenicol CMB Centre for Marine Bio-Innovation COG Cluster of Orthologous Group (of proteins) CV crystal violet Da dalton DCM dichloromethane DESI Desorption Electrospray lonisation DGGE Denaturing Gradient Gel Electrophoresis DM desferroxamine mesylate DMSO dimethylsulphoxide DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate EASAG European Academies Science Advisory Council ECI Electrospray Ionization EDTA ethylene diamine tetraacetic acid, trisodium salt EPS Extracellular Polymeric Substances ETAG European Technology Assessment Group EtBr ethidium bromide EU European Union g gram GC Gas Chomatography HCl hydrochloric acid HGT horizontal gene transfer HK histidine kinase HMW high molecular weight HFLC High Performance Liquid Chromatography HTS High Throughput Screening IT ion trap k kilo (10^) kb kilo base pairs KEGG Kyoto Encyclopedia of Genes and Genomes Km kanamycin 1 litre LBIO Luria-Bertani (medium) LC Liquid Chromatography LGM light green morphotype m milli (10*^) M molar MA Marine agar (Difco 2216) MB Marine broth (Difco 2216) MetOH methanol MFC Major Facilitator Superfamily MgCl2 magnesium chloride min minute MLST Multilocus Sequence Typing MMM Marine Minimal Medium MS Mass Spectroscopy n nano (10'^) NaAc sodium acetate NaOH sodium hydroxide NCBI National Centre for Biotechnology Information NMR Nuclear Magnetic Resonance NRPS non-ribosomal peptide synthetase NSS Nine Salts Solution OD optical density ORF open reading frame p pico(10-^') PCR Polymerase Chain Reaction PFGE Pulsed Field Gel Electrophoresis PKS polyketide synthase QS quorum sensing RP reversed phase rpm revolutions per minute RR response regulator RT room temperature SCUBA Self Contained Underwater Breathing Apparatus Sm streptomycin T6SS type VI secretion system TCA trichloroacetic acid TCRR two component response regulator TDA tropodithietic acid TFA trifluoroacetic acid TIGRFAM Institute of Genome Research Family (database) TLC Thin Layer Chromatography TPL tyrosine phenol lyase TTC 2,3,5-triphenyl-tetrazolium chloride UNS W University of New South Wales UV ultra violet V volt VNSS Vaatanen Nine Salts Solution WCPN Waltham Centre for Pet Nutrition WHO Worid Health Organization wt wild type YPA Yeast Peptone Agar TABLE OF CONTENTS ORIGINALITY STATEMENT 2 ACKNOWLEDGEMENTS 3 LIST OF PUBLICATIONS 4 CONFERENCE PRESENTATIONS 6 ABSTRACT 8 LIST OF ABBREVIATIONS 9 TABLE OF CONTENTS 12 LIST OF FIGURES 20 LIST OF TABLES 24 CHAPTER ONE Literature Review 25 I A. Preamble 25 1.2. The need for novel antimicrobial compounds 25 1.2.1. The "golden age" of antibiotic discovery 26 1.2.2. Antibiotic resistance 27 1.3. Antibiotics - natural products 29 1.3.1. Microorganisms as producers of biologically active natural products 29 1.3.2. Natural products: primary and secondary metabolites 30 1.3.3. Regulation of secondary metabolism in microorganisms 31 1.3.3.1. Two-component response regulators 33 1.3.3.2. Role of quorum sensing in the regulation of bioactives production in bacteria 34 1.4. Exploring the under-explored — Marine microorganisms as a source of new drugs 35 1.4.1. Marine surface-associated bacteria 36 1.5. The challenges of bioactive natural product development from marine epibiotic microorganisms 39 1.5.1. Improving the culturability and production of bioactives from marine microorganisms 41 1.5.2. De-replication 44 1.5.3. Purification and structure elucidation of bioactive compounds 46 1.6. Search for alternatives to natural products - is there an alternative? 47 1.7. Decline in antibiotic research. Back to the future or are we heading to pre-antibiotic era? 49 1.8. Aims 51 CHAPTER TWO Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs 53 2.1. Introduction 53 2.2. Materials and methods 55 2.2.1. Isolation of bacteria 55 2.2.2. Screening of isolates for antimicrobial (AM) activity 55 2.2.3. Partial 16S rRNA gene sequencing of marine isolates shown to have AM activity 56 2.2.4. Determination of phylogenetic affiliations 57 2.3. Results 57 2.3.1. Isolation of surface associated bacteria and their screening for AM activity 57 2.3.2. Phylogenetic and morphological characterisation of AM producing isolates 58 2.4. Discussion 62 CHAPTER THREE The role of iron in the expression of antimicrobial activity of two phylogenetically distant bacterial isolates, U156 and D245 69 3.1 Introduction 69 3.2 Materials and methods 71 3.2.1 Strains and media used 71 3.2.2. Near full length 16S rRNA gene sequencing 72 3.2.3. Sequential extraction 72 3.2.4. Assessment of extracts AM activity 72 3.2.5. Effect of cultivation conditions on the expression of AM activity 73 3.2.6. Characterisation of crude bioactive extracts using Thin Layer Chromatography (TLC) 74 3.2.6.1. TLC Bioautography Overlay Assay (TLC-BOA) 74 3.2.6.2. TLC Chrome Azurol Sulfonate assay (TLC-CAS) 74 3.2.6.3. Assessment of the AM activity of a known siderophore 74 3.2.7. Comparison between production of iron-binding compounds and AM activity 75 3.2.7.1. Presence of iron-binding compounds and AM activity in the cells of isolates U156 and D245 grown on various media 75 3.2.7.2. Correlation between the presence of iron-binding compounds and AM activity in cells of isolate U156, grown on MA, with the addition of various concentrations of iron chelator 76 3.2.7.2.1. Determination of the optimal concentration of iron chelator in the growth medium 76 3.2.7.2.2. Assessment of the iron binding and AM activities of isolate U156 in relation to the availability of iron in the media 76 3.2.7.2.3. Determination of the total protein content 77 3.2.8. Effect of the addition of iron on the expression of AM activity by isolates U156 and D245 78 3.3. Results 78 3.3.1. Near full length 16S rRNA gene sequencing 78 3.3.2. Extraction and the assessment of AM activity 78 3.3.3. Effect of cultivation conditions on the production of AM compounds 81 3.3.4. Initial separation and characterisation of bioactive crude extracts, obtained from isolates U156 and D245: TLC, TLC-CAS and TLC-BOA 82 3.3.5. Assessment of the possibility that the AM compounds in extracts of isolates U156 and D245 are siderophores 84 3.3.5.1. Drop-plate AM activity assay of the siderophore desferrioxamine mesylate (DM) 84 3.3.5.2. Production of iron-binding compounds and the expression of AM activity in the cells of isolates U156 and D245 grown on various media 85 3.3.5.3. Production of iron-binding compounds and expression of AM activity in the cells of isolate U156 grown on MA with different concentrations of the iron chelator 87 3.3.5.3.1. Determination of the optimal concentration of iron chelator in the growth medium 87 3.3.5.3.2. Assessment of the iron binding and AM activities of isolate U156 in the presence of various concentrations of an iron chelator 88 3.3.6. Effect of iron on the expression of AM activity by isolates U156 and D245 90 3.4. Discussion 91 CHAPTER FOUR Production of violacein by Microbulbifer sp. D250 97 4.1. Introduction 97 4.2. Materials and Methods 98 4.2.1. 16S rRNA gene sequencing and phylogenetic identification of the isolate D250 98 4.2.2. Extraction and identification of the antimicrobial (AM) purple pigment produced by isolate D250 as violacein, 98 4.2.2.1. Extraction and the assessment of AM activity of the extracts 98 4.2.2.2. Purification and identification of violacein from the crude bioactive extract ofD250 99 4.2.3. Transposon mutagenesis and generation of violacein deficient mutants D250-dVl and D250-dV2 100 4.2.3.1. Random transposon mutagenesis with mini-Tn5-Km^ 100 4.2.3.2. Random transposon mutagenesis using mini-Tn-lO-Km'^ 101 4.2.4. Panhandle PCR and sequencing of the DNA regions flanking the transposon 101 4.2.4.1. Extraction of genomic DNA 101 4.2.4.2. Panhandle PCR 102 4.2.5. Phenotypic characterisation of transposon mutants 103 4.2.5.1. Growth assessment 103 4.2.5.2. Analysis of pigmentation 103 4.2.5.3. Assessment of AM activity 104 4.2.5.4. Assessment of biofilm forming ability 104 4.2.5.5. Formation of cell aggregates in liquid cultures 105 4.3. Results 105 4.3.1. 16S rRNA gene sequencing and phylogenetic identification of isolate D250 105 4.3.2. Extraction and identification of the antimicrobial purple pigment produced by isolate D250 105 4.3.2.1. High Performance Liquid Chromatography (HPLC) 106 4.3.2.2. Liquid Chromatography - Mass-Spectrometry (LC-MS) 106 4.3.3. Transposon mutagenesis and generation of violacein deficient mutants D250-dVl (dVl) and D250-dV2 (dV2) 109 4.3.4. Phenotypic characterisation of dVl and dV2 mutants 109 4.3.4.1. Colony morphological and growth characteristics 109 4.3.4.2. Analysis of pigmentation Ill 4.3.4.3. AM activity assessment Ill 4.3.5. Analysis of the DNA sequences flanking the transposon insertion sites ..112 4.3.6. Assessment of biofilm forming ability of isolate D250 and the violacein-deficient mutants dVl and dV2 113 4.3.7. Formation of cell aggregates in liquid cultures of isolate D250 and the violacein-deficient mutants dVl and dV2 114 4.4. Discussion 116 CHAPTER FIVE Production of tropodithietic acid (TDA) and phenol by the epiphytic alpha- proteobacterium D323 and related sponge associated bacteria 122 5.1. Introduction 122 5.2. Materials and Methods 123 5.2.1. Strains and media used 123 5.2.2. Near-full-length 16S rRNA gene sequencing and phylogenetic identification of the isolate D323 124 5.2.3. Testing of the isolate D323 cell free spent culture medium for AM activity 124 5.2.4. Extraction of the cell free spent culture media 125 5.2.4.1. Liquid-liquid extraction 125 5.2.4.2. Precipitation 125 5.2.5. Disc diffusion assay for the assessment of AM activity 125 5.2.6. Gas Chromatography - Mass Spectrometry (GC-MS) of isolate D323 crude bioactive extract 126 5.2.7. Fractionation of isolate D323 CE and the assessment of the possible AM activity of phenol 126 5.2.8. Thin Layer Chromatography (TLC) and TLC - Bioautography Overlay Assay (TLC-BOA) 127 5.2.9. Assessment for the production of phenol and TDA by D323 in marine minimal medium (MMM) in the presence and absence of L-Tyrosine 127 5.2.10. Purification and identification of the AM compound produced by isolate D323 and the related sponge associated bacteria CCSH21, CCSH24 andNWOOl 128 5.2.10.1. Purification 128 5.2.10.2. Structural elucidation of antimicrobial compound present in CEEs.. 128 5.2.10.2.1. Liquid Chromatography - Mass Spectrometry (LC-MS) 129 5.2.10.2.2. Nuclear Magnetic Resonance (NMR) 129 5.3. Results 129 5.3.1. Phylogenetic identification of isolate D323 129 5.3.2. AM activity assessment of cell free spent medium of isolate D323 130 5.3.3. AM activity assessment of CEE and DBP obtained from isolate D323 spent medium 131 5.3.4. Purification and RP-HPLC analysis of tropodithietic acid (TDA) in the CE extracts of isolates D323, CCSH21, CCSH24, and NWOOl 133 5.3.5. Structure elucidation and identification of the antimicrobial compound present in isolate D323 CEE as tropodithietic acid (TDA) 134 5.3.5.1. Liquid Chromatography - Mass Spectrometry (LC-MS) 134 5.3.5.2. Nuclear Magnetic Resonance (NMR) 137 5.3.6. Analysis of CEEs by Gas-Chromatography - Mass Spectrometry (GC-MS) and Thin Layer Chromatography (TLC) 137 5.3.7. Fractionation of isolate D323 CEE and the assessment of the inhibitory activity of phenol 141 5.3.8. Production of phenol and TDA by D323 in marine minimal medium (MMM) in the presence and absence of L-tyrosine 143 5.3.9. Assessment of TDA, purified from isolate D323 cell free supernatant, for the AM activity against marine epibiotic strains 144 5.4. Discussion 146 CHAPTER SDC Construction, screening and analysis of a large inser DNA (fosmid) library using genomic DNA from cultured antimicrobial producing isolates 152 6.1. Introduction 152 6.2. Materials and methods 154 6.2.1. Extraction and size-selection of genomic DNA 154 6.2.1.1. Pulsed Field Gel Electrophoresis (PFGE) 154 6.2.1.2. Recovery of DNA fragments from agarose gel 154 6.2.2. Construction of the fosmid library 155 6.2.2.1. End-repair of the size-selected genomic DNA 155 6.2.2.2. Ligation of insert DNA into the fosmid vector 156 6.2.2.3. Packaging 156 6.2.2.4. Infection of the E. coli host 156 6.2.2.5. Determination of number of clones for complete genome coverage. 156 6.2.2.6. Storage of library clones 157 6.2.2.7. Screening of the clones for antibacterial activity 157 6.2.3. Extraction and purification of fosmid DNA from bioactive producing clones 157 6.2.4. Sequencing and analysis of bioactive producing fosmid sequences 158 6.2.5. Identification of the strains from which bioactive producing fosmids originated 159 6.3. Results 160 6.3.1. Size-selection of genomic DNA 160 6.3.2. Construction and screening of the fosmid library for the antibacterial activity 162 6.3.2.1. Determination of number of clones in the library necessary for a complete coverage of six bacterial genomes included in the library 162 6.3.2.2. Screening of the library clones for antibacterial activity 162 6.3.3. Sequencing and analysis of selected fosmids 164 6.3.4. Identification of the parental strains from which fosmids originated 166 6.3.5. Analysis of fosmid sequences 168 6.3.5.1. Fosmids originated from isolate D250 168 6.3.5.1.1. Fosmid O-Gll 168 6.3.5.1.2. Fosmid fl2-Al 168 6.3.5.1.3. Fosmids fl5-E10, f20-G8, and f23-H6 168 6.3.5.2. Fosmids originated from isolate D323 169 6.3.5.2.1. Fosmid f9-E12 169 6.3.5.2.2. Fosmid flO-D3 169 6.3.5.2.3. Fosmid fl4-D9 169 6.3.5.2.4. Fosmid fl6-B12 169 6.3.5.3. Fosmids originated from isolate U95 170 6.3.5.3.1. Fosmid fl9-F 10 170 6.4. Discussion 170 CHAPTER SEVEN General Discussion 177 7.1. The potential of marine epibiotic bacteria as a source for future drug discovery 178 7.2. Importance of environmental conditions in the expression of bioactive compounds 179 7.3. Intracellular localization of bioactive compounds: A possible adaptation to the marine environment 180 7.4. Antimicrobial compounds produced by bacteria may have multiple ecological functions 181 7.5. Identification of microorganisms: 16S rRNA based phylogeny and phenotypic characterization 182 7.6. Large DNA insert libraries based on the genomic DNA from cultured isolates, known for the production of bioactive compounds, enhances the hit rate of positive clones and provides an insights into the genes involved in the their production 185 7.7. Concluding remarks.. 187 REFERENCES 188 APPENDIX 1 233 APPENDIX II 234 APPENDIX III 236 LIST OF FIGURES Figure 1-1. Timeline of the antibiotic-resistance development in S. aureus with the introduction of new antibiotics, which illustrates the emergence of bacterial resistance towards the antibiotic after its introduction. MRSA - methicillin-resistant S. aureus, VRSA - vancomycin-resistant S. aureus 28 Figure 1-2. General procedure for the discovery of biologically active natural compounds, such as antimicrobials, of microbial origin. The procedure starts with the isolation of microorganisms from the environment, for example, from the surfaces of marine eukaryotes, followed by their antimicrobial activity screening and the identification of the producer organism. The bioactive compound is then purified and the chemical structure elucidated. Production optimization can be performed to maximize the yield of the desired compound for fiirther in vivo trials and product development. (Clip art images provided by Open Clip Art Library (www.openclipart.org) are used in the figure) 40 Figure 2-1. Maximum likelihood tree constructed in Arb using the aligned partial 16S rRNA gene sequences (-700 nucleotide positions) of the antimicrobial isolates obtained from the surfaces of U. australis and D. pulchra. Sequences from the current study are highlighted in boldface, while close relatives from GenBank are shown in italic. The phyla to which the strains belong are presented on the right. Maximum parsimony bootstrap values (1000 resamplings) are given for major nodes. The scale bar indicates the number of substitutions per nucleotide position 60 Figure 2-2. Ratio of AM producing isolates belonging to different phylogenetic groups obtained from the surface of U. australis (a) and D. pulchra (b) 61 Figure 3-1. Drop-plate AM activity screen of of isolates U156 (a) and D245 (b) cell extracts obtained during the sequential extraction against S. aureus. Extracts are marked as follows: H- hexane, DCM - dichloromethane. Et Ac - ethylacetate, MetOH - methanol. The blank solvent controls are indicated by red 79 Figure 3-2. Changes in colony morphology observed for isolates U156 (a) and D245 (b), during different periods of cultivation (in days) 82 Figure 3-3. TLC-CAS assay (a) and TLC-BOA (b) of the crude methanol extracts of isolates U156 and D245. Spots on the TLC plate are shown as seen under UV light. Spots, showing both iron-binding and AM activities, are outlined by red dashed ovals 83 Figure 3-4. Results of the AM activity assessment of the pure siderophore, DM, using drop-plate assay. "S" indicates spots, where DM was applied, "K" is the solvent control (methanol / deionised water, 1:20) 84 Figure 3-5. Isolates U156 (a) and D245 (b) grown on various media. Media are indicated by numbers that correspond to those in Table 3-2 86 Figure 3-6. Results of the liquid CAS assay of methanol extracts of isolate D245 cells grown on various media, as indicated in Table 3-2. "+" - positive control, - negative control. Similar results were also observed for the corresponding extracts of the cells of isolate U156 86 Figure 3-7. Colorimetric titration of MB medium by addition of 2,2-D. Abosrbance of the red complex, formed as a result of binding of the iron present in the MB medium by 2,2-D was measured at 430 nm 88 Figure 3-8. Comparison of the iron binding activity (a) and the AM activity (b) of a crude methanol extract of isolate U156 grown on MA, 5x diluted MA (5xd MA), and MA supplemented with 0.1 mM 2,2-D (same as 2D in this figure), 0.2 mM 2,2- D, and 0.3 mM 2.2-D. Error bars indicate the standard deviations of four replicates. AM activity is presented as a percentage of the activity relative to the activity of the cells grown on MA (100 %). All values are normalised for 1 mg of total protein.. 89 Figure 3-9. Colony morphology of isolate U156 and D245 grown on VNSS agar solid medium (a), iron supplemented VNSS agar solid medium (b), and MA solid medium (c) 90 Figure 4-1. HPLC chromatogram of isolate D250 semi-purified cell methanol extract. The compound eluted at 6.5 - 7.5 minutes, representing the dominant peak, was collected and was chemically identified as the purple pigment violacein. The UV spectrum of the peak is given in the upper right comer 107 Figure 4-2. LC-ESr-MS characterisation of the purple pigment produced by Microbulbifer sp D250 as violacein, (a) - ESI ion current chromatograms obtained in positive ion mode, (b) - results of MS/MS showing the fragmentation pattern of violacein, (c) - some of the putative fragments derived from violacein in MS/MS and their corresponding masses in parentheses. All mass values are rounded up to the nearest whole numbers. The chemical structure of violacein is shown in (b). 108 Figure 4-3. Isolate D250 (wt) and its transposon mutants D250-dVl (dVl) and D250- dV2 (dV2) 110 Figure 4-4. Growth curves of isolate D250 wild type (wt) and its transposon mutants D250-dVl (dVl) and D250-dV2 (dV2). Strains were grown for 30 hours in MB media. Optical densities were measured at 600 nm at various time points during growth. Error bars represent standard deviations between three replicates 110 Figure 4-5. Drop-plate AM activity test of crude methanol cell extracts of isolate D250 (wt) and its violacein-deficient transposon mutants 250-dVl (dVl) and 250-dV2 (dV2) against N. cams OH73. Methanol (M) was used as a negative control 112 Figure 4-6. Formation of biofilm by isolate D250 and its transposon mutants 250-dVl and 250-dV2 as measured by crystal violet (a) and safranin (b) staining. Error bars represent standard deviations between three replicates 114 Figure 4-7. Heterogenous cell aggregates formed in the liquid cultures of isolate D250. Images show the long thick sinuous cylindrical structures (a) surrounded by thinner interweaving threads (b); (c, d) - cylindrical structures at higher magnification formed by densely packed cells containing violacein as evident by the deep purple colour; (e, f) - regions surrounding these structures, under high magnification with more loosely packed cells/matrix with less violacein content, as evident by the lighter purple pigmentation 115 Figure 4-8. Cells of transposon mutants 250-dVl (a) and 250-dV2 (b) grown in liquid cultures and showing the presence of non-pigmented planktonic cells 116 Figure 4-9. Putative model of TypA itypA) and violacein regulated biofilm formation in isolate D250. TypA upregulates the production of violacein in planktonic cells (a, b). Initially, at low concentrations, violacein, acting as a regulator, stimulates the formation of biofilm (b, c). After the establishment of biofilm, QS mechanisms become effective and enhance the biosynthesis of violacein, allowing it to rapidly reach an inhibitory concentration and act as an antibiotic (d) 121 Figure 5-1. Neighbour-joining phylogenetic tree showing the isolates used in this study (in red) and their close neighbours from GenBank related to isolate D323 (in black) and P. inhibens (in blue) 130 Figure 5-2. Disc-diffusion assay of crude extract (CEE) and dark brown precipitate (DBP) obtained from isolate D323 spent medium against N. canis OH73. EA - blank ethylacetate control, NaOH - blank O.IM solution of NaOH 132 Figure 5-3. RP-HPLC chromatogram of the purified TDA obtained from isolate D323 showing a single dominant peak and its corresponding UV spectrum 134 Figure 5-4. LC-ESf-MS spectra of TDA purified from isolate D323 135 Figure 5-5. LC-APCI-MS spectra of TDA purified from isolate D323 136 Figure 5-6. ^^C (a) and ^H (b) NMR spectra of TDA obtained from CEE of isolate D323. TDA purified from isolate D323 was dissolved in d6-DMS0 (for '^C NMR) and d6-benzene (for ^H) prior to measurements. The spectrum was obtained on Bruker DPX 300 MHz spectrometer. The chemical structure of TDA is given in (a). ^^C NMR spectroscopic data for TDA obtained from isolate D323 in this study, and the data previously reported for TDA by Liang (2003), are compared in the table included in (a) 138 Figure 5-7. GC-MS analysis of isolate D323 CEE (a) and corresponding MS spectrum of phenol (b). Identical masses are indicated by dashed arrows, the mass of the parental molecule (phenol) is indicated by the red arrow 139 Figure 5-8. TLC (a) and TLC-BOA (b) results of the crude extract obtained from isolate D323 (323); CCSH21 (21); CCSH24 (24); NWOOl (NW); PAnhibens T5 (P.i.) and its TDA-deficient mutant P. inhibens T5-3 (P.Í.-3), all grown in MB. Purified TDA (TDA) and phenol (PS, 0.1 mg/ml, in ethylacetate) were used as standards 140 Figure 5-9. TLC and TLC-BOA of isolate D323 crude ethylacetate extracts obtained at acidic (CI), neutral (C2) and alkaline (C3) conditions, as well as the fractions (Fl- F6) obtained via the fractionation of the CI extract, indicated by a red arrow. The ethylacetate extract of acidified MB medium was used as a negative control (B). The pink arrow indicates the position of phenol on the TLC plate after developing it in ethylacetate : hexane (5:1) mixture. The red arrow indicates the crude CEE extract (CI in this figure) 142 Figure 5-10. Disc-diffusion assays of isolate D323 crude ethylacetate extract of neutral (ph7 crude extract) and acidified (pH2 crude extract) spent medium, its fractions F1-F6, as well as ethylacetate solutions of commercially available pure phenol (Sigma) 142 Figure 5-11. TLC (a) and TLC-BOA (b) results of the crude extract obtained from isolate D323 grown in liquid MMM (323) as well as in MMM supplemented with L-Tyrosine (323 Tyr). Purified TDA (TDA) and phenol (PS, 0.1 mg/mL in ethylacetate), were used as standards. 3M - crude extract obtained from blank MMM medium 143 Figure 5-12. Putative model of the interaction between isolate D323 and the eukaryotic host. Possible symbiotic relationship could include the protection of the host surface, as well as the producer bacterium, via secretion of AM compound TDA by D323 in return for nutrients available on the surface of the host. Phenol, also produced by D323, could possibly be used by the host organism as a precursor for various phenolic compounds 151 Figure 6-1. Analytical PFGE of genomic DNA obtained from AM producing isolates. Ml - MidRange I PPG Marker, M2 - EcolHindlll marker, 95 - genomic DNA of isolate U95, 140 - genomic DNA of isolate U140, 156 - genomic DNA of isolate U156, 250 - genomic DNA of isolate D250, 295 - genomic DNA of isolate D295, 323 - genomic DNA of isolate D323 161 Figure 6-2. Agarose gel electrophoresis of HMW DNA (35-40 kB) recovered from the gel slices after preparative PFGE. Ml - MidRange I PFG Marker, M2 - EcolHindlll marker, 95 - genomic DNA of isolate U95, 140 - genomic DNA of isolate U140, 156 - genomic DNA of isolate U156, 250 - genomic DNA of isolate D250, 295 - genomic DNA of isolate D295, 323 - genomic DNA of isolate D323 161 Figure 6-3. E. coli clones carrying the fosmids grown in the presence of L-arabinose. The arrow indicates a clone 20-G8 expressing dark purple pigmentation 163 Figure 6-4. The pigment expressing E. coli clones 15-ElO (a), 23-H6 (b), 20-G8 (c), and 19-FlO (d) growing on petri dishes containing LB 10 agar supplemented with chloramphenicol (12.5 |xg/ml) and L-arabinose (0.02 % w/v) 163 Figure 6-5. Antibacterial activities of violacein producing E. coli fosmid clones 23-H6 and 20-G8, originated from the genome of isolate D250, against S. aureus 163 Figure 6-6. Percentage of genes belonging to each COG functional groups present in P. tunicata whole genome and 8 different fosmids obtained in the current study 165 Figure 6-7. Agarose gel-electrophoresis showing the amplification products obtained using the primers specific for the fosmids f3G-ll (1); f9-E10 (2); flO-D3 (3); fl2- A1 (4); fl4-D9 (5); fl5-E10. f20-G8, f23-H6 (6); fl6-B12 (7); fl9-F10 (8); and the genomic DNA of the isolates U95 (a), U140 (b), U156 (c), D250 (d), D295 (e), and D323 (f). The GeneRuler 100 bp DNA Ladder Plus (Fermentas) was used as a size marker (M). Bands, containing amplification products, are highlighted with arrows 167 LIST OF TABLES Table 2-1. Phenotypical and phylogenetical characteristics of antimicrobial producing isolates obtained from the surfaces of marine algae U. australis and D. pulchra... 59 Table 3-1. Inhibition of canine tooth surface colonizers by crude methanol extracts, obtained from cells of isolates U156 and D245 grown on MA solid medium 80 Table 3-2. Presence of AM and iron-binding activities in the methanol extracts of isolates U156 and D245 grown on different solid media 85 Table 4-1. Restriction enzymes used for panhandle-PCR 102 Table 4-2. The absorbance of the methanol extracts obtained from the same cell biomass of isolate D250 and its mutants (wet weights), measured at 595 nm, corresponding to the absorption maximum of violacein. Extract of E. col i cells was used as a reference in spectroscopic measurements 111 Table 5-1. AM activity of cell free spent medium of isolate D323 liquid culture against primary tooth surface colonising bacteria provided by WCPN 131 Table 5-2. Antibacterial activities of the CEE extract and the DBP precipitated fraction against various clinical strains 133 Table 5-3. Inhibitory activity of TDA produced by isolate D323 against various marine epibiotic bacteria 145 Table 6-1 Primer pairs used for fosmid identification and their expected product lengths 159 Table 6-2. Summary of the identification of the parental strains from which fosmids originated 166 CHAPTER ONE Literature review 1.1. Preamble This thesis describes an investigation of marine microorganisms as producers of biologically active compounds such as antimicrobials. An initial aim was the discovery and development of novel antimicrobials though the project has broader implications with respect to the ecology of antimicrobial producing marine eukaryote-associated bacteria. 1.2. The need for novel antimicrobial compounds The pre-antibiotic era was characterised by the lack of antibiotics and hence, the lack of adequate means for the treatment of numerous infectious diseases. The ground- breaking discovery of penicillin and other antibiotics, which came to be widely used in clinical practice, brought great benefit and contributed to the increase of the average human life expectancy from 47 years in 1900 to 74 years (in men) and 80 years (in women) in 2000 in the US (Lederberg 2000). Furthermore, in 1941 Skinner and Keefer reported a shocking 82 % mortality among 122 consecutive patients who had been treated for Staphylococcus aureus bacteremia before antibiotics were available (Skinner and Keefer 1941), as contrasted to 20-40 % mortality rates being reported in the post- antibiotic era (Mylotte et al. 1987; Shurland et al 2007). The use of antimicrobials is not limited to clinical applications. Currently antimicrobials, such as, for example, chitosan based products, triclosan and various bacteriocins, are widely used in hand washing products, toothpastes and in the food industry to increase the shelf life of various products (Stephen et al. 1990; Waaler et al. 1993; Barkvoll and Rolla 1994; Bhargava and Leonard 1996; Gould 1996; Jones et al. * Parts of this chapter have been published in: Penesyan A, Kjelleberg S and Egan S (2010). "Development of novel drugs from marine surface associated microorganisms." Marine Drugs (special issue) 8(3): 438-459. 2000; Haas et al 2005; Galvez 2007; Dutta et al 2009). By comparison to chemical disinfectants, these natural products are generally less toxic and also provide great advantages, such as biodegradability, biocompatibility as well as chemical and physical versatility (Gould 1996; Dutta et al 2009). Nevertheless, the use of antibiotics for the treatment of diseases historically remains the main focus of antibiotic research. Ever since the discovery of penicillin there have been many attempts to find novel antimicrobials due to the inevitability of development of bacterial resistance towards the widely used antibiotics. In the past years, much of the effort in that direction was focused on terrestrial sources. Nowadays, more than ever before, the exploration of new and under-explored sources becomes extremely important in the process of finding biologically active compounds ("bioactives") with novel chemical structures. 1.2.1. The "golden age" of antibiotic discovery The history of antibiotics began in the early 20^^ century with the observation, made by Alexander Fleming in 1928, that the mould Penicillum notatum could effectively prevent the growth of the bacterium Staphylococcus aureus (Fleming 1929; Sheehan 1982). Subsequently, the inhibitory compound - penicillin, was isolated and described by Florey and Chain in the 1940s thus becoming the first antibiotic to be widely used by the public in the treatment of infectious diseases. The discovery of penicillin and other antibiotics opened a new era in the treatment of infectious diseases, which was later described as the "golden age" (1940 - 1962) of antibiotic research (Singh and Barrett 2006). Discovery of other antimicrobials soon followed and included widely used antibiotics such streptomycin, chloramphenicol, terramycin and others (Cassell 1995). As a result, for the first time many common infectious diseases could be cured. Moreover, those first antibiotics played a pivotal role in the treatment and prevention of infections during the World War II (Lemer 2004). In fact, those drugs were so successful that they were considered as the ultimate cure, the "miracle drugs" for which the medical world was craving in the first half of the 20^^ century. As a result of the initial success of antibiotics, infectious diseases were widely considered as permanently and irreversibly defeated. The following statements made during the "golden age" of antibiotics cleariy illustrate the general notion that dominated at that time: "... experts agree that by year 2000, viral, and bacterial diseases will have been eliminated" (Time, February 1966) "... even diseases have lost their prestige, there aren't so many of them left. Think it over... no more syphilis, no more clap, no more typhoid... antibiotics have taken half the tragedy out of medicine" (Louis-Ferdinand Celine ,1894-1961). "... we had essentially defeated infectious diseases and closed the book on infectious diseases" (US Surgeon General, W.H. Stewart in a testimony to the US Congress in 1969). However, those hopes were not destined to be fulfilled. We are currently well beyond the year 2000, but the book on infectious diseases is far from being closed. The problem can be encompassed in two words: "antibiotic resistance". 1.2.2. Antibiotic resistance With the increased use of antibiotics more and more pathogenic bacteria have developed mechanisms of resistance towards the inhibitory effect of antimicrobials. Bacteria have been present on Earth for several billion years, during which they have encountered a variety of bioactive compounds, including antibiotics, and, consequently, have developed effective mechanisms of resistance (Hancock 2007; Demain and Sanchez 2009). In the environmental setting, antibiotic resistance provides a big ecological advantage for the resistant organism, allowing it to grow in the presence of a high load of antibiotics and to utilize resources in the absence of competitors, which are killed by the antimicrobials (Martinez and Baquero 2002; Fajardo et al. 2009). Consequently, despite the initial effectiveness, most antibiotics have a limited lifetime of use, and, soon after their introduction, they will select for the small population of the pathogen, which has either intrinsic or acquired mechanisms of resistance (Alanis 2005). For example, the evolution of antibiotic resistance in S. aureus - the first bacterium shown to be affected by penicillin by Fleming, clearly illustrates the pattern of the emergence of antibiotic resistance (Figure 1-1). penicillin methicillin vancomycin ^^ Zj^ox, > Daptomycin • 1940 1950 1960 1970 1980 1990 2000 > p-lactam resistant S. aureus MRSA VRSA Figure 1-1. Timeline of the antibiotic-resistance development in S. aureus with the introduction of new antibiotics, which illustrates the emergence of bacterial resistance towards the antibiotic after its introduction. MRSA - methicillin-resistant S. aureus, VRSA - vancomycin-resistant S. aureus. From its introduction into clinical practice in 1940s penicillin was successful in the treatment of S. aureus - related infections. However, the emergence of resistance towards the p-lactams, class of antibiotics to which penicillin belongs, led to the introduction of methicillin in 1960 (Figure 1-1.). Methicillin was effective up until the spread of methicillin-resistant S. aureus (MRSA), therefore, another antibiotic - vancomycin was developed in 1986. The latter was widely used until the emergence of vancomycin-resistance S. aureus in the 1990s. Since then new antibiotic preparations have been approved for the treatment of S. aureus infections, such as Synercid and Zyvox (also known as Linezolid) in the year 2000, and Daptomycin, which was approved by the US Food and Drug Administration (http://www.fda.gov) in 2003, (Walsh 2003); most likely, they will soon need to be followed by yet other antibiotic preparations. In fact, the Zyvox resistant clinical strain of S. aureus was isolated as early as 2001 (Tsiodras et al. 2001), and the failure of the staphylococcal infection treatment due to the Daptomycin resistance was already reported in 2005 (Skiest 2006). As apparent from the example of S. aureus, the possibility of finding the ultimate antimicrobial, which would be always effective in the treatment of the pathogenic strains, seems to be rather an unrealisticideal. In fact, it was estimated, that as of 2004, more than 70 % of pathogenic bacteria were resistant to at least one of the currently used antibiotics (Katz et al. 2006). Indeed, mankind seems to be involved in a continuous struggle against bacterial resistance, requiring the constant development and supply of novel antimicrobials to tackle pathogens (Hancock 2007; Projan 2008). As Dr Allison McGeer, the Director of Infections Control at Mount Sinai Hospital in Toronto, was quoted as saying in 2003: "Although the news is good, our biggest threat right now is complacency. Antibiotic resistance is a problem that will always be with us. If we don't maintain our vigilance, it will come back and bite us." 1.3. Antibiotics - natural products This section will discuss some of the major characteristics of antimicrobial compounds: their main sources, the nature of antimicrobial compounds, and the regulation of their production. 1.3.1. Microorganisms as producers of biologically active natural products. The number of natural products, discovered from various living organisms including plants, animals and microbes, to date exceeds 1 million (Berdy 2005), with the majority (40-60 %) derived from terrestrial plants. Of these natural products, 20-25 % possess various bioactive properties including antibacterial, antifungal, antiprotozoal, antinematode, anticancer, antiviral and anti-inflammatory activities. Plants and plant extracts have been used for the treatment of human diseases for millennia, and their use has been recorded in the most ancient archaeological sources. In contrast, the exploration of microorganisms as producers of therapeutical agents only began in the 20th century (Monaghan and Tkacz 1990). However, despite this relatively short history, nearly 10 % of all currently known biologically active natural products are of microbial origin. These include the majority of antibiotics, clearly demonstrating the potential of microorganisms as an emerging source for the production of biologically 29 active products. Indeed, by the 20th century microbially derived bioactives had become the foundation of modem pharmaceuticals (Capon 2001). For example, the production of antimicrobials is observed in 30-80 % of actinomycete and fungal strains screened in various studies (Basilio et al 2003; Pelaez and Genilloud 2003). Moreover, mathematical models predict that the number of undiscovered antibiotics from actinomycetes could be in the order of 10^ (Watve et al 2001). 1.3.2. Natural products: primary and secondary metabolites The word "antibiotic" was originally referred to as "natural products with antimicrobial activity" (Strohl et al 2001; Overbye and Barrett 2005). Natural products are defined as compounds obtained from living organisms (Lefevre et al. 2008). These compounds are mainly divided into two categories: primary and secondary metabolites. Primary metabolites are common amongst almost all living organisms and are generally involved in the building of the biomass and energy generation. They include amino acids as building units of the various proteins, fatty acids as components of lipid structures, and the heterocyclic purine and pyrimidine bases as elements of nucleic acid structures that carry the genetic code (Haslam 1994; Lefevre et al 2008). In contrast, secondary metabolites are generally thought to serve to improve the survival and fitness of the producer organism (Williams et al 1989). Secondary metabolites are secreted or, otherwise, accumulated by many different microorganisms, which is one of the characteristics of secondary metabolism; the accumulation of primary metabolites is considered rather as an exception (Haslam 1994). Moreover, as opposed to primary metabolites that are common for many organisms, secondary metabolites are often species specific and extremely diverse. Those characteristics make them an excellent source for biologically active compounds, and, indeed, the majority of bioactives identified to date are secondary metabolites (Larsen et al 2005; Lefevre et al 2008). Because secondary metabolites are not considered to be absolutely essential for the organism, but rather assist in its fitness, particularly under stressful conditions, the biosynthesis of these compounds is strictly regulated in response to environmental conditions. This often complex regulatory network prevents the waste of energy towards the production of the particular secondary metabolite and ensures that the compound is only produced in circumstances, when it may provide sufficient advantage to the producer organism and, thus, may compensate for the loss of energy and resources (Haslam 1994). 1.3.3. Regulation of secondary metabolism in microorganisms Though the regulation of primary and secondary metabolism is different, nevertheless, both are closely linked in terms of precursor supply and nutrient regulation, both at the metabolic and genetic levels. It is a widely accepted that bioactive secondary metabolites are formed from ordinary primary metabolites (Haslam 1994). Moreover, all antibiotics are synthesised from the limited number of precursor metabolites that are used for synthesis of all cellular constituents. These precursors are subsequently converted to the great variety of chemical structures found in nature (Rokem et al 2007). Biosynthesis of secondary metabolites occurs via several intermediate stages and is controlled by many different processes and molecular mechanisms. Regulation, both positive and negative, is achieved at all stages, beginning from the transport of necessary nutrients into the cell, as well as the regulation of biosynthesis of various precursors. Various parameters have been shown to play a role in the production of different secondary metabolites. The control mechanisms are usually unique for the given organism. Moreover, even if similar regulatory systems are present in different organisms, they do not necessarily work in exactly the same fashion (Rokem et al. 2007), which makes it difficult to generalise regulatory patterns. The genes for the production of secondary metabolites in the genomes of microorganisms are usually clustered and are coordinately regulated by pathway-specific transcriptional activators (Takano et al 1992; Gramajo et al 1993; Keller and Hohn 1997; White and Bibb 1997; Aceti and Champness 1998; Guthrie et al 1998; Takano et al 2005). The specific regulators may, in their turn, be under the control of hierarchically higher global regulators, transcription factors encoded by genes unlinked to the gene clusters and located in separate parts of the genome (Fox and Howlett 2008; Ou et al 2009). These genes are involved in regulation of multiple physiological processes within the cells and may respond to the changing environmental conditions, such as temperature and pH, or the presence or absence of particular nutrients (Yu and Keller 2005; Hoffmeister and Keller 2007) Among the parameters that affect the formation of bioactive secondary metabolites, the effect of nutrients has been subject to much attention in the recent decades. Generally, the availability of certain carbon sources has been shown to stimulate or otherwise repress the production of bioactives. Secondary metabolite production is highly influenced by the regulatory mechanism termed Carbon Catabolism Repression (CCR). CCR allows bacteria to switch from one carbon source to another in a sequential manner, ensuring that the best carbon source (the one which most rapidly supplies the carbon and energy) is utilized first; at the same time, the pathways, allowing the utilisation of alternative carbon sources, are usually inhibited (Sanchez and Demain 2002). As a result, in media that contain several carbon sources, the rapidly used carbon source, such as glucose, is preferentially used, leading to rapid cell growth, but often, almost no secondary metabolite production. For example, organisms, such as Streptomyces lividans and Janthinobacterium lividum show enhanced levels of production of bioactives, actinorhodin and violacein respectively, when glycerol is used as a carbon source, and inhibition of production in the presence of glucose (Kim et al. 2001; Pantanella et al 2007).. Nitrogen source is also known to affect the production of bioactive secondary metabolites in various organisms. Nitrogen regulation is very complex and has been studied in both bacteria (Reitzer 2003) and fungi (Marzluf 1997). It was found to differentially regulate gene transcription when the preferred nitrogen source is replaced by non-preferred. For example, the change of nitrogen source from ammonia or glutamate to alanine have resulted in global changes in the physiology in the case of the yeast Saccharomyces cerevisae (Usaite et al 2006). However, the exact molecular mechanisms, by which different nitrogen sources may influence the production of secondary metabolites, are not well established. As early as the 1990s the concentration of phosphate in the growth media was also documented to influence the biosynthesis of secondary metabolites (Asturias et al 1990; Liras et al 1990). Often low phosphate concentration stimulates their production (Rokem et al 2007). In recent years the involvement of two-component response regulators (discussed below in section 1.3.3.1) in the phosphate regulation, such as, for example, PhoR-PhoP, have been shown. These two-component regulators consist of a the membrane sensor protein kinase (PhoR) which senses phosphate scarcity, and a response regulator (PhoP) which binds DNA and controls the transcription of genes belonging to the so-called pho boxes, many of which are involved in the biosynthesis of secondary metabolites (Sola-Landa et al 2003; Martin 2004; Sola-Landa et al 2005; Ghorbel et al. 2006; Rodriguez-Garcia et al. 2009). Two component response regulators are further discussed in section 1.3.3.1. Understanding of the complex regulatory networks, functioning in bioactive producing organisms, would greatly assist in designing new strains and cultivation conditions for maximal output of bioactives (Nielsen 1998; Nielsen 2001; Thykaer and Nielsen 2003; Gunnarsson et al. 2004). Such methods include the supply of certain precursors, inhibition of alternate divergent pathways leading to the production of bi- products by stimulating or inhibiting cetain key enzymes in the biosynthetic pathway (Rokem et al. 2007), as well as amplification of the genes and gene clusters, involved in the biosynthesis of the bioactive metabolite (Smith et al. 1989). For example, up to 16 copies of the penicillin gene clusters are known to be present in the high-producer strain Pénicillium chrysogenum BW1890 (Smith et al. 1989). 1.3.3.1. Two-component response regulators Two-component response regulators (TCRRs) are among the major mechanisms that are involved in the regulation of secondary metabolite production in bacteria as a response to changing environmental conditions. They represent the dominant signal transduction pathways in prokaryotes (Galperin 2005), are rarely found in plants and yeasts and are absent in animals (Watanabe et al. 2008). Typically, TCRRs consist of an autophosphorylating protein (a histidine kinase, HK), that is responsible for transferring a phosphoryl group onto an effector protein (a response regulator, RR), thus modulating its activity. Moreover, the HK and RR of a TCRR are usually encoded by a pair of adjacent genes (Whitworth and Cock 2009). Both these are multi-domain proteins: the HK is noted for containing a sensory ('input') domain at the N-terminus, which responds to changes in environmental stimuH, as well as a transmitter domain; while the RR possesses an N-terminal receiver domain in conjunction with C-terminal effector ('output') domain. Certain stimuli from the environment may be sensed by the input domain that leads to the activation of the transmitter domain of HK, which, in its turn, leads to autophosphorylation of HK. The phosphorylated transmitter domain can interact with the receiver domain of their cognate RR to form the phorphotransmitter-receiver domain complex. The latter facilitates transfer of the phosphoryl group from the transmitter His residue onto a conserved Asp residue within the receiver domain of RR. Phosphorylation of the RR receiver domain then alters its interaction with the output domain, up- or down-regulating the activity of the effector (Gao et al 2007; Whitworth and Cock 2009). Besides being known to regulate the production of secondary metabolites, the TCRRs are also involved in regulation of almost all known aspects of bacterial behaviour, such as virulence, pathogenicity, nodulation, motility, nutrient uptake, metabolic regulation, and proliferation (Whitworth and Cock 2009). Consequently, better understanding of TCRRs would provide tools to attenuate bacterial behaviour, such as the production of desired secondary metabolites, both in vitro and in vivo. 1.3.3.2. Role of quorum sensing in the regulation of bioactives production in bacteria One of the unique mechanisms, by which the production of bioactive compounds in bacteria is regulated, is via the now well characterised quorum sensing (QS) system. This form of gene regulation in bacteria enables them to coordinately express specific phenotypes in response to population density (Sauer et al 2002). It is widely accepted that QS plays a central role in the regulation of the production of many bacterial biologically active compounds, such as toxins and virulence factors. QS is achieved via the secretion and recognition of cell-to-cell communication signalling autoinducers, such as A^-acyl homoserine lactones (AHLs) of gram-negative bacteria. These signalling molecules are produced by bacteria and diffused out of the cells, but may only be recognised when they reach a certain threshold concentration as a direct function of the number of the producing bacteria present. The detection of these signals by bacteria leads to changes in gene expression (Waters and Bassler 2005). Thus, QS regulated production of bioactives ensures that those metabolites are only produced when sufficient numbers of producer cells are present to be able to achieve the certain effect. In the case of pathogens it also prevents the triggering of the host defence system before adequate cell numbers are achieved to cause substantial damage (Rice et al 2005; Dong et al 2007). 1.4. Exploring the under-explored - Marine microorganisms as a source of new drugs In the past, the search for bioactive natural product was mainly focused on terrestrial environments (Berdy 2005). Given that the ocean covers more than 70 % of the Earth's surface, marine environments remain one of the greatest unexplored natural product sources. In the past, it was presumed that the marine environment was a "desert" with scarcity of Hfe forms (Zobell 1946). However, it is now clear that the oceans are thriving with tremendous diversity of living microorganisms, with cell counts of 10^-10^ cells per millilitre (Rheinheimer 1992; Fenical and Jensen 2006) and levels of species diversity and richness predicted to exceed many of the Earths rainforests (Holler et al 2000; Haefner 2003; Stach and Bull 2005; Sogin et al 2006). This microbial diversity is presumed to translate into metabolic diversity resulting in the potential for new bioactives to be discovered. Indeed, in the past decades we have witnessed an increase in the number of marine natural products, a large proportion of which are of microbial origin (Blunt et al 2009) (MarinLit database. University of Canterbury: http://www.chem.canterburv.ac.nz/marinlit/marinlit.shtml). For example, compared to the preceding year, in 2007 there was a significant increase (38%) in the number of new marine microbially derived compounds (Blunt et al 2009). In addition to structural variety, bioactives obtained from marine microorganisms are known for their broad range of biological effects, which include antimicrobial, antiprotozoan, antiparasitic, antitumor (Lichstein and Van de Sand 1945; James et al 1996; Andrighetti-Frohner et al 2003; Feling et al 2003; Matz et al 2004; Franks et al 2005; Ratnayake et al 2007; Fremlin et al 2009) as well as antifouling activities which prevent the surface-settlement of various marine organisms (Dash et al. 2009; Xiong et al. 2009; Xu et al 2010). Many of these compounds are also noted for their high potency, which could be related to the need to overcome the dilution of allelochemicals in the seawater (Haefner 2003; Zhang et al 2005). Use of bioactive producing marine eukaryotes in large-scale production faces many difficulties (Uria and Piel 2009), mainly due to the fact, that, in many cases, the eukaryotic organism is killed in the process of obtaining the bioactive. In addition, many of these eukaryotes cannot be cultured in laboratory, but need to be hand-picked by SCUBA diving (Molinski et al. 2009). It also raises the issue of sustainability of these organisms in the nature. In contrast, many bioactive producing marine microorganisms can be easily cultured and manipulated in bioreactors and, therefore, represent the best renewable source of biologically active compounds (Sarkar et al. 2008). 1.4.1. Marine surface-associated bacteria The marine environment is a complex ecosystem with an enormous diversity of different life forms often existing in close associations. Among these, microorganism- eukaryote associations have gained significant attention in the past decade (Egan et al. 2008). The surfaces of all marine eukaryotes are covered with microbes that live attached in diverse communities, often embedded in a matrix and forming a biofilm. The microbial consortia, living on various eukaryotes, differ significantly from each other and from the microorganisms living in the surrounding seawater (Rohwer et al. 2001; Rohwer et al 2002; Bourne and Munn 2005; Dobretsov et al 2005; Enticknap et al 2006; Longford et al 2007; Martinez-Garcia et al 2007; Perez-Matos et al 2007; Santiago-Vazquez et al 2007; Webster and Bourne 2007). For example, a DGGE based comparison of the microbial community composition of the coral Montastraea franksi and the surrounding seawater revealed almost no overlap (Rohwer et al 2001). Likewise, Longford et al (Longford et al 2007) found only two (out of a total of one hundred) bacterial species that were common to three different marine sessile eukaryotes. Moreover, host specificity has also been illustrated by studies that have shown the presence of unique stable communities living on geographically distant individuals belonging to the same species (Taylor et al 2004; Webster and Bourne 2007). In contrast to free living planktonic microorganisms, which often encounter fluctuations in environmental conditions that require quick short-term adaptive responses, surface associated microorganisms supposedly have developed more specialised and stable adaptations, specific to the microenvironment created by a particular host. The high level of specificity of microbial communities on various marine eukaryotes highlights the existence of close cross-relationships between microbial epibionts and their eukaryotic hosts. Li fact, some epibiotic microorganisms have been shown to be essential for the normal life and development of the eukaryote, for example, being involved in the development of host morphology (Provasoli and Pintner 1977; Provasoli and Pintner 1980; Nakanishi et al 1996; Nakanishi et al. 1999; Matsuo et al 2003). Host specific bacteria have also been shown to be vertically transferred from the parental eukaryotic organism to its offspring, which indicates the importance of these bacteria for the host. Such inheritance of members of the microbial community has been reported to occur in sponges (Enticknap et al 2006; Schmitt et al 2007; Sharp et al 2007), bivalves (Cary 1994) and ascidians (Hirose and Fukuda 2006). While the exact nature of the relationship between the microorganisms and their hosts remains unclear, it has been hypothesized that the microbial partners construct chemical microenvironments with the eukaryotic host and live in syntrophy, participating in cycling of nutrients, as well as preventing prédation of the host via the production of bioactive molecules (Sharp et al 2007). Marine surface associated bacteria are often metabolically linked with their host. For example, epiphytes belonging to the Roseobacter lineage are known to degrade the algal osmolyte dimethylsulphoniopropionate (DMSP) yielding the climate-relevant gas dimethylsulphide, and are regarded as major players in sulphur cycling in the ocean (Buchan et al 2005; Wagner-Dobler and Biebl 2006). In contrast, some marine eukaryotes heavily rely on the metabolites produced by their microbial symbionts to survive. For example, some marine sponges use the carbon produced by their associated photosynthetic cyanobacteria (Wilkinson 1979) and may even rely on their autotrophic cyanobacterial symbionts to provide more than 50 % of their energy requirements, which allows them to grow in low-nutrient environments (Wilkinson 1983). Close metabolic associations between microorganisms and their host can make it difficult to reveal which partner organism is responsible for the production of a particular metabolite. As a result, several bioactive products, previously ascribed to the eukaryotes, have later been found to be produced by associated microorganisms (Stierle et al. 1988; Kobayashi and Ishibashi 1993; Unson and Faulkner 1993; Oclarit et al. 1994; Unson et al. 1994; Bewley et al. 1996; Schmidt et al. 2000; Schmidt 2005; König et al. 2006). For example, the microbial origin of the cytotoxic compound bryostatin was demonstrated by the identification of polyketide synthase genes, involved in its biosynthesis, in the genome of the bryozoan bacterial symbiont "Candidatus Endobugula sertula" (Sudek et al. 2007). Moreover, it was proposed that microbially derived bryostatin, found on the larvae of bryozoan Bugula neritina, serves to defend the larvae against potential predators (Lopanik et al. 2004). The interactions between the epibiotic microorganisms and their host, in which microorganisms are thought to acquire nutrients from the eukaryote, while the host benefits from the wide range of bioactives produced by its associated microorganism, seems to be widespread in the marine environment (Harder 2009). For example, the gamma-proteobacterium Pseudoalteromonas tunicata, known for the production of several bioactive compounds, is proposed to play a role in defending the host against surface colonisation by producing antimicrobial, antilarval and antiprotozoan compounds (Holmstrom et al. 1998; Egan et al. 2001; Egan et al. 2002; Franks et al. 2006). Likewise, the surfaces of the healthy embryos of the lobster Homarus americanus are covered almost exclusively by a single gram-negative bacterium that produces an antifungal compound highly effective against the fungus Lagenidium callinectes, a common pathogen of many crustaceans (Gil-Tumes and Fenical 1992). The production of antimicrobials by epiphytic microorganisms could also give the producers a distinct advantage in competition with other surface-dwelling microbes. This is especially important given the fierce competition that exists on the surfaces of marine living organisms that are relatively rich in nutrients compared to seawater, and, therefore, are attractive for numerous microorganisms (Egan et al. 2008). The fact that many marine microorganism-host associations are based on metabolic or chemical interactions may explain the abundance of bioactive producing bacteria on marine living surfaces (Lemos et al 1985; Hentschel et al 2001; Muscholl- Silberhom et al. 2008; Penesyan et al 2009; Wilson et al 2009). Nevertheless, the vast biotechnological potential of marine epibiotic microorgansims remains mostly unexplored (Egan et al 2008). Consequently, a better understanding of the ecological challenges and the underlying mechanisms involved in such interactions should accelerate the search for novel bioactives. 1.5. The challenges of bioactive natural product development from marine epibiotic microorganisms The general procedure for the isolation of natural products from marine epibiotic microorganisms includes several essential steps. The process begins with the isolation of microorganisms from the environment. Often, in the past, the isolation of microorganisms has been a random process. However there is now a growing recognition that the source of microbial samples can be important for increasing the success rate of bioactive discovery (Burgess et al 1999; Egan et al 2008; Penesyan et al 2009). Thus, as discussed above, due to the various and often chemically mediated interactions that occur between microorganisms and their host and between members of the epibiotic community, isolation of microorganisms from marine living surfaces can significantly increase the chances of obtaining bioactive producing strains. After growing the microorganisms in the laboratory on nutritional media, the screening of individual isolates for biological activity is performed, for example, based on the inhibition of growth of microorganisms surrounding the test organism in the case of antimicrobials. The phylogenetic and phenotypic identification of the bioactive producing organism is then performed as the first de-replication, to ensure that the organism has not been previously used for a particular activity and, subsequently, to maximise the possibility of finding a novel bioactive compound. The extraction and purification of biologically active compounds are then carried out, followed by chemical structure elucidation. At this stage a second de-replication can be done to exclude already known compounds. Once novel compounds are identified, the various growth conditions of the producer organism can be assessed to optimize their production. Finally, compounds are assessed for use in the treatment of certain diseases (Pelaez 2006; Singh and Barrett 2006) and in a variety of industrial settings (Figure 1-2). C> Identification of Isolation of Assessment of the isolates / microorganisms from isolates for AM De-replication at the the environment activity producer level V In vivo evaluation, Extraction and identification clinical trials and Production of bioactive compounds / commercialization optimization De-replication at the compound level Figure 1-2. General procedure for the discovery of biologically active natural compounds, such as antimicrobials, of microbial origin. The procedure starts with the isolation of microorganisms from the environment, for example, from the surfaces of marine eukaryotes, followed by their antimicrobial activity screening and the identification of the producer organism. The bioactive compound is then purified and the chemical structure elucidated. Production optimization can be performed to maximize the yield of the desired compound for further in vivo trials and product development. (Clip art images provided by Open Clip Art Library (www.openclipart.org) are used in the figure). 1.5.1. Improving the culturability and production of bioactives from marine microorganisms The limited ability to culture the majority of environmental strains represents a major bottleneck in classical culture-based screening programs for microbial derived bioactives, including those from marine surfaces. It is estimated that the majority (98-99 %) of microorganisms cannot be cultured by traditional techniques (Staley and Konopka 1985; Ward et al. 1990; Pace 1997). Nevertheless, marine living surfaces may provide an advantage as, in some cases, a higher percentage of eukaryote associated microorganisms can be readily cultured (Jensen et al. 1996). Being able to grow the organism in vitro provides great advantages, such as better access to its physiology (Tripp et al. 2008; DeLong 2009). This may allow the manipulation of different growth parameters to achieve the maximum yield of various products and for their large-scale production via fermentation. Among strategies to improve the culturability of microorganisms, those that attempt to grow the organisms under conditions that mimic the physical and chemical parameters of their natural environment, have been the most successful. For example, by using specialised environmental chambers, Kaeberlein et al. (2002) could successfully culture up to 40% of of all microbial cells present in a marine environmental sample. The close associations, often present between marine eukaryotic organisms and their microbial epibionts, clearly impose conditions that are difficult to replicate with standard laboratory procedures. It has, therefore, been proposed that the development of suitable culturing techniques for such organisms should involve conditions that reflect the microenvironment created by their host (Osinga et al. 2001). This approach has proven successful for the isolation of the sponge associated bacterium Oscillatoria spongeliae (Hinde et al. 1994), for which hyperosmotic medium, resembling the osmolarity of the sponge mesophyle, was used to cultivate the organism. Cultivation conditions, such as temperature, aeration, pH of the media, incubation time and media composition, can affect the production of the desired metabolite, and, therefore, must be taken into account and fine-tuned (Boeck et al 1971; Aharonowitz 1980; Gotoh et al. 1982; Shimada et al. 1986; Saitoh et al. 1993; Betina 1994; Barberel and Walker 2000; Pfefferie et al. 2001; Schimana et al. 2002). Usually this requires a producer strain to be grown in the conditions optimal for the production of the active compound. These conditions can differ significantly from the optimal growth conditions of the strain, hi some cases, the producer organism is grown under a variety of conditions in parallel and the differences in metabolic spectra are assessed (Minas et al. 2000; Knight et al 2003; Bills et al. 2008). Marine surface associated microorganisms may also require conditions that resemble their native environment in order to produce the maximum amount of bioactives. For example, several studies have shown an increase in the production of antimicrobial compounds when the surface associated bacteria were grown, in vitro, to form surface attached biofilms (Yan et al 2002; Matz et al 2008; Sarkar et al 2008). In addition, Okazaki et al (1975) have shown that marine isolate SS-228 was able to produce the antibiotic compound only when the growth medium was supplemented with powdered Laminaria seaweeds, common in the habitat from which strain SS-228 was obtained. It is now becoming clear that knowledge of a microorgansims' natural habitat, including the specifics of the host organism, can improve production of microbial derived bioactives. Sequence information obtained via the sequencing of the environmental DNA ("metagenome") can greatly assist in understanding the metabolic potential of the organisms present in the environment, and, thus, guide the development of specialised cultivation conditions (Lorenz et al 2002; Handelsman 2004). For example, Tyson et al (2005) successfully cultured Leptospirillum ferrodiazotrophum by developing a selective isolation strategy. The predicted nitrogen fixing capability of this organism, based on the sequence information of an acid mine drainage biofilm community, underlined the development of that method. Over the past decade genomics has emerged as an alternative to directly culturing microorganisms for the isolation of new bioactives. In particular, functional metagenomics was first developed to access the biotechnological potential of unculturable microorganisms. In this approach the DNA obtained from the environment ("environmental DNA") is inserted into a host organism, such as E. coli, and a functional screen of libraries is performed to detect the desired activity in the clones (Osbume et al 2000; Daniel 2004; Handelsman 2004; Handelsman 2005; Langer et al 2006; Sleator et al. 2008). Some of the successes of this approach were the discovery of terragine A (Wang et al 2000), bioactive N-acyl-tyrosine derivatives (Brady and Clardy 2000) as well as indirubin (MacNeil et al 2001) from the soil metagenomes. Functional metagenomics can provide an insight into the genes and gene clusters involved in the production of certain metabolites, and, thus, provide information about the possible biosynthetic pathway leading to the metabolite (Uria and Piel 2009). Such an approach has been used by Burke et al. (2007) to propose the biosynthetic pathway of the antifungal compound tambj amine produced by the marine bacterium Pseudoalteromonas tunicata. Recently the same approach was successful in identifying two positive clones from a P. tunicata genome library, with different modes of action against the nematode Caenorhabditis elegans (Ballestriero et al., unpublished data), hi addition, a functional metagenomics approach provides an advantage for further purification of the bioactive compound produced by a clone, since the "extra" metabolite can be relatively easily pinpointed by using the non-bioactive-producing clone as a reference (Lefevre et al. 2008). However, despite some success, currently the hit rate of using metagenomic functional screening to obtain bioactive producing clones generally remains low, in the order of 1 in 10000 (Brady and Clardy 2000; Brady et al. 2004), or even as low as 1 in 730000 (Henne et al. 2000) clones screened. This low hit rate is mainly a result of the limited ability of host expression strains to express compounds of foreign origin (Schloss and Handelsman 2003; Handelsman 2005; Pelaez 2006). Therefore, it is possible that with improvements of such strains the hit rate for positive clones in metagenomics functional screens will increase. For example, recent studies have demonstrated that the use of a variety of host expression strains can assist in the expression of the desired metabolite (Butzin et al. 2010; Sarovich and Pemberton 2007; Diaz et al. 2008; Li et al. 2009). Specifically, these strains are often chosen based on possible similarities with the producer bacteria (if known), such as, for example, the similarities in codon usage, as well as the presence of specific machinery necessary for the production of particular metabolites (Butzin et al. 2010; Diaz et al. 2008). Alternatively, shotgun sequencing of environmental DNA and subsequent data analysis has the potential to identify genes encoding new structures of known compound classes, e.g. polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) usually involved in production of bioactive secondary metabolites (Foerstner et al. 2008). For example, recent analysis of the genome of P. tunicata has revealed the presence of nine NRPS genes (Thomas et al 2008). Two products of these NRPS with predicted biological activity have been recently identified in the laboratory via heterologous expression, and their presence was confirmed in the original strain of P. tunicata by varying its conditions of growth (Blasiak and Clardy 2010). Moreover, the availability of sequence data from a variety of microorganisms has further highlighted the importance of developing culturing conditions that would be suitable for the production of bioactives. There are now several examples of genes, involved in the biosynthesis of bioactives, found in non-bioactive producing organisms, suggesting that, given suitable growth conditions, these organisms have the potential to produce bioactive metabolites. For example, the genome of the myxobacterium Stigmatella aurantiaca DW4/3-1 showed the presence of multiple PKS/NRPS gene clusters, which had not been previously observed in this organism (Silakowski et al 2001). In another example, the previously unknown potential for the production of the antitumor compound terrequinone A in the fungus Aspergillus nidulans, was demonstrated (Bok et al 2006). 1.5.2. De-replication In the early years of antibiotic discovery the selection of antibiotic producer strains was based on morphology rather than genotypes, resulting in redundancy in many natural product extract libraries (Handelsman et al 1998; Fim and Jones 2000). The initial success in the discovery of many antibiotic compounds from natural sources included thousands of compounds being described within a few decades. However, the lack of a systematic approach often resulted in the frequent re-discovery of known compounds. Therefore, it is important to put considerable effort into the de-replication process for early detection of both the known producer organisms, as well as the known bioactive compounds. Currently, 16S rRNA gene sequencing (Lane 1991) and phenotypic characterisation can serve for the identification of producer organisms, and, hence, may reveal whether a given microorganism or its close relatives have been previously known to produce certain bioactives, in order to focus the efforts on organisms with as yet uncharacterised activity. Concerning the early detection of known bioactive compounds, advances in the development of new chemical analysis techniques, coupled with a database evaluation, can serve as tools for rapid detection of known compounds requiring only small quantities of sample and/or minimal efforts in sample preparation (Williams and Scrivens 2005; Mitova et al. 2008; Nielen et al. 2009). Hence, they may greatly assist in preventing the waste of resources, which would otherwise be necessary for scale-up and characterisation of bioactive compounds. The exploration of relatively unexplored environments can also assist in finding novel microorganisms and chemical structures, and, hence, minimise the re-discovery of known compounds. The oceans have proven to be a habitat for many unique microbes (Jensen et al. 2005), such as, for example, the recently discovered marine genera Salinispora and Marinophilus (Jensen et al. 2005; Maldonado et al. 2005). In addition, some of the members of these groups were found to produce structurally novel bioactive metabolites. For example, salinosporamides, a family of compounds with cytotoxic activity, were successfully isolated from Salinispora tropica (Williams et al. 2005). Likewise, the structurally novel compounds marineosins (Boonlarppradab et al. 2008) and largazole (Taori et al. 2008) have been recently isolated from marine bacteria belonging to actinomycete and cyanobacterium respectively. Recently discovered marine bioactive natural products including compounds with unique structures are reviewed in (Hill 2009). This supports the idea that the unique chemical and physical parameters of the marine environment can lead to the evolution of life forms that could also produce metabolites with novel chemical scaffolds (Jensen and Fenical 1994; Williams 2009). Given the diversity and uniqueness of marine microbial communities living in association with different eukaryotes, the exploration of various new marine living surfaces could also become a source of yet unknown organisms producing novel bioactive metabolites. 1.5.3. Purification and structure elucidation of bioactive compounds Despite their potential, full characterisation of marine microbially derived bioactives, as well as the development of extraction and purification strategies, can be a long and laborious process requiring a great deal of manual work, with little room for automation (Monaghan et al. 1995; Pelaez 2006). Purification and structure elucidation of mass-limited sample material is considered a major bottleneck. This is usually because the compound of interest often represents less than 1 % of the crude extract, which, in most cases, is a mixture of hundreds of different compounds. Therefore, every extract has its unique combination of "contaminants" necessitating a specific approach; as a result, development of the purification strategies remains largely experimental (Koehn and Carter 2005). Furthermore, obtaining adequate quantities of bioactive compounds, necessary for structure elucidation and evaluation, usually requires extensive optimization of conditions and scale-up (Strobel 2002). To facilitate the chemical characterisation, analytical methods are constantly being developed and improved, one of the major lines of improvement being the possibility of using small quantities of sample and easy sample preparation. For example, the recently developed Ultra High Performance Liquid Chromatography (UPLC) coupled to high resolution mass spectrometers (HRMS), as well as capillary probe nuclear magnetic resonance spectrometers have greatly assisted the process of natural product discovery from mass limited samples (Mitova et al 2008; Frenich et al. 2009; Rowe et al. 2010). Likewise, the newly developed Desorption Electrospray Ionisation Mass Spectrometry (DESI-MS) technique allows for the rapid detection of the compounds requiring minimal effort to be spent on sample preparation (Williams and Scrivens 2005; Nielen et al. 2009). These techniques may soon eliminate, or, by some estimates, have already eliminated the purification and chemical characterisation step as a major drawback in natural product discovery (Cardellina 2006; Bull and Stach 2007; Lam 2007; Bugni et al. 2008; Koehn 2008). 1.6. Search for alternatives to natural products - is there an alternative? The challenges of natural product research have resulted in a search for an alternative to bioactive product development. Combinatorial biosynthesis has emerged as one of the alternatives and is defined as "the application of genetic engineering to modify biosynthetic pathways to natural products in order to produce new and altered structures using nature's biosynthetic machinery" (Floss 2006). It involves the use of genes from different biosynthetic pathways, in various combinations, in order to generate libraries of hybrid structures. However, in practice, this approach is rather problematic. Firstly, it involves the construction of various mutant organisms and, therefore, is very labour-intensive and costly. Secondly, it often relies on the low substrate specificity of enzymes in the biosynthetic pathways, which is not always the case as many enzymes are rather specific (Floss 2006). High-throughput screening (HTS) of synthetic chemical libraries is also regarded as an alternative to bioactive discovery and the development of combinatorial chemistry has allowed for smaller, more drug-like libraries to be screened against defined macromolecular targets. Furthermore, an increase in the availability of genomic data has provided more potential targets for these screens (Dougherty et al. 2002; Koehn and Carter 2005; Dougherty and Miller 2006; Monaghan and Barrett 2006; Alksne and Dunman 2007; Alvarez and Vicente 2007; Payne et al. 2007). However, the first libraries of chemically synthesised compounds provided more quantity than quality; some produced more than million compounds, but were a disappointment, as they yielded very low numbers or no active compounds (Walsh 2003; Koehn and Carter 2005). For example, GlaxoSmithKline (http://www.gsk.com) have recently disclosed the results of a six-year campaign to discover broad-spectrum antibiotics that was abandoned because of the limited chemical diversity of their synthetic screening libraries (Payne et al 2007). These approaches obviously failed to fulfil initial expectations (Muller-Kuhrt 2003; Newman et al 2003; Projan 2003; Butler 2004; Norrby et al 2005; Overbye and Barrett 2005), and are unlikely to substitute the benefits of natural product development. In contrast to chemical libraries, bioactives of natural product origin provide a diversity and a structural complexity, with densely packed functional groups allowing maximum selectivity and interaction with the target (Singh and Barrett 2006; Larsen 2006; Larsson et al. 2007). Such complexity makes the chemical synthesis of these compounds extremely difficult. Despite some success with the total synthesis of some marine natural products, such as, for example, dinoflagellate toxins azaspiracids and brevetoxins (Nicolaou et al 2006; Nicolaou et al. 2006; Haywood et al. 2007; Crimmins et al. 2009; Tillmann et al. 2009), the antibiotic compound uncialamycin produced by marine Streptomyces (Nicolaou et al. 2007); however, in most cases, chemical synthesis of many natural products cannot be achieved by modem techniques (Verdine 1996; Henkel et al. 1999; Feher and Schmidt 2003; Newman et al. 2003; Tulp and Bohlin 2004; Bull and Stach 2007). It has been suggested that the success of natural compounds is due to the fact that they have undergone natural selection and, therefore, are best suited to perform their activities (Nisbet and Moore 1997; Muller-Kuhrt 2003; Koehn and Carter 2005). Thus, further research on bioactive natural products may provide a source of new chemical structures that can guide the design of novel chemical compounds (Breinbauer et al. 2002; Nicolaou et al. 2009), as well as reveal yet unknown modes of action (Urizar et al. 2002). Despite all the challenges and complications, natural products remain an indispensable and unparalleled source of biologically active compounds, such as antimicrobials. Thus the research into the diversity of bioactive natural products justifies the resources invested, due to the lack of equivalent alternatives in synthetic compounds (Koehn and Carter 2005; Nicolaou et al. 2009) The majority of antibiotics currently used in clinical practice are of natural product origin (Newman et al. 2003; Singh and Barrett 2006; Von Nussbaum et al. 2006). For example, 70 out of the 90 antibiotics marketed in the years 1982-2002 originated from natural products (Newman et al. 2003). Notably, the quinolones or fluoroqinones, one of the most successful classes of synthetic antibiotics, are also based on the structure of the natural product quinine (Demain and Sanchez 2009). In fact, chemical modifications based on a natural product scaffold is a widely used approach in modifying the chemical and physical properties of the molecule, thus making it useful for a particular pharmacological application (Walters et al. 1999; Leeson et al. 2004). For example, Jenkins et al. (2009) have recently synthesised four new chemical scaffolds useful for drug development based on novel spiro structures of a number of bioactive natural products such as the histrionicotoxins, isolated from the skin of the Colombian poison dart frog, Dendrobates histrionicus (Jenkins et al. 2009). Likewise, the chemically synthesised analogs of epothilones - compounds produced by myxobacterium Sorangium cellitlosum (Bollag et al. 1995), have shown an increased potency against tumour cells, compared to the original natural product (Nicolaou et al. 2000; Nicolaou et al. 2006). 1.7. Decline in antibiotic research. Back to the future or are we heading to pre-antibiotic era? "...If the world fails to mount a more serious effort to fight infectious diseases, antimicrobial resistance will increasingly threaten to send the world back to a pre- antibiotic age. " Dr Gro Harlem Brundtland, Director General of World Health Organisation (WHO Press Release, 12 June 2000) Unfortunately, after the initial excitement in bioactive natural product discovery, research was soon abandoned primarily due to the perceptions that the few developed antibiotics would be sufficient to combat microbial disease into the future. This perception was clearly misguided with estimates showing that 25 % of annual deaths worldwide are caused by infectious diseases (WHO 2004). Nevertheless, this trend has continued during the last decades with a lack of investment from the pharmaceutical industry because of the limited financial returns (Morens et al. 2004). The pharmaceutical industry has generally accepted a "treat rather than cure" position, preferring to invest in drugs for chronic diseases and lifestyle drugs which provide a long-term revenue, as opposed to antibiotics, where the length of treatment is generally rather short (Projan 2003; Shlaes et al. 2004; Norrby et al. 2005; Overbye and Barrett 2005; Femandes 2006; Bull and Stach 2007; Gyssens 2008; Projan 2008; Demain and Sanchez 2009). In addition, the re-discovery of already known antibiotics has created a notion, that the bioactives of natural product origin are becoming exhausted. However, that is undoubtedly wrong as more than 900 new compounds were discovered only in 2005 from marine organisms, including bacteria (MarinLit database) (Bull and Stach 2007). Finally, the development of antibiotics often faces tough government regulations which may delay new drugs entering the market (Livermore 2004). Thus, the period of time between the initial discovery of the compound to its entering the market takes on average 10 years, which means, that the antibiotics launched today are the products of drug discovery research projects initiated a decade ago (Koehn and Carter 2005). If the current trend remains, as soon as within another decade we may face the situation which existed in the pre-antibiotic era with no effective cure against resistant pathogens. This realisation highlights the urgency of getting the pharmaceutical companies back to the antimicrobials research. As was stated by Dr. Richard Wenzel in 2004 (Wenzel 2004): " ...Good public companies need to be profitable and know the cost of disease, but great companies also aspire to serve and to know the value of health " There is a hope and recently there seem to be a revival in the interest towards the bioactive natural products, partially, due to the realisation of the grid-lock of the situation and the necessity of new compounds in the race against bacterial resistance. Thus, two European Union (EU) expert groups have recently recommended to EU policy-makers that more funds should be invested in research to tackle antibiotic resistance (EASAC. 2007; ETAG 2007). Hopefully, this and similar policies will help to revitalize research into the development of novel biologically active compounds from nature in the near future. 1.8. Aims This thesis describes the potential of targeted isolation of marine epiphytic bacteria in the production of antimicrobial compounds in order to provide more sources of novel bioactive compounds. The initial aim of this project was to obtain antimicrobial compounds from marine eukaryote-associated bacteria. While the area of use for antimicrobials is widespread, including human medicine, industrial and household cleaners, food, and agriculture, the current project, partially funded by the Waltham Centre for Pet Nutrition (WCPN), was focused on the development of such products for applications relating to animal oral health. Dental problems in companion animals are a growing concern. Dental diseases can be very painful and affect the quality of a pet's life. According to the American Veterinary Dental Society, more than 80 % of dogs suffer from such as severe oral disease as periodontal disease by age two (Wiggs 1997). Many oral pathologies, such as dental caries, periodontal disease etc. are plaque-related. Dental plaque is a microbial biofilm formed by organisms tightly bound to a solid substrate and each other. The colonization of the tooth by plaque forming bacteria and their build up can lead to painful periodontal disease and potentially more serious complications such as cardiovascular disease, pulmonary infections, kidney and liver damage (DeBowes 1998; Kuramitsu et al. 2001; Genco et al. 2002; Mojon 2002). Therefore, the prevention of bacterial growth and colonization on the teeth can subsequently prevent the development of many oral diseases and other complications. Thus, to fulfil this objective, the antimicrobial extracts obtained in this study were tested against the range of bacteria provided by the WCPN and specified as primary tooth colonisers (Elliott et al 2006); they are listed in Table 3-1 Historically most of the antibiotic research was focused on the effect of antibiotics on their target organism, and, as a result, the role that these compounds play for the producer organisms was largely underexplored. The current thesis attempts to fill in that gap by addressing some of the ecological roles that these bioactive compounds may be designed to perform in their natural settings and, thus, to broaden our understanding of naturally produced antibiotic compounds Chapter 2 of this thesis provides further details concerning the antimicrobial producing marine bacterial epiphytes. It describes the isolation of marine surface associated bacteria, their screening for antimicrobial activity, as well as discusses their phylogenetic affiliations. Chapter 3 is focused on the antimicrobial producing isolate U156, annotated as Microbulbifer sp. U156, as a representative of a range of isolates obtained in this study, that shared similar colony morphology and 16S rRNA gene sequences and were found to be associated with both algae U. australis and D. pulchra (light green morphotype, LGM). The results obtained for isolate U156 are further compared with the properties exhibited by phenotypically similar, but phylogenetically distant isolate D245, annotated as Pseudovibrio sp. D245. The key role of iron in the expression of antimicrobial activity by both isolates U156 and D245 is demonstrated. Chapter 4 deals with the antimicrobial activity of isolate D250, annotated as Microbulbifer sp. D250, which, despite the high 16S rRNA gene similarity with the LGM bacteria, was found to have different colony morphology and was able to produce violacein. Moreover, a possible ecological role of violacein in the producer organism was proposed. Chapter 5 describes the production of the antibiotic compound tropodithietic acid (TDA) as well as phenol by the D. pulchra surface-associated isolate D323 and related sponge-associated bacteria. It also highlights the broad-spectrum activity of TDA against various marine epibionts, which could be used as a defence strategy. Chapter 6 describes the construction and analysis of a fosmid library using the genomic DNA from a collection of antimicrobial producing isolates obtained in this study. It demonstrates the use of the ftinctional genomics approach, such as cloning and functional screening of the clones, in an attempt to reveal the genes involved the production of biologically active compounds with antimicrobial activity. Chapter 7 provides a general discussion, outlining the main conclusions of this work and possible directions for future research. CHAPTER TWO^ Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs 2.1. Introduction Phylogenetic studies of epiphytic bacteria provide a fascinating insight into the complex bacterial communities that are associated with different marine plants and animals. Often, the community composition of surface attached microorganisms differs significantly from the bacterial population of the surrounding seawater, thus providing an additional argument for the importance of bacterial-host associations (Enticknap et al 2006). During their lifetime, marine eukaryotes produce various metabolites, which coupled with the differences in their surface architecture, provide unique conditions that may select for the best adapted microorganisms, thus, leading to a highly specific relationship between epibiotic microorganisms and their hosts (Longford et al. 2007; Webster and Bourne 2007). The eukaryotic host surface may provide a relatively rich source of nutrients for host-associated microbes, and may, therefore, result in a series of highly competitive interactions within the epibiotic community (Burgess et al. 1999; Armstrong et al. 2001). In such an environment, the production of antimicrobial (AM) compounds can serve as a powerful tool for microorganisms to outcompete other surface colonisers. Consequently, the specifics of marine epiphyte - host associations outlined above has the potential of delivering a rich source of undiscovered natural compounds. ^ Parts of this chapter have been published in: Penesyan A, Marshall-Jones Z, Holmstrom C, Kjelleberg S and Egan S (2009). "Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs: Research article." FEMS Microbiology Ecology 69(1): 113-124. Studies of surface-associated microbial communities of marine algae have provided a great deal of knowledge regarding chemical interactions with their host and between members of the microbial community. The red alga Delisea pulchra, used in this study as a source of epiphytic bacteria, is known for its ability to defend itself against surface colonisation via the production of quorum-sensing inhibitory furanone molecules and, thus, is able to influence its surface-associated bacterial population (Maximilien et al. 1998; Manefleld et al 1999). The community composition of D. pulchra surface attached bacteria has previously been investigated using culture independent methods (Longford et al. 2007). The latter authors described high phylum-level diversity among D. pulchra epiphytes, with Proteobacteria being the most common group, followed by Bacteroidetes. However, little is known about the diversity of the epiphytic bacteria on D. pulchra that are able to produce bioactive compounds. In contrast, the green alga Ulva australis (closely related to Ulva lactuca) is considered as a relatively chemically poor alga with little known chemical mechanisms of protection against colonisers and is hypothesised to rely on its surface-associated bacterial community to provide that role (Egan et al. 2000; Holmstrom et al. 2002). In a previous culture-based study, Egan et al (2000) identified a number of Pseudoalteromonas strains from the surface of U. australis having antimicrobial activity. Interestingly, subsequent studies using culture independent methods indicated that Pseudoalteromonas species do not represent the dominant members of the surface community (Tujula et al. 2006; Longford et al. 2007). These findings suggested that a more extensive culture-based analysis of the surface-associated community of U. australis may yet identify other bioactive-producing bacteria that belong to a variety of phylogenetic groups. In this chapter it is demonstrated, that twelve percent of cultured bacterial isolates belonging to various phylogenetic groups, from two common temperate algae, displayed antimicrobial activity. Moreover, a single bioactive morphotype, phylogenetically related to Microbulbifer sp., was dominant among the bioactive producing strains from D. pulchra. Hence, primarily being focused on the characterisation of the antimicrobial producing bacteria, this study was intended to fill the gap in our current understanding of the diversity of culturable bioactive producing epiphytic bacteria from two marine temperate algae, D. pulchra and U. australis. 2.2. Materials and methods 2.2.1. Isolation of bacteria The alga Ulva australis was collected from intertidal rocky shores at Shark Point near Sydney (33°5r09"S, 151°16'00"E) on the east coast of Australia; Delisea pulchra was collected by SCUBA from nearby Bare Island (33°59'38"S, 151°14'00E") at the depth of 6-10 meters. Algal specimens were rinsed three times with sterile seawater to remove planktonic and loosely attached microorganisms. Five grams of seaweed tissue were placed into a sterile Falcon tube and 10 mL of sterile seawater were added and vortexed for 5 minutes to detach the surface associated bacteria. The seawater, containing the epiphytic bacteria was serially diluted and spread over the surface of petri dishes, containing rich Marine Agar (MA) medium (37.4 g/1 Marine Broth, Difco 2216), as well as oligotrophic (Goodman et al 1993) NSS Agar (Appendix lb), both containing 15 g/1 agar (Agar-agar, Research Organics). Plates were incubated at 25°C for 48 hours. Individual colonies were picked and streaked onto fresh media until pure cultures were obtained. Cultures were stored in 30 % glycerol solutions at -80 °C. Colony morphologies, such as colour and shape, of all the isolates obtained in this study were recorded based on their appearance when grown on MA solid medium. 2.2.2. Screening of isolates for antimicrobial (AM) activity An overlay assay was used for antimicrobial (AM) activity screening against indicator microorganisms, both gram-positive {Streptococcus suis OH78, Waltham Centre for Pet Nutrition (WCPN) culture collection. Staphylococcus aureus 31, Centre for Marine Bio-Innovation (CMB) culture collection) and gram-negative {Neisseria canis OH73, WCPN culture collection) bacteria, as well as fungus Candida albicans (CMB culture collection), all of which are considered to be opportunistic pathogens of humans and animals (Samaranayake and MacFarlane 1982; Sanford and Higgins 1992; Archer 1998; Huang et al 2005; Elliott et al. 2006). Briefly, 10 ^L of overnight liquid cultures of marine isolates, grown in Marine Broth (MB) liquid medium (Difco 2216), were dropped on fresh MA plates. Seeded plates were incubated at 25°C for 96 hours and subsequently overlaid by soft medium, containing 0.7 % agar and inoculated with overnight liquid culture of indicator strain. The following media were used for overlay: Luria-Bertani (LB 10) for S. aureus and N. canis (Appendix la). Brain Heart Inftision (Brain Heart Infusion powder: 37 g/1, Oxoid Ltd) for S. suis, and Yeast Peptone Agar (YPA) for the growth of C. albicans (peptone: 20 g/1, yeast extract: 10 g/1, glucose: 20 g/1). To allow for the growth of indicator strains overlaid plates were incubated for 24 - 48 hours at 37°C {S. aureus, S. suis and N. canis) and 30°C (C. albicans). Inhibitory activity was observed as a zone of clearance around the colony of the marine isolate. Some of the isolated, i.e. Pseudoalteromonas tunicata, have previously been reported to have an antimicrobial activity (James et al. 1996), and, therefore, acted as controls. 2.2.3. Partial 16S rRNA gene sequencing of marine isolates shown to have AM activity Bacterial genomic DNA was extracted according to the XS DNA extraction protocol (Tillett and Neilan 2000). Briefly, isolates were grown overnight in MB. Liquid cultures (2 mL each) were centrifuged for 5 minutes at 21250 x g at room temperature, and the supematants discarded. One millilitre of freshly made XS buffer (1% potassium ethyl xanthogenate (Fluka); 100 mM Tris-HCl, pH 7.4; 20 mM EDTA, pH 8; 1% sodium dodecylsulfate; 800 mM ammonium acetate) was added to each pellet and incubated at 70°C for 1 hour. After incubation supematants were transferred to a new sterile tube and one volume of isopropanol added and gently mixed. After 5 minutes of incubation at room temperature, the mixture was centrifuged at 21250 x g for 15 minutes. Pelleted DNA was washed with ice-cold 70% ethanol and resuspended in molecular biology grade water (Eppendorf). Electrophoresis of the DNA obtained was performed using 1% agarose gels (Agarose, Low Electroendoosmosis, Roche) in 1 x TBE buffer (Appendix He) to check for the quality and to quantify the extracted genomic DNA. Amplification of thel6S rRNA gene was performed in 20 |iL reaction mixtures containing 50 ng of extracted genomic DNA, 2 |aL REDtaq buffer (Sigma), 2.5 mM each dNTP (Roche), 12.5 pmol of each primer (Sigma), 5 |ig of 10 % BSA (New England Biolabs) and molecular grade water (Eppendorf). The 16S rRNA primers used were F27 (5'-GAGTTTGATCCTGGCTCAG-3') (Lane 1991) and R1492 (5- ACGGTTACCTTGTTACGACTT-3') (Stackebrandt and Liesack 1993), which are universal primers designed to amplify the near-full length of 16S rRNA gene sequence. One unit of REDtaq Polymerase (Sigma) was added at the Hot Start, after the initial thermal ramp. The PGR conditions were 94°C for 3 min, then 25 cycles each of 1 min at 94°C, 1 min at 50°C, and 2 min at 72°C. A final extension step of 72°C for 6 min was then performed. Amplified 16S rRNA gene fragments were purified with a QIAquick Spin PGR purification kit (Qiagen GmbH), and used as sequencing templates. Sequencing was carried out based on the BigDye terminator cycle sequencing reaction mix (Applied Biosystems), using F27 primer, then analysed on the Applied Biosystems 3730 DNA Analyzer sequencing system at the UNSW Automated DNA Sequencing Facility (UNSW ADSF). 2.2.4. Determination of phylogenetic affíliations Sequences obtained from UNSW ADSF were edited using SeqEd and Lasergene SeqMan softwares (DNAStar). Homology searches were performed using sequences of ~ 700 nucleotides and close relatives were determined in GenBank database using the Basic Local Alignment Search (BLAST) algorithm (Altschul et al. 1997), available through the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). Sequences were aligned using the FastAlign function of the alignment editor incorporated into ARB software package (Ludwig et al. 2004) and then manually curated. Phylogeny calculations were performed with FastDNAML function, also included in the ARB, according to the maximum likelihood method. 2.3. Results 2.3.1. Isolation of surface associated bacteria and their screening for AM activity Three hundred and twenty five bacterial isolates were obtained from the surfaces of the marine algae D. pulchra and U. australis in pure cultures. Among these, 39 isolates, or 12 % of all isolates obtained, were found to have an AM activity against at least one indicator strain used in this study (Table 2-L). 2.3.2. Phylogenetic and morphological characterisation of AM producing isolates Phylogenetic analysis revealed that the majority of AM producing isolates obtained from the surfaces of both algae belonged to phylum Proteobacteria, with representatives of classes Alpha- and Gamma-proteobacteria (Figure 2-2). Other isolates included phyla Actinobacteria, isolated from U. australis, and Firmicutes and Bacteroidetes isolated from the surface of D. pulchra (Figure 2-2). Among the AM producing isolates, 18 isolates were found to form identical light green coloured colonies when grown on MA and showed an identical spectrum of activity against the target strains used in the current study (Table 2-1).. These isolates were also found to have highly similar 16S rRNA gene sequences, and, therefore, were assigned a single morphotj^e designated a "light green morphotype" (LGM). Isolate D250 was also found to share a similar 16S rRNA gene sequence with the LGM bacteria (Figure 2-1), however, it formed deep-purple coloured colonies on MA solid medium, therefore, it was not be assigned to LGM due to its different phenotype. On the contrary, isolate D245 showed similar morphology to the LGM, but was not assigned to the same morphotype due to the absence of a close phylogenetic relationship with the LGM bacteria. For all other isolates, the similarity in the 16S rRNA gene sequence was also reflected in the similarities in colony morphology (Table 2-1). Table 2-1. Phenotypical and phylogenetical characteristics of antimicrobial producing isolates obtained from the surfaces of marine algae U. australis and D. pulchra AM activity against "/o Isolate Origin Colony morphology Closest relative in GenBank identity S. 5. N. c. suis aureus canis albicans U5 U. australis orange with white Shewanella waksmanii, AY 170366 100 + edges, raised Ull U. australis dark green, raised Pseudoalteromonas. tunicata 100 + ++ + ++ DQ005908 U15 U, australis brown, raised Phaeobacter inhibens, AY 177712 100 ++ - +-H- - U16 U. australis dark orange, raised Roseobacter sp. PI23, EU195952 99 ++ - +++ - U35 U. australis bright yellow, raised Micrococcus luteus, AJ717367 100 + + - - U49 U. australis brownish gray, raised Shewanella japónica, AF500078 100 + - +++ - U56 U. australis brownish green. Ruegeria sp. S0Emb9, AM709695 100 + + - - raised U82 U. australis dark green, convex Rhodobacteraceae bacterium 97 + + + _ 11.5.052CC10, EU276977.1 U95 U. australis dark green, convex Rhodobacteraceae bacterium 97 + + + - 11.5.052CC10, EU276977.1 U107 U. australis pink, raised Roseobacter sp. DG942, 99 + + - - AY258088 U114 U. australis bright yellow, raised Micrococcus luteus, EU071594 100 + + - - U115* U. australis light green, raised Mucus bacterium 25, AY654767 100 ++ ++ ++ + U121 U. australis brownish green. Ruegeria sp. S0Emb9, AM709695 99 + + - - raised U140 U. australis bright yellow, raised Micrococcus luteus, EU071594 99 • + + - U141 U. australis orange, convex Vibrio sp. Y4tang, EF187013 99 + - + - U156* U. australis light green, raised Mucus bacterium 25 AY654767 99 ++ ++ ++ + D203 D. pulchra yellow convex Bacillus pumilus, EF512718 99 - + + + D214* D. pulchra light green, raised Mucus bacterium 25 AY654767 99 -H- ++ -H- + D241* D. pulchra light green, raised Mucus bacterium 25 AY654767 99 ++ ++ ++ + D242* D. pulchra light green, raised Mucus bacterium 25 AY654767 99 -H- ++ -H- + D243 D. pulchra brown, raised Phaeobacter inhibens, AY 177712 100 -H- - +-H- - D245 D. pulchra light green, raised Photobacterium sp. 4.5.0412CS7, 99 ++ ++ ++ + EU276996 D249* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 -H- ++ ++ + D250 D. pulchra dark purple, raised Mucus bacterium 25, AY654767 99 ++ ++ + + D252* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ ++ + D256* D. pulchra light green, raised Mucus bacterium 25, AY654767 100 ++ ++ -H- + D258* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ ++ + D259* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ -H- + D261* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ ++ + D262* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ ++ + D263* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 -H- ++ ++ + D264* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ -H- + D295 D. pulchra yellow, flat Flavobacteriaceae bacterium 98 4-H- + -H- + SW058, AF493683 D299* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ -H- + D304 D. pulchra brown, raised Roseobacter sp. 27-4, AJ536669 100 ++ - +++ - D310* D. pulchra light green, raised Mucus bacterium 25, AY654767 99 ++ ++ ++ + D316* D. pulchra light green, raised Mucus bacterium 25, AY654767 100 -H- ++ -H- + D319* D. pulchra light green, raised Mucus bacterium 25, AY654767 100 -H- ++ -H- + D323 D. pulchra brown, raised. Alpha proteobacterium 1413, 100 ++ ++ +-H- - distinctive rubbery DQ888851 consistency no inhibition observed, '+': inhibition zone was up to 2 mm, '++': inhibition zone was 2-4 mm, '+++': inhibition zone was more than 4 mm * - isolates assigned to LGM Roseobacter sp. 27-4, AJ536669 Phaeobacter gallaeciensis BS107. Y13244 l80late_0304, FJ440984 ISOiate_D243, FJ440970 lsolate_U15, FJ440952 Phaeobacter inhibens, AY177712 r Roseobacter sp. DG942, AY258088 ' lsolate_U107, FJ440959 L| lsolate_U82, FJ440957 < l80late_U95, FJ440958 Ruegeria sp. SOEmbQ, AM709695 l80late_U121, FJ440962 Alpha- lsolate_U56, FJ440956 proteobacteria Rtiodobacteraceae bacterium 11.5.052CC10. EU276977 alpha proteobacterium S0GA1. AJ244780 Isolate. U16, FJ4409S3 77% Roseobacter sp. P123, EU195952 sponge bacterium Isolatel, AY948382 alpha proteobacterium 1105, DQ888838 alpha proteobacterium CRA 8L, AY562564 lsolate_D323, FJ4409(t8 sponge bacterium Isolates, AY948383 81% alpha proteobacterium MBIC3368. AF218241 alpha proteobacterium 1413, DQ888851 lsolate_D319, FJ440987 Pseudomonas sp. C127, DQ005892 lsolate_D316. FJ440986 lsolate_D252, FJ440974 l80late_D256, FJ44097S Microbulbifer cystodytense, AJ620879 mucus bacterium 25, AY654767 lsolate_U115, FJ440961 lsolate_D263, FJ440980 lsolate_D310, FJ44098S lsolate_0299, FJ440983 lsolate_U156, FJ440965 lsolate_D261. FJ440978 lsolate_D242, FJ440969 l80late_D241, FJ440968 lsolate_D2S8, FJ440976 lsolate_D214, FJ440967 lsolate_D264, FJ440981 Gamma- lsolate_D259, FJ440977 proteobacteria lsolate_D250, FJ440973 ^ uncultured bacterium ebne 33F10, EU183928 l8olate_D262, FJ440979 lsolate_D249. FJ440972 68% 81%' Pseudomonas sp. ilCUa, AM180745 l8olate_0245, FJ440971 Photobacterium sp. 4.5.0412CS7, EU276996 Isolate U141,FJ440964 73% Vibrio sp. Y4tang, EF187013 I l8olate_U11,FJ440951 79% ' Pseudoalteromonas tunicate, DQ005908 I— Shewanella japónica, AF500078 80% ' l80late_U49, FJ440955 I Shewanella waksmanii, AY170366 79% ' lsolate_U5, FJ440950 lsolate_U114, FJ440960 lsolate_U3S, FJ440954 81% Micrococcus luteus, EU071594 Actinobacteria lsolate_U140, FJ440963 8i%[ lsolate_D203, FJ440966 ' Bacillus pumilus, EF512718 Firmicutes 81% r Flavobacteriaceae bacterium SW058, AF493683 1 l80late_D295, FJ440982 Bacteroidetes 0.10 Figure 2-1. Maximum likelihood tree constructed in ARB using the aligned partial 16S rRNA gene sequences ('-TOO nucleotide positions) of the antimicrobial isolates obtained from the surfaces of U. australis and D. pulchra. Sequences from the current study are highlighted in boldface, while close relatives from GenBank are shown in italic. The phyla to which the strains belong are presented on the right. Maximum parsimony bootstrap values (1000 resamplings) are given for major nodes. The scale bar indicates the number of substitutions per nucleotide position. Firmicutes 4% Bacteroidetes 4% Alpha-proteobacteria 13% Gamma-proteobacteria\ 79% Actinobacteria 19% Alpha-proteobacteria 43% Gamma-proteobacteria 38% Figure 2-2. Ratio of AM producing isolates belonging to different phylogenetic groups obtained from the surface of U. australis (a) and D. pulchra (b). 2.4. Discussion The high proportion of antimicrobial producing epiphytic isolates (12 %) demonstrated in this study is in agreement with the literature, where the relative ratio of antimicrobial producing culturable isolates obtained from different marine eukaryotes were reported to be 16.9 % from intertidal seaweeds (Lemos et al, 1985), 11.3% (Hentschel et al. 2001) and 15.2 % from sponges (Muscholl-Silberhom et al. 2008), and even as high as 35 % from various species of seaweeds and invertebrates (Burgess et al. 1999). These numbers are substantially higher compared to the proportion of bioactive producing bacteria isolated from other habitats (Ivanova et al. 1998; Burgess et al. 1999), amounting to, for example, 7 % in seawater planktonic communities (Zheng et al. 2005). Furthermore, it was recently demonstrated that bacteria growing in a surface attached biofilm have an increased production of chemical defence mechanisms compared to planktonic growth (Matz et al. 2008). This observation may represent an adaptive response to compensate for the loss of the ability to escape prédation when attached to a surface. Phylogenetic analysis demonstrated that the majority of antimicrobial isolates obtained from the surfaces of both algae belonged to phylum Proteobacteria (Figure 2-2), with representatives of classes Alpha- and Gamma-proteobacteria (Figure 2-2), in agreement with a previous report showing a high percentage (63 %) of epibiotic Proteobacteria (Longford et al. 2007). The majority of the Gamma-proteobacteria in the current study (19 out of 24) were isolated from the surface of D. pulchra and were found to be phylogenetically closely related to Microbulbifer sp. (Figure 2-1) In addition to sharing a similar 16S rRNA gene sequence (Figure 2-1), eighteen of these isolates were found to have a similar spectrum of activity and colony morphology, forming light green circular colonies when grown on MA medium (LGM). The only exception was the isolate D250 (Figure 2-1, GenBank accession no. FJ440973) which, while similar in 16S rRNA gene sequence, was found to have a different colony morphology forming large circular colonies with a distinctive purple colour when grown on the same medium (Table 2-1). These results highlight the importance of considering the phylogeny in conjunction with the phenotypic characteristics for the identification of isolates. Interestingly, species phylogenetically closely related to LGM were also reported to be abundant on other marine living surfaces, such as corals, green and red algae and ascidians (Peng et al 2006). Moreover, in almost all habitats these isolates may carry an important ecological function. For example, an LGM related species (Figure 2-1, GenBank accession no. AY654767) was reported to be among the most abundant bacteria associated with bleached and azooxanthellae (lacking the endosymbiotic algae) coral Oculina patagónica (Koren and Rosenberg 2008), as opposed to the healthy coral (Koren and Rosenberg 2006). A similar trend was recently found for sponges, where related bacteria (Figure 2-1, GenBank accession no. EUl83928) were observed in diseased individuals, in contrast to the healthy sponges that lacked those organisms (Webster et al 2008). The LGM-related isolate (Figure 2-1, GenBank accession no. AM 180745) was also shown to cause the return of the aberrant morphology of axenic plantlets of the green alga Ulva Unza to normal (Marshall et al. 2006). In addition, a phylogenetically related species (Figure 2-1, GenBank accession no. DQ005892) was reported to induce the settlement of sea urchin Heliocidaris erythrogramma larvae (Huggett et al. 2006). Future studies would be needed to reveal the ecological role that LGM related bacteria may play as members of the D. pulchra bacterial community. According to the 16S rRNA gene sequence, isolate Ull (GenBank accession no. FJ440951) was found to be 100 % identical to the surface associated bacterium Pseudoalteromonas tunicata. Pseudoalteromonas species, especially pigmented strains, are well known for their ability to produce various biologically active compounds, including antimicrobials (Lovejoy et al. 1998; Holmstrom et al. 2002; Longeon et al. 2004; Dobretsov et al. 2006; Zheng et al. 2006). They are considered as significant sources for novel drug discovery (Bowman 2007), however, in the current study this genus was represented by only one isolate. The low abundance of Pseudoalteromonas species detected in this study is in accordance with recent data that suggests that Pseudoalteromonas species may represent less than 1.6 % of the microbial population 3 2 associated with marine algae, such as U. lactuca, occurring at densities of < 10 cells cm" (Skovhus et al. 2004). However, despite their apparent low abundance, these bacteria may still prove to be effective competitors and play an important ecological role in preventing host surfaces from subsequent colonisation. In support of this view Rao et al. (2007) demonstrated, that even at concentrations as low as 10^ - 10^ cells/cm, P. tunicata was effective in inhibiting the settlement of algal spores and marine flingi, showing that low density microbial colonisers can have a significant ecological impact. The most abundant alpha-proteobacterial group obtained in this study was affiliated with the Roseobacter clade - a phylogenetically coherent, but physiologically heterogeneous group of marine bacteria. They often exist as epiphytes and are among the first and most successful surface colonisers, some of which are known to produce various antimicrobial compounds (Brinkhoff et al. 2004; Bruhn et al 2005). Roseobacters are abundant in the marine environment and may represent almost 25% of all microbial communities in coastal waters and polar regions (Wagner-Dobler and Biebl 2006). This ratio correlates with the results of the current study showing that 26.3% of all isolates obtained belongs to the Roseobacter group. Two of those isolates, U82 and U95 (GenBank accession nos. FJ440957 and FJ440958 respectively), may represent a novel genus within that group with lower than 97 % 16S rRNA gene sequence identity to known Roseobacters. Another alpha proteobacterium, isolate D323, obtained from the surface of D. pulchra was phylogenetically related to the Alpha-proteobacterium MBIC3368 with 98% identity in 16S rRNA gene sequence (Figure 2-1, GenBank accession no. AF218241). Related bacteria have been isolated from different marine sponges and are largely regarded as sponge specific bacteria (Muller et al. 2004; Muscholl-Silberhom et al. 2008). Moreover, they are reported to be the stable, dominant symbiotic bacteria isolated from the healthy sponge Rhopaloeides odorabile (Webster and Hill 2001) and in at least six other marine sponges (Thiel and Imhoff 2003; Lafi et al. 2005; Muscholl-Silberhom et al. 2008) while being absent in the diseased R. odorabile and in surrounding seawater (Webster et al 2002; Enticknap et al. 2006). This may suggest the presence of a highly specific symbiotic relationship between isolate D323 related bacteria and their sponge host. Moreover, Enticknap et al. (2006) demonstrated that these alpha-proteobacteria can be transferred from the parent sponge to its offspring through larvae, providing an additional clue towards the stability of this association. However, the physiological nature of the putative symbiosis is not clear. Future advances in this area may also help in the understanding the relationship between isolate D323 and its algal host D. pulchra. The Bacteroidetes phylum is represented by isolate D295, belonging to class Flavobacteria. Culture independent techniques indicated that Bacteroidetes are among the most abundant bacteria found to be associated with marine eukaryotes (Longford et al. 2007), and may comprise up to 14% of all bacterial populations in coastal waters (Zhang et al 2007). Marine Bacteroidetes usually belong to classes Flavobacteria and Sphingobacteria and are also found in freshwater, soil and sediments. They are ubiquitous heterotrophs noted for their ability to degrade complex lignocellulosic plant materials (Lydell et al. 2004). This feature may explain their abundance on surfaces of marine seaweeds, which can provide a continuous source of nutrients for these organisms. Nevertheless, the phylum Bacteroidetes is not well represented among cultured isolates (Lydell et al. 2004), which correlates with this study showing only one cultured isolate out of 39 belonging to that group. However since the current study is focused on isolation of antimicrobial bacteria, the limited number of Bacteroidetes isolates obtained may be a reflection of a scarcity of AM producing marine epiphytic bacteria belonging to that phylum. Early studies estimated that the ratio of gram-positive bacteria among all the bacteria in the ocean to be as low as 5 % (Zobell 1946). However, later investigations demonstrated that this number and the diversity of marine gram-positive bacteria may be considerably higher (Jensen et al. 2005; Stach and Bull 2005). Despite being frequently isolated from the marine environment, there is a lack of understanding of the physiology and ecology of marine gram-positive bacteria (Gontang et al. 2007), even though many of them, e.g. Actinomycetales have been extensively studied for their biotechnological potential in production of antibiotic compounds, with 50 % of known microbial antibiotics derived from actinomycete bacteria (Fenical and Jensen 2006). In the current study gram-positive bacteria are represented by the phyla Actinobacteria, which includes isolates U35, U114 and U140, and Firmicutes, represented by isolate D203. Isolate D203 was phylogenetically related to Bacillus pumilus species, with 99% identity in the 16S rRNA gene sequence. Numerous Bacillus species have been detected in the marine environment and are known for their ability to produce bioactive compounds. Bacilli are ubiquitous in various environments and, owing to their capability to survive under vastly different conditions, in early studies they were not regarded as being indigenous to certain habitats (Claus and Berkeley 1986). However, in the past decade exclusively marine halophilic Bacillus species, such as B. salexigens have been described (Garabito et al. 1997). Interestingly, according to some observations, ubiquitous strains of B. subtilis and B. pumilus remain the most abundant surface- associated bacilli found in marine environment (Ivanova et al 1999). All three Actinobacteria described in this study (isolates U35, U114 and U140) were isolated from U. australis. With 16S rRNA gene sequence similarity of at least 99%, they were all found to be phylogenetically related to Micrococcus luteus strains (Figure 2-1), ubiquitous bacteria found in both marine and terrestrial environments. Despite their widespread occurrence, there is limited information concerning marine Micrococcus strains possessing AM activity. In one of the few reports, Bultel-Ponce et al. (1998) described an AM producing marine sponge-associated M luteus. Recently Lo Giudice et al (2007) found that almost 73 % of AM producing isolates obtained from various Antarctic marine sources belonged to Actinobacteria, more than 37 % of which were affiliated with the family Micrococcaceae. This clearly indicates the antimicrobial producing capability of marine Micrococcaceae strains, which otherwise might have been underestimated. Small collections of bacterial isolates from the surface of U. australis have been previously obtained in several studies (Rao et al. 2005; Burmolle et al. 2006; Tujula 2006). These studies identified isolates belonging to Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria, with the Proteobacteria being the most commonly isolated group. A similar pattern was also demonstrated among the collection of bacterial isolates from the surface of D. pulchra (Longford 2008). Data from the culture- independent studies of the surface associated communities of these algae generally agreed with the distribution pattern demonstrated with culture based methods with some exceptions. For example, members of Verrucomicrobia and Chloroflexi were identified on the surface of D. pulchra only via a culture independent approach (Longford 2008) and representatives of Firmicutes were successfully cultured from U. australis though not found in clone libraries from the same alga (Tujula 2006). Interestingly, while the current study was primarily focused on the bioactive producing bacteria, the phylogenetic distribution of isolates is similar to previous studies not specifically aimed at AM producing members (Tujula 2006; Longford 2008). Representatives of nearly all major phylogenetic groups that were previously shown to be commonly associated with U. australis and D. pulchra were obtained in the present study of antimicrobial producing epiphytic bacteria. Thus, production of antimicrobials is not restricted to a certain bacterial group but instead appears to be widespread across different bacterial phyla. On the species level, however, there was little overlap phylogenetically among the isolates obtained from the two marine algae D. pulchra and U. australis, with the only strains isolated from both organisms being the "mucus bacterium 25" related isolates (GenBank accession no. AY654767) assigned to LGM and isolates affiliated with Phaeobacter inhibens (Table 2-1). Such lack of overlap amongst bacterial species associated with various marine eukaryotes was also described in a culture-independent study (Longford et al. 2007) and can serve as an additional argument towards the specificity of bacteria-host associations in marine environments. Despite the emergence of metagenomics as a potentially powerful tool in natural product discovery, current metagenomic methods, which include sequence-driven analysis and functional screening of environmental clone libraries, have yielded limited results in the discovery of novel bioactive compounds (Schloss and Handelsman 2003; Handelsman 2005). Indeed, the rates of obtaining positive bioactive producing clones in metagenomic libraries have been very low; often fiinctional metagenomics requires screening of tens of thousands clones and may only yield few or no bioactive producing clones (Brady and Clardy 2000; Henne et al. 2000; Gillespie et al 2002; Brady et al 2004). While the development of advanced expression hosts and screening methods may improve heterologous production and hit rates for novel bioactives, to date culture based approaches remain arguably the most powerftil resource in exploring novel bioactives of bacterial origin. A high percentage of epiphytic isolates possessing AM activities also highlights the biotechnological potential for targeted isolation of marine eukaryote associated bacteria. Products from such microorganisms may prove to be a valuable source of fiiture novel drugs. Some of the AM producing isolates, mentioned in the current chapter, namely isolates U156, D245, D259 and D323, became subjects of detailed studies and their biological activities were ñirther characterised. Isolate U156 was selected for ñirther characterisation as a representative of LGM, isolate D245 was used for comparison with the secondary metabolite profile of isolate U156 due to their phenotypic similarities (Chapter 3). In contrast, isolate D250, despite the phylogenetic similarity with LGM, was phenotypically different and was, therefore, chosen for fiirther characterisation (Chapter 4). Finally, isolate D323 was selected for chemical characterisation due to its exceptionally potent broad-spectrum antimicrobial activity as well as the literature data suggesting its important ecological role as a marine eukaryote-associated symbiont. Isolate D323 is further discussed in Chapter 5. CHAPTER THREE The role of iron in the expression of antimicrobial activity of two phylogenetically distant bacterial isolates, U156 and D245 3.1 Introduction Thirty-nine antimicrobial (AM) producing bacteria were isolated from the surfaces of marine algae during the course of the current project (Chapter 2). This chapter focuses on an investigation of isolate U156, which is a representative of the light green morphotype (LGM) - range of isolates, having similar colony morphology and 16S rRNA gene sequence (Figure 2-1.); and isolate D245 - a strain showing a production of green pigment similar to LGM, but phylogenetically distant (Figure 2-1). LGM isolates and isolate D245 were previously shown to have an identical spectrum of AM activity against both gram-positive and gram-negative bacteria, as well as the yeast C. albicans (Table 2-1). The LGM bacteria were among the few strains to be isolated from both Ulva australis and Delisea pulchra, and were shown to be dominant AM producing isolates obtained from D. pulchra in this study (Table 2-1). Strains phylogenetically closely related to LGM, were previously found on other marine living surfaces, and shown to play significant ecological roles. For example, similar isolates from the alga Ulva compressa were found to mediate the return to normal from an aberrant morphology of axenic plantlets of a related green alga Ulva Unza (Marshall et al 2006). Related isolates were also reported to be among the most abundant bacteria found associated with the azooxanthellae (lacking the endosymbiotic algae) Oculina patagónica coral (Koren and Rosenberg 2008) but were not observed on healthy coral (Koren and Rosenberg 2006). Interestingly, as was shown by Koren and Rosenberg (2008), the other bacteria, found to be abundant on the same diseased corals, were phylogenetically closely related to isolate D245. Likewise, Webster et al. (2008) found bacteria, which were related to both isolates U156 and D245 on diseased, but not healthy individuals of the sponge Rhopaloeides odorabile. Li addition, Huggett et al. (2006) described isolates closely related to U156 and D245 as high inducers of sea urchin larvae settlement from the surface of the coralline alga Corallina officinalis. As was mentioned above, isolate D245 displayed colony morphology similar to the LGM, also producing a green pigment. It was found to have an identical spectrum of AM activity to that of isolate U156 and other LGM related bacteria, when tested against a range of target strains (Table 2-1, Table 3-1). Taking into consideration these similarities, this chapter aims to characterise and compare the AM activities of these isolates and reveal the effects of environmental conditions on their expression. Over the last two decades there has been a considerable interest in the role of trace elements in various ecosystems, in particular, in the marine environment. Currently, iron is regarded as one of the most important elements, as its availability directly correlates with the viability of phytoplankton and, thus, affects the primary productivity in the ocean (Martin and Fitzwater 1988; De Baar et al. 1990; Price et al. 1991). Due to its redox properties, iron can act as a cofactor of many metabolic processes in the cell. However, the availability of iron in many aquatic environments is estimated to be as low as 10'^^ M, mainly due to the formation of water insoluble ferric oxide (Fe^^). This concentration is significantly lower than 10'^ M, the intracellular concentration of iron, necessary for bacterial growth (Harvie et al. 2005). Bacteria have, therefore, developed specialised systems to ensure a constant supply of iron. Such adaptive systems include siderophores - small molecules that act as iron scavengers and can successfully bind Fe^^, followed by their uptake by the cell. Subsequently, Fe^^ is reduced to soluble Fe^^ by ferri-siderophore ferri-reductases within the bacterial cell (Ratledge and Dover 2000). However, the high concentration of ferrous ion in the cell can also be toxic, leading to oxidative stress. Therefore, some bacteria employ specialized ferritin-like iron storage compounds, which can isolate and store excessive iron within the cavities formed by their molecules (Andrews et al. 2003; Carrondo 2003). The results described in this chapter suggest that bioactive compound(s) produced by isolates U156 and D245 possess a high affinity for iron; fiirthermore, iron was found to play a key role in the expression of the AM activity by both isolates U156 and D245. The results also demonstrated that isolates U156 and D245 exhibit identical behaviour in the expression of the AM activity, i.e. the activity was expressed at the early stage of cultivation on solid MA medium, while no activity was observed in either isolate grown in various liquid media, indicating the importance of attached growth for the expression of these bioactive compounds. 3.2 Materials and methods 3.2.1 Strains and media used Isolates U156 and D245 were grown in different solid and liquid media suitable for the growth of marine bacteria, including liquid MB (Difco 2216), VNSS (Appendix Ic) (Marden et al 1985), and solid MA and VNSS agar, the last two containing 1.5 % agar-agar (Research Organics). Target strains used to explore the inhibitory activity of isolates U156 and D245 crude extracts, are listed in Table 3-1. They were described as primary tooth surface colonisers responsible for the development of dental diseases in animals (Elliott et al. 2006) and were provided by the Waltham Centre for Pet Nutrition (WCPN), UK. These bacteria were grown in liquid BHI medium (Brain Heart Infusion powder: 37 g/1, Oxoid) and on supplemented CBA solid medium (Appendix Id) (Elliott et al 2006). Staphylococcus aures 31 (CMB culture collection) was grown in liquid LB 10 broth (Appendix la) or on solid LB 10 agar (LB 10 with addition of 1.5 % agar-agar) and was routinely used as an indicator strain for the detection of inhibitory activity in drop- plate assays, as well as in the TLC-BOA experiments described below (section 3.2.6.1). 3.2.2. Near full length 16S rRNA gene sequencing To obtain a near-full length 16S rRNA gene sequence of isolates U156 and D245, in addition to the primers F27 and R1492, described in Chapter 2, the intermediate primers F530 (5'-GTGCCATC-CAGCCGCGG-3') and R903 (5'- CCGTCAATTCCTTTRAGTTT-3') were also used to sequence the 16S rRNA gene PGR amplification products (Vossbrinck et al 1993) as described in section 2.2.3, resulting in four sequences for each of the isolates U156 and D245. All four 16S rRNA gene sequences, derived from the same isolate, were aligned using the SeqMan software incorporated into the Lasergene software package (DNAStar). The consensus near-fiill- length 16S rRNA gene sequences were used for the phylogenetic comparisons discussed in this chapter. 3.2.3. Sequential extraction Overnight liquid cultures of isolates U156 and D245 were dropped onto petri dishes filled with MA (10|il drops, 30-40 drops per petri dish) and allowed to grow and form colonies for up to 7 days. Colonies were scrapped from the surface of the solid medium and collected in fresh glass vials. The agar surrounding the colonies was cut using a razor blade and transferred to separate vials. Both the colonies and the surrounding agar were freeze dried under reduced pressure. Following drying, a sequential extraction was performed using solvents in order of increasing polarity (namely: hexane, dichloromethane, ethylacetate, methanol, and water). The amount of solvent used corresponded to the original volume of the sample before freeze-drying. Briefly, hexane was added first to the dry sample, mixed, incubated for 10 min. After incubation the sample was centrifuged and the first extract was collected into a glass vial. The pellet was air-dried before the next solvent was added and the procedure repeated. 3.2.4. Assessment of extracts AM activity The standard disk-diffusion assay (Bauer et al. 1966) was found to have a limited sensitivity for the assessment of the AM compound produced by isolates U156 and D245, possibly due to limited diffusibility of the bioactive compound. Therefore, the drop-plate assay was adopted as being more sensitive for the detection of the AM activity for both isolates. Briefly, each solvent extract was dropped (7 ^1) directly onto petri dishes containing solid LB 10 medium, the blank solvent was used as a negative control, and allowed to air dry. Plates (petri dishes) were thereafter spread by the target strain S. aureus and incubated overnight at 37°C. Inhibition was observed as zones of clearance in the area where the extract was spotted. Using the drop-plate inhibitory assay bioactive extracts, obtained from the isolates U156 and D245, were further tested for the spectrum of AM activity against a variety of primary canine tooth colonizers, provided by WCPN (UK) and listed in Table 3-1. 3.2.5. Effect of cultivation conditions on the expression of AM activity To study the effect of growth conditions on the expression of AM activity, the AM activity of the isolates U156 and D245 extracts was assessed after growing them in different media and for different time periods. Overnight liquid cultures of isolates U156 and D245 were dropped (10 fil drops, 30-40 colonies per petri dish) onto different solid media, including MA, marine minimal medium (MMM) (Neidhard et al 1974), and VNSS, all containing 15 mg/ml agar. Colonies were incubated for 4-14 days at room temperature. Samples of cells were harvested from plates at different time points during the incubation and freeze-dried. Solid media samples, underlying and surrounding the colonies, were also cut out and freeze dried separately for further extraction. Methanol, corresponding to the original volume of the samples before freeze-drying, was applied to powdered dry samples, mixed and incubated for one hour at room temperature. The crude extracts were then collected and assessed for AM activity in the drop-plate assay (section 3.2.4). To further compare the expression of AM activities, isolates U156 and D245 were grown in MB and VNSS broth, both static and shaken, for up to 7 days at room temperature. Cells and cell free spent media, collected on days 1, 3, 5, 7 were separated; the supematants were concentrated under reduced pressure, while the cell pellets were freeze-dried and extracted with methanol. Both the concentrated spent medium and the cell extracts were tested for AM activity in the drop-plate assay (section 3.2.4). 3.2.6. Characterisation of crude bioactive extracts using Thin Layer Chromatography (TLC) Thin Layer Chromatography (TLC) was used for the initial separation of the bioactive extracts obtained from isolates U156 and D245. The crude methanol extracts and blank solvent controls were applied to TLC plates (Merck silica gel 60 F254), and developed in chloroform/methanol (4:1) solution. Spots were identified under UV light. 3.2.6.1. TLC Bioautography Overlay Assay (TLC-BOA) TLC-BOA was used to localise the bioactive compounds on TLC plates after development, employing the protocol by Hamburger and Cordell (1987). Developed TLC plates were sterilized under UV light for 15 minutes and then transferred into sterile petri dishes, where they were overlaid with molten (45°C) LB 10 (Appendix la), containing 0.7% agar-agar, and 0.025% (w/v) of 2,3,5-triphenyl-tetrazolium chloride (TTC, Sigma), and inoculated with an overnight liquid culture of the indicator strain S. aureus 31. Inhibitory activity was observed after 24 hours of incubation at 37°C, as a colourless transparent halo surrounded by a red coloured overlay that indicated bacterial growth. 3.2.6.2. TLC Chrome Azurol Sulfonate assay (TLC-CAS) Chrome Azurol Sulphonate (CAS) reagent was prepared as described by Schwyn and Neilands (1987) and gently applied using a cotton swab on top of developed TLC plates to reveal the presence of iron-binding compounds. Iron binding activity was detected based on the change of colour of the CAS reagent, from blue to orange, within 30 minutes after application. 3.2.6.3. Assessment of the AM activity of a known siderophore To assess whether a commercially available siderophore is able to prevent the growth of the target organism, 2 mg/ml solution of desferrioxamine mesylate (DM, Sigma) in a methanol/deionised water mixture (20:1), was applied in the drop-plate overlay inhibitory assay, previously used for the inhibitory activity assessment of the extracts (section 3.2.4.). Briefly, the DM solution was drop-plated on petri dishes (plates) containing LB 10 agar, and allowed to air dry. A blank methanol/water mixture was used as a negative control. Plates were thereafter gently spread by the overnight culture of the indicator strain S. aureus 31, using a sterile cotton swab; the duplicate plates with dry samples was overlaid by molten (45°C) LB 10, containing 0.7% agar-agar, and inoculated with an overnight liquid culture of S. aureus 31. All seeded plates were incubated overnight at 37°C. 3.2.7. Comparison between production of iron-binding compounds and AM activity 3.2.7.1. Presence of iron-binding compounds and AM activity in the cells of isolates U156 and D245 grown on various media To reveal a possible correlation between the presence of iron-binding compounds and the AM activity, isolates U156 and D245 were grown on different solid media, including MMM, with and without the addition of iron, and also supplemented with either 0.1 mM of the iron chelator - 2,2-Dipyridyl (2,2-D), or 4,4-Dipyridyl (4,4-D) (Sigma). The latter was used as a control, as a compound similar to 2,2-Dipyridyl, but lacks the iron-chelating ability. It was previously shown that neither of these compounds interfered with bacterial growth at a concentration of 0.1 mM (Stelzer et al. 2006). Cells were harvested from the plates, freeze dried and extracted with methanol. Extracts were tested for AM activity in the previously described drop-plate inhibitory assay (section 3.2.4), and for the presence of iron-binding compounds (iron- binding activity) - using the modified CAS liquid assay (Schwyn and Neilands 1987; Payne 1994). For the CAS liquid assay, equal volumes of the methanol extracts and the CAS reagent were mixed and incubated at room temperature for 30 min. Change of colour of the CAS reagent from blue to orange, after addition of the extract (1:1) and incubation at room temperature for 30 minutes, was indicative of the presence of iron-binding compounds in the extracts. A mixture of the CAS reagent and methanol (1:1) was used as a negative control, and a 2 mg/ml methanol solution of the siderophore DM and the CAS reagent (1:1) - as a positive control. Correlation between the presence of iron-binding compounds and AM activity in cells of isolate U156, grown on MA, with the addition of various concentrations of iron chelator 3.2.7.2.1, Determination of the optimal concentration of iron chelator in the growth medium To experimentally estimate the iron binding capacity of the iron chelator 2,2-D in MB and, subsequently, in MA medium, the iron in MB was colorimetrically titrated by addition of 2,2-D, based on the formations of red complex between ferrous iron and 2,2- D (Hill 1930). The development of the red colour was monitored by measuring the absorption of the complex at 430 nm. The tolerance of isolate U156 towards increasing concentrations of 2,2-D and 4,4- D was determined by growing it on MA containing different concentrations of 2,2-D and 4,4-D, ranging from 0.1 mM to 1 mM, in 0.05 mM increments. The highest concentration of the iron chelator, that could sustain bacterial growth, was considered as the maximum level of tolerance. 3.2.7.2.2, Assessment of the iron binding and AM activities of isolate U156 in relation to the availability of iron in the media An overnight liquid culture of isolate U156 was dropped (10 jil drops, 40 drops per plate) on MA, 5 fold diluted MA (to retain the salinity, sodium chloride was added to its original concentration in MA), as well as on MA supplemented with either 0.1 mM, 0.2 mM or 0.3 mM of 2,2-D. Cells were grown at RT for 4 days. Colonies were harvested from the surface of the solid media, weighted, freeze dried and extracted with methanol. AM activities of methanol extracts were measured by serially diluting the extracts in methanol and testing them in the drop-plate inhibitory assay (section 3.2.4.). Maximum dilution of the extract showing AM activity was used as a measure of AM activity, thus, the AM activity was quantitatively expressed as values, corresponding to the maximum dilution of the extract that had an AM activity. The presence of iron binding compounds was measured in the modified CAS liquid assay (section 3.2.7.1.) Iron binding activity of the extracts was determined based on the absorbance of mixtures of the CAS reagent and extracts, measured at 450 and 630 nm. 3,2.7,2.3, Determination of the total protein content All results in section 3.2.12. were normalized with respect to the protein content. To determine the total protein content of the cells, isolate U156 was grown on the various modified MA media (section 3.2.7.2.2.) and the wet weight determined. To prepare cell lysates, cells of isolate U156, grown on solid media, were collected and washed in phosphate buffer (PBS, Appendix le). The total bacterial biomass in samples was precipitated by incubation in 0.6 M solution of trichloroacetic acid (TCA) at -20°C overnight, after which the samples were centrifliged at 21250 x g for 15 minutes, the supematants careftilly removed and discarded. Sodium hydroxide solution (NaOH, 0.66 M) was added to each cell pellet and incubated at 80°C for 20 minutes. Samples were mixed by vortexing every five minutes. Following the incubation, the samples were cooled and diluted 5 fold in 0.66 M NaOH. Standard solutions of protein bovine serum albumin (BSA, Sigma) were prepared in 0.66 M NaOH. Total protein content of samples was quantified using the Bicinchoninic Acid (BCA) Protein Assay Reagent Kit (Pierse Biotechnology) according to the manufacturer's protocol (Smith et al 1985; Wiechelman et al 1988). Briefly, diluted samples and BSA standard solutions at various concentrations, as well as protein extracts of isolate U156 cells, were added to 96 well plates, 10 [iL in each well. The BCA working reagent was prepared and added to the samples, 200 \iL into each well. The mixtures in the plates were incubated at 3TC for 30 minutes, and the absorbance of each well measured at 560 nm using an automated plate-reader (Wallac). The 0.66 M solution of NaOH in the same 96 well plate was used as a blank reference. A standard curve depicting the correlation between the known concentrations of BSA and the absorbance was constructed and used to determine the concentration of protein in the samples of isolate U156 cells grown on various media. The amount of protein in each sample was related back to the wet weight of cells before extraction and used to normaUze the results of AM activity and iron-binding activity (section 3.2.7.2.2.) for 1 mg of protein content. 3.2.8. Effect of the addition of iron on the expression of AM activity by isolates U156 and D245 To assess whether the addition of iron can stimulate the expression of the AM activity in isolates U156 and D245, the isolates were grown on solid VNSS medium, previously shown not to result in bioactivity, and iron-supplemented VNSS containing 10 times more iron compared to normal VNSS. After 5 days, the cells were harvested from the plates, freeze dried and extracted with the amount of methanol equated to the original volume of the cells harvested from the solid media. The AM activity of methanol extracts was assessed using the drop-plate method (section 3.2.4). 3.3. Results 3.3.1. Near full length 16S rRNA gene sequencing Results of near full length 16S rRNA gene sequencing confirmed the phylogeny previously presented in chapter 2 (Figure 2.1). 3.3.2. Extraction and the assessment of AM activity Among the various solvent extracts obtained during the sequential extraction of the cells grown on MA and of the surrounding agar media, AM activity was observed only in the green-coloured crude methanol extracts derived from both isolates U156 and D245, which might be indicative of the similarities in the physiochemical properties of their AM compounds. No activity was observed in the agar surrounding the AM producing cells (Figure 3-1). Crude methanol extracts of isolates U156 and D245 cells showed inhibitory activity against 11 out of 16 tooth surface colonising strains tested. These belonged to the following genera: Neisseria, Pasteurella, Porohyromonas, Bacteroides, and Prevotella (Table 3-1). Both isolates U156 and D245 showed identical spectra of activity. Figure 3-1. Drop-plate AM activity screen of of isolates U156 (a) and D245 (b) cell extracts obtained during the sequential extraction against S. aureus. Extracts are marked as follows: H- hexane, DCM - dichloromethane, EtAc - ethylacetate, MetOH - methanol. The blank solvent controls are indicated by red. Table 3-1. Inhibition of canine tooth surface colonizers by crude methanol extracts, obtained from cells of isolates U156 and D245 grown on MA solid medium. Inhibition by Target strains (WCPN) U156 extract D245 extract Streptococcus australis OH 116 - - Streptococcus gallinacous OH 19 - - Streptococcus suis OH 78 - - Pasteurella dagmatis OH 258 + + Naisseria canis OH 73 + + Neisseria canis OH 255 + + Porphyromonas cangingivalis OH 87 + + Porphyromonas canoris OH 64 + + Porphyromonas macacae OH 77 + + Porphyromonas gulae OH 67 + + Porphyromonas gulae OH 168 + + Porphyromonas endontalis OH 167 + + Bacteroides steroris OH 186 + + Prevotella heparinolyticum OH 188 + + Actinomyces naeslundii OH 69 - - Actinomyces hordeovulneris OH 273 - - + - inhibitory activity observed - - no inhibitory activity observed I I - grown and tested aerobically ] - grown and tested anerobically 3.3.3. Effect of cultivation conditions on the production of AM compounds To assess the effect of the different cultivation conditions, such as the composition of the growth medium and the period of cultivation, on the expression of AM activity of isolate U156 and D245, both isolates were grown in various media and for different periods of time. The results showed a potent AM activity in the extracts of the cells that grew on MA solid medium. No activity was observed from the agar media surrounding the AM producing cells, suggesting that the AM compound is localised intracellularly and may not be secreted. The activity was absent in the extracts of the cells of both isolates grown on VNSS agar or on MMM agar solid media, nor was any AM activity observed in all the liquid cultures, including cells grown in liquid VNSS and MB. Isolates U156 and D245 showed identical behaviour in terms of the expression of AM activity in response to the media composition. The production of bioactive compounds by isolates U156 and D245, grown on MA medium, was also shown to depend on the period of cultivation. After prolonged cultivation (more than 2 weeks), no activity was detected in either the cell extracts or in the surrounding media of U156 and D245. The AM activity of the cells of both U156 and D245 was shown to correlate with colony morphology: bioactive producing cells of the isolate D245 formed dense, circular, raised and slightly concave colonies with distinctive concentric green-coloured circles; and isolate U156 formed green coloured circular colonies. After prolonged cultivation 2 weeks), the colony morphology of the isolates gradually changed, colonies became more smooth and homogenic, and the green colour disappeared; the latter also corresponded to a loss of AM activity (Figure 3-2). a day 4 day 8 day 14 Figure 3-2. Changes in colony morphology observed for isolates U156 (a) and D245 (b), during different periods of cultivation (in days). 3.3.4. Initial separation and characterisation of bioactive crude extracts, obtained from isolates U156 and D245: TLC, TLC-CAS and TLC-BOA The TLC profiles of the U156 and D245 isolates crude methanol extracts were found to be similar (Figure 3-3), producing 8 major spots when developed on TLC plates in the chloroform/methanol mixture. As a step towards a preliminary characterisation of the AM compound, present in methanol extracts of isolates U156 and D245, the developed TLC plates were treated with the CAS reagent (Schwyn and Neilands 1987). CAS is routinely used for the assessment of the iron-binding activity of various compounds, such as siderophores - compounds produced by some bacteria, which act as iron scavengers owing their high affinity towards iron. The assay is based on the removal of ferric ion from the deep blue CAS ferric complex to yield a brightly orange coloured free dye (Schwyn and Neilands 1987; Neilands 1993). Here it was used for the initial characterisation of AM compounds synthesized by isolates U156 and D245 and, hence, to check for such activity in the partially separated crude extracts on the developed TLC plates. Spots were identified on TLC which showed the change of colour, characteristic for iron-free CAS (Figure 3-3). TLC-BOA identified spots on developed TLC plates with AM activity against the target strain. Surprisingly, spots exhibiting AM activity in TLC-BOA were also found to possess affinity to iron that enabled them to bind iron present in the CAS reagent. It suggests, that the compound located in that spot may possess both iron-binding ability and also be responsible for the AM activity of the crude extract. The TLC-CAS and TLC- BOA profiles for crude methanol extracts of isolates U156 and D245 were, once again, found to be similar (Figure 3-3). ) C) o U156 ©245 (a) (b) Figure 3-3. TLC-CAS assay (a) and TLC-BOA (b) of the crude methanol extracts of isolates U156 and D245. Spots on the TLC plate are shown as seen under UV light. Spots, showing both iron-binding and AM activities, are outlined by red dashed ovals. 3.3.5. Assessment of the possibility that the AM compounds in extracts of isolates U156 and D245 are siderophores 3.3.5.1. Drop-plate AM activity assay of the siderophore desferrioxamine mesylate (DM) Experiments were performed to assess whether pure commercially available siderophore is able to prevent cell growth in the inhibitory assay, previously used for the detection of the AM activity in the crude extracts. Resuhs of the experiments showed no AM activity in the spot where DM was applied (Figure 3-4), suggesting that the iron binding activity of a siderophore, which might lead to iron depletion, is not sufficient to the inhibit the target strain. Figure 3-4. Results of the AM activity assessment of the pure siderophore, DM, using drop-plate assay. "S" indicates spots, where DM was applied, "K" is the solvent control (methanol / deionised water, 1:20). 3.3.5.2. Production of iron-binding compounds and the expression of AM activity in the cells of isolates U156 and D245 grown on various media Comparison between production of iron-binding compounds and AM activity in the cells grown on various media revealed that all cell extracts tested showed the presence of iron-binding compounds, whereas only cells of isolates U156 and D245 grown on MA had an AM activity. This also correlated with the green colour of the cells (Figure 3-5). No AM activity could be detected in the extracts of cells grown on MMM that were, however, shown to possess iron-binding activity (Table 3-2, Figure 3-6). Thus, no direct correlation was observed between the presence of iron binding activity and AM activity. Both isolates U156 and D245 showed similar results (Table 3-2). Table 3-2. Presence of AM and iron-binding activities in the methanol extracts of isolates U156 and D245, grown on different solid media. Media Description AM Iron Binding Activity Activity* 1 MMM - Fe MMM without addition of FeS04 x 7H2O inactive positive 2 MMM + Fe MMM with addition of FeS04 x 7H2O inactive positive 3 MMM - Fe + 2D MMM without addition of FeS04 x 7H2O inactive positive and with addition of 2,2 Dipyridyl 4 MMM - Fe + 4D MMM without addition of FeS04 x 7H2O inactive positive and with addition of 4,4-Dipyridyl 5 MMM + Fe + 2D MMM with addition of FeS04 x 7H2O and inactive positive 2,2 Dipyridyl 6 MMM + Fe + 4D MMM with addition of FeS04 x 7H2O and inactive positive 4,4 Dipyridyl 7 MA + 2D MA with addition of 2,2 Dipyridyl active positive 8 MA Marine Agar active positive 9 MA + 4D MA with addition of 4,4 Dipyridyl active positive * As assessed in CAS liquid assay Figure 3-5. Isolates U156 (a) and D245 (b) grown on various media. Media are indicated by numbers that correspond to those in Table 3-2. Figure 3-6. Results of the liquid CAS assay of methanol extracts of isolate D245 cells grown on various media, as indicated in Table 3-2. "+" - positive control, - negative control. Similar results were also observed for the corresponding extracts of the cells of isolate U156. 3.3.5.3. Production of iron-binding compounds and expression of AM activity in the cells of isolate U156 grown on MA with different concentrations of the iron chelator Despite the similarities observed between isolates U156 and D245, the latter was found to have variable levels of expression of the green pigment and AM activity, even when grown on the same medium. Therefore, only the isolate U156 was used in the quantitative experiments described in this section. 3.3,5.3.1, Determination of the optimal concentration of iron chelator in the growth medium Addition of 0.1-0.3 mM of the iron chelator 2,2-D to the MA did not affect the growth of isolate U156, whereas no growth was observed at a concentration of 0.35 mM and higher. 4,4-D had no effect on cell growth up to a concentration of ImM Colorimetric titration of MB by 2,2-D showed that addition of 2,2-D to a final concentration of 0.4 mM led to complete binding of the iron present in the medium (Figure 3-7). Taking into account the cells' tolerance to the presence of iron chelator, the 0.3 mM was chosen as the maximum concentration of the chelator, suitable for growth, and, at the same time, leading to a near-complete binding of the iron present in MA. Therefore, concentrations of 0.1 mM, 0.2 mM and 0.3 mM of 2,2-D in MA were used for further comparative experiments to study the effect of iron availability on the expression of AM activity by isolate U156. Figure 3-7. Colorimetrie titration of MB medium by addition of 2,2-D. Absorbance of the red complex, formed as a result of binding of the iron present in the MB medium by 2,2-D, was measured at 430 nm. 3.3,5,3,2, Assessment of the iron binding and AM activities of isolate U156 in the presence of various concentrations of an iron chelator The increase in iron binding activity of the cell extract was observed as an increase in the absorbance of the CAS mixture with the extract at 430 nm that correlated with the formation of the orange-red complex. As can be seen from Figure 3-8, the iron binding activity of cells of isolate U156 was increased slightly in the presence of 0.3 mM iron chelator (Figure 3-8a). In contrast, the addition of the chelator resulted in more than 5-fold decrease in the AM activity of the cell extract (Figure 3-8b). 1.5 1.25 (a) c mo ''I- 8 • MA 1 0.75 • 5xd MA no.l mM 2D < 0.5 • 0.2 mM 2D • 0.3mM2D 0.25 (b) • MA • 5xd MA OO.l mM2D • 0.2mM 2D • 0.3 mM 2D Figure 3-8. Comparison of the iron binding activity (a) and the AM activity (b) of a crude methanol extract of isolate U156 grown on MA, 5x diluted MA (5xd MA), and MA supplemented with 0.1 mM 2,2-D (same as 2D in this figure), 0.2 mM 2,2-D, and 0.3 mM 2.2-D. Error bars indicate the standard deviations of four replicates. AM activity is presented as a percentage of the activity relative to the activity of the cells grown on MA (100 %). All values are normalised for 1 mg of total protein. 3.3.6. Effect of iron on the expression of AM activity by isolates U156 and D245 To confirm the role of iron in the expression of AM activity, isolates U156 and D245 were grown on iron-supplemented VNSS solid media. The colonies acquired the characteristic green colour (Figure 3-9), with crude extracts having AM activity against the target strain S. aureus. As shown, no colour and no activity was observed for cells grown on normal VNSS, in agreement with previous results (section 3.3.3.)» thus demonstrating the need for a high iron concentration in the media for the production of the AM compound by isolates U156 and D245. U156 D245 a Figure 3-9. Colony morphology of isolate U156 and D245 grown on VNSS agar solid medium (a), iron supplemented VNSS agar solid medium (b), and MA solid medium (c). 3.4. Discussion Bacteria assigned to the light green morphotype (LGM), including isolate U156, were previously found to possess AM activity against various microorganisms (Chapter 2, Table 2-1). Moreover, the spectrum of activity of the isolate U156 crude methanol extract was shown to be similar to that of the phylogenetically distant isolate D245, as demonstrated when both extracts were tested against a broad range of tooth surface bacteria (Table 3-1). TLC analysis of the crude bioactive methanol extract of isolate U156 further showed a striking similarity to the extract of isolate D245 in the localisation of the spots, observed under UV light, as well as in the localisation of the spot showing inhibitory activity in TLC-BOA (Figure 3-3). This study demonstrated that the expression of AM activity by both isolates, U156 and D245, was strongly influenced in a similar fashion by growth conditions, such as media composition and the period of cultivation. The impact of cultivation conditions on the production of secondary metabolites is well known and is discussed in more detail in Chapter 1. In fact, the AM activity of both isolates was observed when they were grown on rich MA, though not expressed when the cells were grown on VNSS agar or in any liquid media, highlighting the importance of attached growth for the expression of AM activity by both isolates. A similar phenomenon was observed by Yan et al. (2002) who demonstrated an enhanced level of antibiotic production in two marine algal- associated Bacillus strains when grown on solid media, as opposed to growth in shaken liquid cultures. The latter is not surprising given the surface-attached lifestyle of these organisms. The difference in media composition also led to the preferential expression of AM activity in the cells grown on MA, but not on VNSS agar. Both media are used for the growth of marine isolates, but MA is considered richer compared to VNSS agar. In particular, MA has a higher iron content, which was found to play a key role in the expression of AM activity by isolates U156 and D245. Antimicrobial and iron binding activities observed for the same spots in TLC BOA (Figure 3-3) were a key to unlocking the growth media requirements for the production of the bioactives. Initially the assumption was that the compound, responsible for the AM activity, might be a siderophore. Siderophores are small iron binding molecules acting as iron scavengers and widespread among bacteria. Due to their high affinity for iron, it was assumed that they might prevent the growth of the target strain via binding the iron present in the media, to an extent which would make it impossible for the target strain to grow (Wang et al 2009). However, the absence of AM activity of the commercially available pure siderophore, desferrioxamine mesylate, in a similar drop- plate inhibitory assay (Figure 3-4) suggested that the iron binding activity of siderophores, presumably leading to iron depletion, may not be sufficient to inhibit the growth of the target strain S. aureus in the drop-plate inhibitory assay used in this study. Therefore, it can be assumed that the AM activity, observed in the extracts of isolates U156 and D245, might not purely be due to the iron binding activity of the compound, but could be achieved via a separate mechanism. Further evidence supporting the suggestion that the AM activity of isolate U156 is not due to it being a siderophore was provided when the correlation between the iron- binding activity of isolate U156, and the availability of iron in the media, was assessed (Figure 3-8a, b). This was based on the theory that the production of siderophores is strictly regulated by the presence of iron in the environment, i.e. their biosynthesis is usually suppressed in the presence of iron and stimulated in iron depleted conditions, where their iron-scavenging ability is of particular importance for cell survival (Neilands 1993; Arahou et al 1998; Calugay et al 2003; Rachid and Ahmed 2005). Experiments designed to compare the AM activity and the iron-binding activity of the isolate U156 grown on various media with different iron availability, showed no direct correlation between AM activity and the availability of iron in the growth medium. However, they did not exclude the presence of siderophores in the extract of isolate U156, independently from the bioactive compound. Specifically, marine amphiphilic siderophores that remain bound to the cell surface via a fatty acid chain, presumably as an adaptation not to release the siderophore molecule into the environment, but rather keep it cell bound, at the same time allowing it to shuttle and supply iron to the cell, have been reported in the literature (Butler 2005; Martinez and Butler 2007). These types of siderophores are usually extracted from the cell pellet, and, therefore, may also be present in the crude extract of the cells of isolates U156andD245. Evidence of the presence of siderophores could also be supported by a slightly increased iron-binding activity in the liquid CAS assay in the extracts of the cells grown under iron depleted conditions, which followed the classical pattern of iron-dependant siderophore regulation (Figure 3-8a). Such siderophores might play an important role in the solubilisation and transport of Fe^^ from the growth media, followed by reduction of Fe^^ to Fe^^ inside the cell (Köster 2001; Andrews et al 2003) However, the sharp decrease of AM activity by U156 when grown in iron depleted conditions (Figure 3-8b) indicated the opposite effect. These observations, coupled with the lack of AM activity of the pure siderophore (Figure 3-4) in the same inhibitory assay, suggests that, even if siderophores are present in the extract, they are not likely to significantly contribute to the AM activity. The results also demonstrate that a high concentration of iron in the growth medium promotes the expression of AM activity by isolate U156, which could be due to another iron binding compound, not considered as a classic siderophore. It is possible that iron is an essential part of the AM compound, and may even be involved in its chemical structure. The correlation of inhibitory activity with the green colour suggests that the AM compound is likely to be a pigment, which is characteristic of iron complexes (Scarpellino 1977; Serres 2002; Takeda 2006; Whited et al. 2006; Xie 2007). A key role of iron for the AM activity was confirmed by the expression of both green colour and AM activity by cells grown on a VNSS medium supplemented with high concentrations of iron (Figure 3-9), as opposed to normal VNSS. Taking into consideration the results showing the accumulation of the green colour and AM activity in cells, followed by the disappearance of both colour and activity (Figure 3-2) with time. It appears that, at the initial stages of growth, when cells are exposed to fresh media with plenty of nutrients including iron, they may store it in the form of the green coloured iron complex, which may also act as an antimicrobial. However, when the nutrients in the media become depleted, cells may access the stored iron to maintain the primary metabolism, which would lead to the disappearance of colour and activity. In such a case, in nutrient and iron rich environments, which can also be attractive to numerous microorganisms, the enhanced expression of AM activity may be of importance for these bacteria, allowing them to effectively compete with other colonisers. Iron storage compounds are widely present in different organisms, including higher eukaryotes, and they include ferritins (found in both bacteria and eukaryotes), bactoferritins (found only in eubacteria), and Dps proteins (present only in bacteria and archea) (Andrews et al. 2003). All three are proteins comprised of multiple subunits, which form a cavity where iron is stored, thus sharing many structural and functional similarities. However, to my best knowledge, no AM activity has been observed for these ferritin-like compounds, suggesting that the AM compound produced by isolates U156 and D245, is different, and could potentially be an unknown type of iron storage compound also possessing an AM activity. The ecological relevance of iron requirement at these high concentrations may be challenging in the light of the common presumption regarding iron limitation in the marine environment. The concentration of iron in the ocean may be as low as 0.02-0.19 nM (Sedwick et al 2005). In such cases, it may often be a growth-limiting factor (Mann and Chisholm 2000; Berman-Frank et al 2001; Kustka et al 2003). However, iron availability in the marine environment may vary significantly taking into consideration also that it can accumulate in the form of iron depositions. Moreover, the close association of microorganisms with iron depositions are widespread in the marine environment. For example, such associations have been found to be present in marine snow (Cowen and Silver 1984; Heldal et al 1996), sediments (Preat et al 2000; Boulvain et al 2001; Fortin and Langley 2005) and epibiotic communities (Gillan et al 2000; Gillan and De Ridder 2001; Gillan et al 2004). Interestingly, an iron- encrusted microbial community was found to be associated with the gastropod Hydrobia ulvae, which, in its turn, is a common macroepibiont of many marine algae, including Ulva species (Anibal et al 2007). Moreover, it has been shown, that exopolymeric substances (EPS) produced by biofilm bacteria can bind various metals, including iron (Geesey et al 1988; McLean et al 1996; Bhaskar and Bhosle 2006; Yu et al 2007). Thus the EPS may be acting as an iron scavenger, possibly due to the richness of anionic groups, such as carboxyls. Therefore, the concentration of iron within the surface biofilm, from where these isolates were obtained, could be substantially higher than the surrounding seawater. In addition, previously reported data also suggest that coastal waters harbour a high abundance of bacteria able to sequester iron in the form of extracellular structures (Heldal et al 1996). Whether the concentration of iron in the particular microenvironment, from which the isolates belonging to the LGM were obtained, is sufficient for the expression of AM activity, remains to be investigated. The similarities in the expression of AM activity of strains U156 and D245 is also of interest in terms of their association with eukaryotic hosts. In addition to being found on the surface of temperate green and red algae, bacteria closely related to both isolates U156 (LGM) and D245 were concurrently found as part of the microbial community associated with corals and sponges. Interestingly these strains were found to be abundant on some diseased corals and sponges, but absent on healthy individuals (Koren and Rosenberg 2006; Webster et al. 2008). Several explanations can be given to that observation. Firstly, these strains may act as active pathogens and cause disease, in which case the AM compound, which may be similar to the compound produced by the phylogenetically distantly related isolate D245, may act as a virulence factor. A second possibility is that, the strains related to U156 and D245 could be be present in many habitats in the "rare biosphere" (Sogin et al 2006; Pedros-Alio 2007) and become abundant on diseased organisms due to the changes in surface conditions, for example, as a result of losing their native defensive symbionts. Recently, the loss of isolate D323 related symbiont NWOOl (further discussed in Chapter 5) by the host sponges was demonstrated as a result of changing the environmental conditions such as temperature (Webster et al 2008); it was followed by a heavy colonisation of the host by various bacteria, including those related to both isolates U156 and D245. Thirdly, there could be a combination of the above two: in some conditions, such as increased temperature (Webster et al 2008), the host can be stressed and less resilient to these bacteria, which, after initial colonisation, may be involved in the development of disease, thus acting as opportunistic pathogens. In conclusion, this study clearly demonstrates many similarities in behaviour, particularly in the expression of AM activity, of isolate U156, as a representative of LGM, and a phylogenetically distant isolate D245. These physiological similarities, coupled with a number of reports showing their coexistence and possibly similar ecological roles (e.g., possible pathogenicity in coral Oculina patagónica (Koren and Rosenberg 2008) and sponge Rhopaloeides odorabile (Webster et al. 2008); induction of larval settlement in Corallina officinalis (Huggett et al 2006)) suggest that these bacteria may complement and interact with each other within the same ecological niche. Future studies will involve the chemical identification and characterisation of the AM compound(s) produced by isolates U156 and D245. However, for this to be achieved, a number of obstacles need to be overcome. For example, the bioactive compound was found to be unstable with the crude extract losing AM activity in the course of a few weeks. This limited stability, coupled with the difficulties in obtaining large amounts of biomass from cells grown on solid media, especially when the time point for harvesting was shown to be crucial, make the up-scaling and chemical characterisation via standard techniques, such as conventional NMR, extremely difficult. Technological advances in the area of chemical purification and characterisation, such as the recently developed technique of capillary probe NMR, which only requires small quantities of the sample (Mitova et al 2008), would be invaluable in this process. In addition, knowledge of the important aspects of cultivation conditions, such as the importance of the attached growth for the expression of antimicrobials by both isolates, as well as the effect of iron concentration in the growth media, described in this study, would greatly assist in the further development of optimal cultivation techniques to enhance the yield of the AM compound produced by these isolates Chemical characterisation of the AM compound(s), produced by phylogenetically distant isolates U156 and D245, would establish whether those bioactives are similar, and, hence, reinforce the possible similarities in the ecological roles of these isolates in the environment. Further studies could also reveal the mechanisms by which iron affects the expression of the AM compound in these bacteria. CHAPTER FOUR Production of violacein by Microbulbifer sp. D250 4.1. Introduction Based on the 16S rRNA gene sequence, isolate D250 appeared to be closely related to the LGM bacteria (Chapter 2, Figure 2-1). However, the colony morphology of D250 was different: while LGM bacteria produced a green pigment when growing on MA, isolate D250 was found to form dark purple coloured colonies (Table 2-1). Numerous purple pigment producing bacteria have been isolated from both freshwater (Logan 1989) and marine environments (Gauthier 1976). In the majority of cases, the pigment has been characterized as violacein - a biologically active compound with a broad spectrum of activity. Violacein has been extensively studied for more than a century. It was first observed in 1882 as the purple pigment produced by the beta-proteobacterium Chromobacterium violaceum (Boisbaudran 1882). Subsequently, it was isolated from C violaceum in 1944 (Strong 1944). Over the last century interest in violacein has been fuelled by reports about its broad range of biological activities, which include antibacterial (Lichstein and Van De Sand 1946; Duran et al. 1994; Leon et al. 2001; Nakamura et al 2003), cytotoxic and antitumoral (Duran et al. 2003; Melo et al 2003; Bromberg et al 2005; Kodach et al 2006), antiviral (May et al 1991; Andrighetti- Frohner et al 2003), antifungal (Becker et al 2009) and antiprotozoal (Matz et al. 2004). Until recently, C. violaceum was regarded as the major source of violacein, however studies over the last 15 years have revealed several other groups of violacein producing bacteria. These include other beta-proteobacteria, such as Janthinobacterium lividum (Shirata et al 1997; Pantanella et al 2007), bacterium RT102 (Nakamura et al 2003), Collimonas sp. (Hakvag et al 2009), and Duganella sp. B2 (Wang et al 2009), as well as several members of the gamma-proteobacteria belonging to the genus Pseudoalteromonas (Franks et al 2005; Yang et al 2007; Wang et al. 2008; Yada et al. 2008). The overall aim of the work described in this chapter was to characterize the AM activity found in the marine bacterium Microbulbifter sp D250. Using analytical chemistry, the antibacterial purple pigment produced by Microbulbifer sp. D250 was identified as violacein. In addition, violacein deficient transposon mutants of D250 were generated and were used to shed light on the regulation of violacein biosynthesis, as well as its ecological role in the producer bacterium, Microbulbifer sp. D250. 4,2. Materials and Methods 4.2.1. 16S rRNA gene sequencing and phylogenetic identification of the isolate D250 Bacterial genomic DNA was extracted according to the XS DNA extraction protocol (Tillett and Neilan 2000) as described in Chapter 2, section 2.2.3. Near full length 16S rRNA gene sequencing was performed and the results were analysed as described in Chapter 3, section 3.2.2. 4.2.2. Extraction and identification of the antimicrobial (AM) purple pigment, produced by isolate D250, as violacein 4.2.2.1. Extraction and the assessment of AM activity of the extracts The previously used sequential extraction method was employed to obtain the extract of isolate D250 (Chapter 3, section 3.2.3). The drop-plate method was used to assess the AM activity of extracts (Chapter 3, section 3.2.4.). Briefly, the extracts were drop-plated on petri dishes containing LB 10 agar, and allowed to air dry. The relevant solvents were used as negative controls. Plates were thereafter gently spread by the overnight culture of the indicator strains N. canis OH73 (WCPN culture collection) and S. aureus 31 (CMB culture collection), using sterile cotton swabs; the duplicate plates with dry samples were overlaid by molten (45°C) LB 10, containing 0.7% agar-agar, and inoculated with an overnight liquid culture of the indicator strain. Plates were incubated overnight at 37°C. AM activity was observed as a zone of clearance on the spot, where the bioactive extracts were dropped. 4.2.2.2. Purification and identification of violacein from the crude bioactive extract ofD250 The crude methanol extract of isolate D250, which showed AM activity, was concentrated under reduced pressure on a rotary evaporator and purified by chromatography using CI8 pre-packed columns (Alltech, lOg). Briefly, the crude bioactive extract, was applied to the preconditioned column and eluted by 100 % water, followed by 10 % methanol (MetOH), 20 % MetOH, 30 % MetOH, 40 % MetOH, 50 % MetOH, and 60 % MetOH solutions in water. After these washing steps the purple fraction, which was retained on the column, was eluted by 100 % MetOH and collected in a fresh vial. This semi-purified sample was again concentrated under vacuum and further purified by reversed-phase high performance liquid chromatography (RP-HPLC) using an Alliance HPLC System comprised of the Waters 2695 Separation Module and the Waters 2998 Photodiode Array Detector. Operation of the HPLC equipment and data collection was controlled by Empower software (Waters). The sample was injected onto the column (Phenomenex, Prodigy ODS3, lOOA, 150 x 4.6 mm, 5 micron), the system started with 100 % water and the concentration of methanol gradually increased until it reached 100 %. The purple coloured fraction was eluted at 6.5 - 7.5 minutes, which corresponded to a dominant peak observed on the chromatogram (Figure 4-1), and tested against S. aureus 31 in the AM activity assay described above (section 4.2.2.1) To identify the purple pigment present in the solution, liquid chromatography electrospray ionisation ion-trap mass spectrometry (LC-ESI-IT-MS) was employed using the LCQ Deca SP lontrap-MS/MS system (Thermo Finnigan) equipped with an autosampler, a quaternary pump, and an electrospray interface. Separation of the components was achieved by RP-HPLC on a Nucleosil CI8 column (3 x 125 mm, particle diameter 5 ^m, Macherey-Nagel, Germany). Twenty microliters of purified concentrated violacein solution was used for injection. The mobile phases used were (A) water acidified with 0.1 % formic acid and (B) acetonitrile at 0.2 ml/min. The gradient program was started with 80 % A and after 3 minutes, the ratio of A decreased to 100 % B via a linear gradient over 13 minutes and maintained there for one minute, until all the violacein was eluted at 13.9 minutes (Figure 4-2). The electrospray voltage was -5 kV with a current of 12 \iA. The sheath gas flow rate was 34 1/min, and the capillary was maintained at 275 Li MS/MS, experiments the mass width for isolation of precursor ions was 1.0 Da, the relative collision energy set at 40 % (arbitrary unit). Chemical structures of violacein and its fragments were drawn using ChemSketch software (ACDLabs). 4.2.3. Transposon mutagenesis and generation of violacein deficient mutants D250-dVl and D250-dV2 To generate mutants of isolate D250, lacking violacein, random transposon mutagenesis was performed using the Tn-5-Km^ and Tn-lO-Km'^ mini-transposon systems. A spontaneous streptomycin resistant mutant of isolate D250 was generated (D250-Sm^) and used as a recipient in both systems. 4.2.3.1. Random transposon mutagenesis with mini-Tn5-Km*^ Transposon mutagenesis with mini-Tn5-Km^, a mini-Tn5 derivative carrying a kanamycin resistance marker (Kristensen et al 1995), was performed by triparental mating. E. coli MG3617 strain was used as a donor, E. coli HBlOl with pRK600 - as a helper. The donor and helper strains were grown in LB 10 containing antibiotics (100 |ig/mL ampicillin (Ap) and 50 ^g/ml kanamycin (Km) for the donor strains, 50 ^ig/ml chloramphenicol (Cm) - for the helper). The recipient (D250-Sm^) was grown in MB supplemented with 200 ^g/ml streptomycin (Sm). Overnight cultures of the recipients, helper, and donor strains were washed twice with fresh media to remove antibiotics and resuspended in 0.1 volumes of 10 mM MgS04. The concentrated recipient, helper, and donor strains were mixed in a 2:1:1 ratio, respectively, and the mixture was dropped on the centre of a sterile filter disc which was then placed on a MA plate. After 12 hours incubation, the bacteria were scraped from the filter, resuspended in 1 ml of MB, and serially diluted. Diluted cells were spread on a MA supplemented with 200 |ig/ml Sm and 100 ^g m/1 Km; the mutants were allowed to grow at room temperature for 48 - 72 hours. Transposon mutants were screened for the loss of purple pigmentation. 4.2.3.2. Random transposon mutagenesis using mini-Tn-lO-Km^ E. coli containing the TnlO based delivery plasmid pLOF/Km, described by Herrero et al (1990), with a Km resistance cassette was used as a transposon donor. Donor cells were conjugated with Sm resistant recipient cells of isolate D250 (D250- Sm^) on filter discs as described above, in 1:1 ratio and incubated for 12 hours at 30 ""C. The conjugation mix was resuspended in 1 mL of MB and serially diluted. The serial dilutions were spread on MA supplemented with 200 |Lig/ml Sm and 100 ^g/ml Km and allowed to grow at room temperature for 48 - 72 hours. Transposon mutants were screened for the loss of purple pigmentation. 4.2.4. Panhandle PCR and sequencing of the DNA regions flanking the transposon 4.2.4.1. Extraction of genomic DNA Bacterial genomic DNA was extracted according to the XS DNA extraction protocol (Tillett and Neilan 2000) as described in Chapter 2, section 2.2.3. Electrophoresis of the DNA obtained was performed in TBE buffer (Appendix He) using 1 % agarose gels (Agarose, Low Electroendoosmosis, Roche) to check for quality and to quantify the extracted genomic DNA. >.-DNA, digested with EcoRI/ Hindlll, was used as a marker. If required, DNA samples were treated with RNAse (Appendix Ila), then extracted with phenol/chloroform/isoamylalcohol (Appendix lib), and ethanol precipitated (Appendix lie). 4.2.4.2. Panhandle PGR Panhandle PGR was used to identify the insertion sites of the transposon, which led to the disruption of specific genes involved in the biosynthesis of violacein. The method for panhandle PGR was adopted from Sibert et al. (1995) with modifications. Briefly, extracted genomic DNA was prepared for panhandle PGR by one-step digestion using a blunt-end generating restriction endonuclease, and ligation of adapters to the digested genomic DNA. In the standard procedure, 1 mg of genomic DNA was digested with 10 U of each of the restriction endonucleases (Roche) listed in Table 4-1, in a 20 ^L reaction volume with the following components: adapter 1 (ADl): 10 pmol, adapter 2 (AD2): 10 pmol, T4 DNA ligase: 2.5 U, ATP: 40 nanomol, buffer solution (Roche) - depending on the endonuclease used. The reaction mixture was incubated at 20''G overnight, and the enzymes inactivated by incubation at l(fC for 10 min. DNA was ethanol precipitated (Appendix LIE) and resuspended in 50 |LIL of molecular grade water (Eppendorf). This "panhandle-ready" DNA was used as a template for panhandle PGR amplification. Table 4-1. Restriction enzymes used for panhandle-PGR. Restriction enzyme Recognition sequence Dr a I TTTjAAA EcoRV GATjATG Hindi GT(T,G)i(A,G)AG Hpal GTTjAGG PvuII GAGjGTG Rsal GTiAG Seal AGTjAGT Sspl AATiATT XmnI GAANNjNNTTG Panhandle PGR amplification was carried out in 20 jiL of reaction mixture containing 1 ^L of "panhandle-ready" DNA, 2.5 mmol of each dNTP (Roche), 10 pmol sequence specific primer, 10 pmol adapter primer 1 (API), 50 nmol MgGb, and 2 f^L lOX REDtaq buffer (Sigma). One unit of REDtaq Polymerase (Sigma) was added at the Hot Start, after the initial thermal ramp. The PGR conditions were 95°G for 3 min, then 30 cycles each of 30 seconds at 95°C and 7 minutes at 68°C. Panhandle PGR products were subjected to agarose-gel electrophoresis. After the electrophoresis, gels were stained in EtBr solution (1 mg/1) for 10 minutes; PGR products were visualised under UV light on the GelDoc transilluminator using the QuantityOne software (Bio-Rad). Amplified panhandle PGR products with a single band were purified with the QIAquick PGR purification kit (Qiagen), whereas single bands were excised from products with multiple bands and purified by the QIAquick gel extraction kit (Qiagen). Purified amplification products were used as sequencing templates. Sequencing was carried out based on BigDye terminator cycle sequencing reaction mix (Applied Biosystems), using pairs of each of the sequence specific primers and API, then analysed on the Applied Biosystems 3730 DNA Analyzer sequencing system at the UNSW ADSF. Homology searches of obtained sequences were performed using the BLAST algorithm {A\Xsc\m\etal 1997). 4.2.5. Phenotypic characterisation of transposon mutants 4.2.5.1. Growth assessment Overnight cultures of isolate isolate D250 (wild type, WT) and the mutants 250- dVl (dVl) and 250-dV2 (dV2) were grown in MB (supplemented by Sm and Km for mutants) for 18 hours. Aliquots of 0.5 mL of each overnight culture, at the same optical density were inoculated into separate 250 mL conical flasks containing 50 mL MB liquid media, in triplicate, and incubated on a horizontal shaker at 27 °G, 180 rpm for up to 30 hours. Absorbance of the cultures was measured using a Novaspec II spectrophotometer (Pharmacia) at 600 nm wavelength. 4.2.5.2. Analysis of pigmentation To obtain a quantitative estimate of the amount of violacein produced by D250 WT and the mutants dVl and dV2, each strain was grown in liquid cultures for 36 hours as described in section 4.2.5.1. The absorption of the methanol extracts obtained from the same cell biomass of each of these strains (wet weights), was measured at 595 nm, which corresponds to the absorption maximum of violacein (Pantanella et al 2007). Methanol cell extract of E. coli, grown in LB 10 broth (Appendix la), was used as a reference. 4.2.5.3. Assessment of AM activity Overnight liquid cultures of transposon mutants dVl and dV2 with altered violacein production were dropped (10 jiL drops) onto MA filled petri dishes, containing Sm (200 |ig/ml) and Km (100 |ig/ml). Approximately 40 drops per dish were applied. Cells were allowed to grow at room temperature for 72 hours. Isolate D250 wt was also grown similarly on MA to be used as a positive control. After 72 hours, cells were careftilly scraped from the agar surface, collected in separate vials and washed in PBS (Appendix le). Samples were freeze dried and extracted with twice the volume of methanol compared to the cells original wet weights before drying. Methanol extracts were dropped onto a surface of fresh MA plates along with blank methanol used as a negative control. After all the solvent had evaporated, each petri dish was overlayed by soft 5 mL LB agar inoculated with 100 ^iL of indicator strains S. aureus 31 and N. canis OH73 (section 4.2.2.1) and incubated at 37 for 24 hours. AM activity was observed as a zone of clearance on the spot where the bioactive extracts were dropped. 4.2.5.4. Assessment of biofilm forming ability A biofilm assay was used to assess the biofilm forming ability of isolate D250 (WT), as well as its violacein deficient mutants dVl and dV2. Briefly, strains were grown in liquid MB overnight. Liquid media used for the mutants was supplemented with antibiotics Sm (200 |ig/ml) and Km (100 |Lig/ml). Ten microliters of overnight cultures were inoculated into 90 ^L of media in each well of the 96 well plates; control wells consisted of blank media. Inoculated 96-well plates were incubated on a horizontal shaker (120 rpm) at RT for 36 hours. After incubation the liquid was removed from the wells and discarded. Wells were washed twice with saline solution and 100 |iL of the crystal violet (CV) solution was added to each well. After 15 minutes of staining, the wells were emptied and washed twice with the saline solution. Stains, retained by the biofilm present in the wells, were extracted by 200 ^L of 100 % ethanol, diluted and transferred to fresh 96 well plates. Absorption of the wells was measured on an automatic plate reader 104 (Wallac) at 550 nm wavelengths; the absorption of the wells, containing blank medium, was used as a reference. To exclude the possible interference of the violacein, present in cell biofilm, with the absorbance measurements of CV stain, due to the similarity of the absorption maxima of violacein and CV, CV was replaced by an alternative stain - safranin in an assay similar to the one used for CV. However, instead of ethanol, safranin, retained by biofilms, was extracted using a mixture of ethanol and acetone (80 : 20 v/v) (Pantanella et al 2007). Absorption of the wells, containing safranin, was measured at 490 nm. 4.2.5.5. Formation of cell aggregates in liquid cultures Overnight cultures of isolate D250 and mutants dVl and dV2 were grown in liquid MB for 48 hours, on a horizontal shaker at 180 rpm. Cells in the liquid cultures were visualized and photographed using a microscope (Olympus U-SPT, Japan) equipped with a camera (Sony DFW-SX900, Japan). 4.3. Results 4.3.1. 16S rRNA gene sequencing and phylogenetic identification of the isolate D250 Near-full-length 16S rRNA gene sequencing confirmed the close phylogenetic relationship of isolate D250 and the LGM bacteria, shown previously, with isolates U156 and D250 sharing an almost identical 16 S rRNA gene sequence and being related to Microbulbifer sp. (Figure 2-1). 4.3.2. Extraction and identification of the antimicrobial purple pigment produced by isolate D250 Methanol extracts of the cells of isolate D250, grown both on solid and liquid media, were found to have an AM activity against the indicator strains S. aureus 31 and N. canis OH73. No activity was found in other solvent extracts nor in the surrounding media. The bioactive methanol cell extract of isolate D250, containing the purple pigment, was used for fiirther purification and characterization of the AM compound. 4.3.2.1. High Performance Liquid Chromatography (HPLC)^ The purple pigment was successfully purified from the crude methanol extract via HPLC and tested positive in the AM assay. The UV spectrum of the compound corresponded to the previously described spectrum of violacein with UV absorbance maxima at 260 and 378 nm (Figure 4-1) (Gauthier 1976; Rettori and Duran 1998) 4.3.2.2. Liquid Chromatography - Mass-Spectrometry (LC-MS)^ The presence of violacein was confirmed by LC-ESI-IT-MS. The molecular ions of violacein were detected as [M+H]^ {m/z = 344) and by a characteristic fragmentation pattern obtained from the source ion in positive ion mode (Rettori and Duran 1998). Consequently, fragment 1, with a mass of 316, could be derived from a parent ion due to the loss of CO from the central ring of violacein, fragment 2 could be a result of CONH loss, and fragment 3 - due to the loss of CONH and NH groups. Fragments 5 and 6 could be formed as a result of the cleavage of the ring system (Figure 4-2). ^ Results, described in this section, were obtained in collaboration with Dr Tilmann Harder ^ Results, described in this section, were obtained in collaboration with Dr David Schleheck 1.00- 0.50- Minutes Figure 4-1. HPLC chromatogram of isolate D250 semi-purified cell methanol extract. The compound eluted at 6.5 - 7.5 minutes, representing the dominant peak, was collected and was chemically identified as the purple pigment violacein. The UV spectrum of the peak is given in the upper right comer. a minutes 316 (J a H ^ "N-X^ ^ H O^ N es H H H H 344 -sQ s Xi CQ 326 299 251 100 120 140 160 180 200 220 240 260 280 300 320 340 Figure 4-2. LC-ESI^-MS characterisation of the purple pigment produced by Microbulbifer sp D250 as violacein, (a) - ESI ion current chromatograms obtained in positive ion mode, (b) - results of MS/MS showing the fragmentation pattern of violacein, (c) - some of the putative fragments derived from violacein in MS/MS and their corresponding masses in parentheses. All mass values are rounded up to the nearest whole numbers. The chemical structure of violacein is shown in (b). 4.3.3. Transposon mutagenesis and generation of violacein deficient mutants D250-dVl (dvl) and D250-dV2 (dV2) To better understand the role of violacein in the AM activity expressed by Microbulbifer sp 250, and to identify genes potentially involved in its biosynthesis and regulation, transposon mutagenesis was performed. Initial conjugation experiments yielded a limited number of transconjugates, therefore, two different transposon systems, mmi- Tn5-Km^ and mini-TnlO-Km , were employed. However, the frequency of mutagenesis, representing the ratio between the number of transconjugates and the number of recipient cells used, for both transposon systems, was found to be as low as 10'^, resulting overall in 483 transconjugates. Transposon mutants (transconjugates) were screened for the loss of purple colour as an indication of the deficiency in violacein production. Two such mutants were obtained; D250-dVl (dVl) and 250-dV2 (dV2), by using Tn5 and TnlO mini-transposon systems, respectively. 4.3.4. Phenotypic characterisation of dVl and dV2 mutants 4.3.4.1. Colony morphological and growth characteristics Transposon mutant dVl was found to form grayish, slightly purple colonies when grown on MA solid medium, while dV2 did not show any purple pigment production (Figure 4-3). All strains reached stationary phase after 22 hours of growth in liquid MB medium. Growth curves obtained for wild type (wt) and both mutants were found not to be significantly different (Figure 4-4), with only minor differences observed in the early exponential growth phase (5-10 hours). Purple pigment production was observed in the wt after 25 hours, at the stationary phase of the growth. No visible pigment production was recorded for either of the mutants in the liquid cultures. Figure 4-3 Isolate D250 (wt) and its transposon mutants D250-dVl (dVl) and D250-dV2 (dV2). OD 15 Time (hours) ^ D250 wt D250-dVl -A^D250d-V2 Figure 4-4. Growth curves of isolate D250 wild type (wt) and its transposon mutants D250-dVl (dVl) and D250-dV2 (dV2). Strains were grown for 30 hours in MB media. Optical densities were measured at 600 nm at various time points during growth. Error bars represent standard deviations between three replicates. 4.3.4.2. Analysis of pigmentation Culture of the dVl mutant showed the presence of only trace amounts of violacein compared to the wild type, as revealed by spectroscopy, whereas no violacein production was observed in dV2, indicating its complete loss of violacein producing capability (Table 4-2). Table 4-2 The absorbance of the methanol extracts obtained from the same cell biomass of isolate D250 and its mutants (wet weights), measured at 595 nm, corresponding to the absorption maximum of violacein. Extract of E. coli cells was used as a reference in spectroscopic measurements. Absorbance 250 wt 32.83 250 dVl 0.03 250 dV2 0 4.3.4.3. AM activity assessment Results of the AM assay of the extracts obtained from D250 wt, as well as its violacein deficient mutants dVl and dV2, revealed no significant decrease in the AM activity of mutant cells despite the lack of violacein (Figure 4-5), suggesting that violacein may not be the only compound with an AM activity produced by isolate D250. Ill Figure 4-5. Drop-plate AM activity test of crude methanol cell extracts of isolate D250 (wt) and its violacein-deficient transposon mutants 250-dVl (dVl) and 250-dV2 (dV2) against N. canis OH73. Methanol (M) was used as a negative control. 4.3.5. Analysis of tlie DNA sequences flanking the transposon insertion sites Sequencing of the panhandle PCR products obtained for the transposon mutants identified the insertion sites of transposons. Since both mutants were deficient in violacein production, it can be assumed, that the genes with a disrupted function, either directly or indirectly, are involved in the biosynthesis of violacein. In dV2 the disrupted gene was similar to vioB encoding for a heme protein / polyketide synthase, which is essential for the production of violacein and included in the violacein biosynthetic cluster of different violacein producing bacteria (August et al. 2000; Brady et al. 2001; Antonio and Creczynski-Pasa 2004). For the mutant D250-dVl, the insertion site was found to be in the gene typA, encoding a ribosome binding GTPase protein TypA, demonstrating, for the first time, the involvement of the typA gene in the production of violacein by isolate D250. Even though the exact mechanism of action of TypA remains unknown, literature data suggest that it is required for growth at low temperature, and low pH (Kiss et al 2004); upregulates the expression of virulence genes (Grant et al 2003), as well as facilitates the host colonization via negatively regulating motility (Farris et al 1998) and modulates the cell surface attachment (Moller et al 2003; DeLivron and Robinson 2008). 4.3.6. Assessment of biofilm forming ability of isolate D250 and the violacein- deflcient mutants dVl and dV2 Taking into account the literature data, suggesting the involvement of TypA in the surface colonization by bacteria, biofilm assay were performed to establish whether TypA is involved in the surface attachment/biofilm formation by isolate D250, which would be observed as the lack of attachment in typA mutant dVL The D250 wild type and the vioB mutant dV2 were used for comparison. To avoid the possible impact of violacein on the absorbance of crystal violet (C V) stained biofilm, due to the similarities in absorption of violacein and CV, an alternative stain, safranin, was also used in a parallel assay. It showed similar trends to the CV assay, suggesting that the violacein, produced in the biofilm in the 96-well plate, does not have a significant impact on the absorbance of the CV stained biofilm (Figure 4-6). By comparison to the wild type, biofilm formation was significantly reduced, not only in the typA disrupted mutant dVl, as was expected, but also in the vioB disrupted violacein-deficient mutant dV2 (Figure 4-6) suggesting the involvement of both TypA and violacein in the formation of biofilm by Microbulbifer sp. D250. 0.50 0.80 0.45 0.70 0.40 0.60 0.35 E i 0.30 lO § 0.25 c 0.40 CD CD n XI)- o 0.20 en S5 0.30 § 0.15 0.20 0.10 0.10 0.05 0.00 0.00 250 wt 250 dV1 250 dV2 250 Wt 250 dV1 250 dV2 Figure 4-6. Formation of biofilm by isolate D250 and its transposon mutants 250-dVl and 250-dV2 as measured by crystal violet (a) and safranin (b) staining. Error bars represent standard deviations between three replicates. 4.3.7. Formation of cell aggregates in liquid cultures of isolate D250 and the violacein-deficient mutants dVl and dV2 Isolate D250 showed the formation of long cylindrical aggregates, visible to the naked eye, in liquid cultures after 48 hours of growth. These structures also showed a high degree of durability and could not be destroyed even after strong agitation, such as vortexing. Under the microscope, the aggregates were found to be heterogeneous, comprised of dark purple cells containing violacein, tightly attached to each other forming long thick highly organized sinuous cylindrical structures (Figure 4-7 a, c, d). Lower violacein content was observed in some parts of these structures, usually at the edges (Figure 4-7 e, f), as well as in the matrix surrounding thee cylindrical structures (Figure 4-7 a, b), as evident by a lighter purple colouration in these areas. These parts also showed less densely packed cells and the abundance of thin interweaving threads (Figure 4-7 b). As opposed to the wild type, no aggregates were seen in liquid cultures of mutants dVl and dV2. Rather, only separate planktonic cells were observed under the microscope (Figure 4-8). Figure 4-7. Heterogeneous cell aggregates formed in the liquid cultures of isolate D250. Images show the long thick sinuous cylindrical structures (a) surrounded by thinner interweaving threads (b); (c, d) - cylindrical structures at higher magnification formed by densely packed cells containing violacein as evident by the deep purple colour; (e, f) - regions surrounding these structures, under high magnification with more loosely packed cells/matrix with less violacein content, as evident by the lighter purple pigmentation. Figure 4-8. Cells of transposon mutants 250-dVl (a) and 250-dV2 (b) grown in liquid cultures and showing the presence of non-pigmented planktonic cells. 4.4. Discussion This study has successfully identified the purple pigment with AM activity, produced by isolate D250, as violacein. As was mentioned above, phylogenetically, isolate D250 was shown to be closely associated with the group of gamma- proteobacteria, all having similar colony morphology forming light green colonies on MA and subsequently called LGM (Figure 2-1). Despite the similarities in the 16S rRNA gene sequences, isolate D250 was found to differ from the LGM bacteria in its phenotype, forming dark purple colonies when grown on the same medium. This fact emphasizes the importance of bacterial culturing that allows for the assessment and identification of physiological differences of bacterial isolates. Transposon mutagenesis of isolate D250 yielded two violacein-deficient mutants, D250-dVl (dVl) and D250-dV2 (dV2). These two mutants, however, differed morphologically; while dV2 showed a complete loss of the purple pigment, dVl had a barely visible light purple pigmentation, which could hint at an altered violacein regulation. Similarities in the dynamics of growth observed for the wild type and the mutants (Figure 4-4) indicate that the deficiency in pigment production is not due to a decline in general fitness of the mutant strains, but may reflect the disruption of specific mechanisms linked to the violacein production. The production of AM compounds other than violacein was observed in isolate D250, as shown by a positive inhibitory effect of both violacein-deficient mutants (Figure 4-5). This demonstrated the presence of multiple antimicrobial compounds in isolate D250, and confirmed the potential of this bacterium as a producer of biologically active compounds. The chemical identification of other secondary metabolites, produced by D250, could be a topic for a future study and could potentially lead to the discovery of structurally novel biologically active compounds. The sequencing of DNA, flanking transposons, indicated that in dV2 the transposon was inserted in the vioB gene that encodes a polyketide synthase, VioB. Disruption in the vioB gene in Chromobacterium violaceium is known to lead to the loss of violacein production (August et al 2000; Balibar and Walsh 2006). This was also demonstrated in the current study, by a complete loss of purple pigment in dV2 mutant. In dVl, the transposon was inserted in a typA - like gene, encoding the ribosome binding GTPase protein TypA. There has been great interest in the gene typA and its homologue bipA in recent years (Krishnan and Flower 2008; Micklinghoff et al 2008; Wang et al 2008). TypA (BipA) was shown to be a regulator required for the adaptation of some bacteria to symbiosis and to stressful environmental conditions (Kiss et al. 2004) and was also found to be involved in the regulation of pathogenicity traits. For example, TypA was shown to negatively regulate flagellar motility promoting the attached growth and colonization, and was necessary for successful invasion of the host (Farris et al. 1998; Scott et al. 2003). TypA was also involved in the upregulation of the expression of genes in pathogenicity islands (Grant et al. 2003) that led to an increase in the resistance towards antimicrobials and enhanced the ability of bacteria to avoid host defense mechanisms (Qi et al. 1995; Farris et al 1998; Barker et al. 2000). The current study showed, for the first time, that TypA may also be involved in the regulation of the production of violacein, a toxic compound with a broad spectrum of growth inhibitory activities. Violacein is often regarded as a virulence factor, providing the producer organism a significant ecological advantage by enabling it to effectively kill its various competitors and predators (Matz et al. 2004). ]n addition, this study also demonstrated, for the first time, that violacein can affect biofilm formation. Based on the observed decrease in biofilm forming ability of both typA (dVl) and vioB (dV2) mutants (Figure 4-6), the involvement of both TypA regulator protein and violacein in biofilm formation is proposed. Biofilms are complex microbial aggregates in which cells grow attached to the surface or to each other in a matrix. This strategy substantially increases the resistance of the cells to the environment by shielding them against damaging environmental conditions, such as UV radiation, and the presence of toxins, as well as promoting a collective defense against predators (Espeland and Wetzel 2001; Hall-Stoodley et al. 2004; Harrison et al 2007). Moreover, given the surface-attached lifestyle of epibiotic isolate D250, the positive effect of violacein on biofilm formation would give the producer organism an ecological advantage allowing it to successfully colonize marine surfaces. In a biofilm mode of life, many bacterial processes are governed by quorum sensing (QS) systems - population dependant gene regulation systems present in a range of bacteria (McClean et al 1997; Blosser and Gray 2000; Juhas et al 2005; Rasmussen and Givskov 2006; Labbate et al 2007; Sokol et al 2007; Irie and Parsek 2008; Wang et al 2008; Willcox et al 2008). Along with various other toxins, the production of violacein in many bacteria was also shown to be controlled by QS (Sainton et al 1992; Bainton et al 1992; Jones et al 1993; McGowan et al 1995; Willcox et al 2008). In gram-negative bacteria QS is often mediated by bacterial signal molecules, such as N- acyl-homoserine lactones (AHLs), which diffuse into the environment, and, after reaching a certain threshold concentration, activate QS regulated genes (Shiner et al 2005; Williams 2007). This method of gene regulation allows bacteria to preserve resources by only expressing QS regulated genes when their population is dense enough to achieve the desired effect, such as to successfully colonize a surface by forming a biofilm or to kill a predator or competitor by collectively releasing toxins. Thus, QS provides advantages for bacteria and contributes to the increased fitness of the population either via i) increasing the damaging ability of the bacterial community, or ii) by increasing the persistence of the population in the presence of unfavorable environmental factors. The QS-controlled biosynthesis of violacein, thus, may serve as an example of the production of a compound which may cause damage in an antagonistic organism, whereas formation of a biofilm, also controlled by QS, is as an example of the mode of life which increases the resistance of bacteria in the environment. Interestingly, Matz et al (2008) have demonstrated that those two manifestations of QS may act in combination. They showed that the production of the bioactive compound violacein was enhanced when cells were grown in biofilms compared to planktonic cultures. For Microbulbifer sp. D250, in particular, the production of violacein was 60 fold higher in the biofilm compared to the planktonic cells. Subsequently, it was concluded that the biofilm mode of life promotes the production of enhanced levels of a toxin, such as violacein, which was regarded as an adaptation to the sessile mode of life, when other means of defense, such as escaping, are limited (Matz et al. 2008). The results presented here indicate, that the production of violacein, besides having a killing effect on other organisms, could also, directly or indirectly, stimulate biofilm formation, delivering an outcome opposite to that previously reported in the literature (e.g. the stimulation of violacein production in biofilms (Matz et al. 2008)). The proposed enhanced biofilm formation by violacein is supported by the decreased biofilm production in D250 violacein deficient mutants (Figure 4-6). A similar effect was observed for the formation of organized cell aggregates in liquid cultures (also called "suspended biofilm"; (Schleheck et al. 2009)) with a distinct heterogeneity, characteristic of biofilm communities (Stewart and Franklin 2008). Moreover, as shown by microscopy, cell aggregates formed by isolate D250 in the liquid culture were not random aggregates, but highly organized structures with a definite pattern of cell aggregation (Figure 4-7). Such "suspended biofilms" were observed for the wild type (Figure 4-7) but not the violacein-deficient mutants of isolate D250 (Figure 4-8). The lack of attachment, as well as the lack of cell aggregation in the absence of violoacein production in strain D250, indicates a dual function of violacein as "a weapon and a signal" (Linares et al. 2006; Fajardo and Martinez 2008) by giving the producer organism the advantage of i) a toxin which can inhibit the growth of competitor microorganisms (weapon), and ii) stimulating biofilm formation thus increasing the resistance of the cells to environmental factors (signal/regulator). There are examples in the literature (Hoffman et al. 2005; Linares et al. 2006), that the role of several antimicrobial compounds is not limited to the growth inhibitory activity. Rather, they can have much broader activities dependent on the concentration of the compound; in low concentration a "signal/regulator" role, involving the modulation of the expression of certain genes, was shown to be prevalent, whereas at high concentrations the mechanisms of cell growth inhibitions dominate and the compound acts as a "weapon" (Linares et al 2006; Fajardo and Martinez 2008). For example, it has been shown, that in low sub-inhibitory concentrations antibiotics such as tobramycin, tetracycline, and norfloxacin can increase biofllm formation in Pseudomonas aeruginosa (Hoffman et al. 2005; Linares et al. 2006). Moreover, tobramycin and tetracycline were also shown to increase the motility and cytotoxicity, respectively, in the same organism (Linares et al. 2006). The current study suggests, for the first time, a dual effect of the bioactive compound violacein which, besides being known for its growth inhibitory activities, may also stimulate biofilm formation. Taking into consideration i) this dual function of violacein, and ii) the involvement of TypA in the production of violacein observed in this study, as well as iii) the role of TypA in the regulation of different pathogeneicity traits, including the stimulation of surface colonization shown in previous studies (Farris et al. 1998; Grant et al. 2003; Scott et al. 2003), it is possible to speculate, that at subinhibitory concentrations violacein may act as a mediator of typA induced surface colonization. Thus, violacein, produced under the control of the typA gene, may serve as a signal/regulator, that at low concentrations stimulates surface colonization/biofilm formation (Figure 4-9), which, as was observed by Matz et al (2008), leads to enhanced levels of violacein biosynthesis in isolate D250, possibly due to the activity of QS systems. The latter allows violacein to rapidly reach its inhibitory concentration within the biofilm and consequently act as a "weapon" against antagonistic organisms, such as protozoa and various surface colonizers (Figure 4-9). violacein (signal/regulator) violacein (antibiotic) Figure 4-9. Putative model of TypA (typA) and violacein regulated biofilm formation in isolate D250. TypA upregulates the production of violacein in planktonic cells (a, b). Initially, at low concentrations, violacein, acting as a regulator, stimulates the formation of biofilm (b, c). After the establishment of biofilm, QS mechanisms become effective and enhance the biosynthesis of violacein, allowing it to rapidly reach an inhibitory concentration and act as an antibiotic (d). In summary, the results described in this chapter demonstrated the production of violacein by Microbulbifer sp. D250, to date, the only violacein producing gamma- proteobacterium outside of the family Pseudoalteromonas. Violacein deficient transposon mutants of D250 were generated. The sequencing of the transposon insertion site, which led to the disruption of violacein production in one of the mutants, pointed to a gene typA previously described to be involved in the regulation of the colonization and pathogenicity. Here, for the first time, typA was linked with the production of violacein. Based on the biofilm forming ability of violacein-deficient transposon mutants, a new model of violacein-regulated biofilm formation was proposed for Microbulbifer sp. D250. The presence of another AM compound was also observed, suggesting the production of multiple bioactives by isolate D250. CHAPTER FIVE Production of tropodithietic acid (TDA) and phenol by the epiphytic alpha-proteobacterium D323 and related sponge associated bacteria 5.1. Introduction In a search for AM producing epiphytic bacteria, isolate D323, obtained from the surface of the red alga Delisea pulchra, showed a pronounced antibacterial activity (Chapter 2) (Penesyan et al 2009). Phylogenetically related bacteria were isolated from the surface of the same alga in a previous study (Longford 2008), suggesting that they are common D. pulchra epibionts. Bacteria, closely related to isolate D323, have also been reported as present in association with different marine sponges in various geographical regions (Thiel and Imhoff 2003; Lafi et al 2005; Muscholl-Silberhom et al 2008) and absent in the surrounding seawater (Enticknap et al. 2006), reflecting their host specificity. Moreover, it is believed that these bacteria could play a significant role in those associations, as they were found to be present in healthy sponges, though absent in diseased individuals (Enticknap et al 2006; Webster et al 2008). Li addition, Enticknap et al (2006) demonstrated that related bacterium NWOOl could be vertically transferred from the parent sponge to the offspring emphasizing their importance for the host. Because of the possible significance of these bacteria for the host organisms, it was considered important to characterise the bioactive compounds produced by the D. pulchra surface-associated bacterium D323, the phylogenetically related sponge isolates CCSH21, CCSH24 obtained from a temperate sponge C. concentrica (P.-Y. Yung, unpublished data), and NWOOl, isolated from a tropical sponge R. odorabile (Webster and Hill 2001; Webster et al 2001). Consequently, tropodithietic acid (TDA) was successfully identified in this study as an AM compound produced by isolate D323 and related sponge-associated bacteria. The potent broad-spectrum activity of TDA against other marine surface colonising bacteria, including strains linked to the development of disease in marine eukaryotes, was demonstrated. It is proposed that the production of TDA by isolate D323 and related eukaryote-associated bacteria may have implications in the defence of the host against unwanted colonisations. 5.2. Materials and Methods 5.2.1. Strains and media used Isolate D323 was obtained from the surface of the marine red alga Delisea pulchra (Chapter 2); isolates CCSH21 and CCSH24 were isolated from the temperate sponge Cymbastela concéntrica (Yung P.-Y., unpublished data) from coastal waters near Sydney, Australia; alpha-proteobacterium NWOOl was isolated from the tropical sponge Rhopaloeides odorabile (Webster and Hill 2001) from the Great Barrier Reef, Australia; Phaeobacter inhibiens T5 was isolated from marine sediment in Wadden Sea, Germany; Phaeobacter inhibiens T5-3 is a spontaneous mutant of isolate T5, lacking pigmentation and deficient in TDA production ability (Brinkhoff et al. 2004). All the strains mentioned above, as well as the marine epibiotic strains used as targets for the assessment of the spectrum of activity of TDA (Table 5-3), were grown in Marine Broth (MB) or Marine Agar (MA) media (both Difco 2216). Batch cultures used for extraction were obtained by inoculating the liquid media with 10% of overnight liquid culture of an isolate in large sterile conical flasks and growing them at 2TC for 24 hours. All liquid cultures were incubated on horizontal shakers for aeration. Neisseria canis OH73 (WCPN culture collection) was routinely used as an indicator strain for the detection of inhibitory activity due to its high sensitivity to TDA. For TLC-BOA (section 5.2.8) the latter was grown on LB 10 solid medium (Appendix la); and on CBA (Appendix Id) when used in the disc-diffusion assay (section 5.2.5). Target strains - tooth surface colonisers (provided by WCPN) were used to explore the inhibitory activity of the spent medium of an overnight liquid culture of isolate D323 and are listed in Table 5.1. These bacteria were grown in liquid Brain Heart Infusion medium (BHI; Brain Heart Inftision powder: 37 g/1, Oxoid) and on CBA solid medium (Appendix Id) (Elliott et al. 2006). Hemin and menadione were filter sterilized and added to both BHI and CBA. Clinical isolates and marine epibiotic strains used to assess the range of antimicrobial (AM) activities were obtained from the Centre for Marine Bio-Innovation (CMB) culture collection. The growth conditions of clinical strains are given in Table 5-2. 5.2.2. Near-full-length 16S rRNA gene sequencing and phylogenetic identification of the isolate D323 Bacterial genomic DNA was extracted according to the XS DNA extraction protocol (Tillett and Neilan 2000) described in Chapter 2, section 2.2.3. Near ftill length 16S rRNA gene sequencing was performed and the results were analysed as described in Chapter 3, section 3.2.2. A neighbour-joining tree was constructed using the ARB software (Ludwig et al. 2004) 5.2.3. Testing of the isolate D323 cell free spent culture medium for AM activity A batch culture (section 5.2.1) of isolate D323 was centrifliged at 15200 x g, 10°C for 5 minutes. The cell pellet was discarded and the cell-free spent medium collected into a fresh vial. The cell free liquid broth, as well as blank MB medium used as negative control, weres dropped (10 jiil) on the surface of a petri dish containing CBA medium and a lawn of overnight culture of the target strains listed in Table 5-1. Inhibitory activity was observed as a zone of clearance in the spot, where the cell-free spent medium was placed. 5.2.4. Extraction of the cell free spent culture media 5.2.4.1. Liquid-liquid extraction To obtain crude bioactive extracts of isolate D323, as well as CCSH21, CCSH24, NWOOl, P. inhibens T5, and P. inhibens T5-3, the cell free spent media of overnight batch cultures were acidified to pH 2 with hydrochloric acid (HCl) and subjected to liquid-liquid extraction with ethylacetate, to give a crude ethylacetate extract, referred to as CEE. The ethylacetate extracts of isolate D323, obtained by extracting the neutral (pH 7) and alkaline (pH 11, achieved by adding NaOH) spent media, were used for comparison in TLC and TLC-BOA experiments (section 5.2.8). 5.2.4.2. Precipitation After liquid-liquid extraction (section 5.2.4.1), the acidified (pH 2) cell free spent culture medium of isolate D323 was incubated at room temperature for 24-48 hours. A dark brown precipitant was collected by centrifugation and washed three times in water, followed by methanol, ethylacetate and hexane. The resultant dry dark-brown precipitant (DBP) was further dissolved in 0.1 M solution of NaOH and assessed for inhibitory activity in a disc-diffusion assay (section 5.2.5) along with a O.IM NaOH as a negative control. 5.2.5. Disc diffusion assay for the assessment of AM activity The disc diffusion assay (Bauer et al. 1966) was used to test the AM activity of the CEE extract (section 5.2.4.1) and its fractions (section 5.2.7), dissolved DBP (section 5.2.4.2) as well as purified TDA. Briefly, 20 |iL aliquots of test solution were applied to filter paper discs (d = 6mm, BioRad) and allowed to air-dry. Dry discs containing samples were placed on petri dishes containing solid CBA medium (section 5.2.1) and spread by a target strain, N. canis OH73. The seeded petri dish was incubated for 24-48 hours. Inhibitory/AM activity was observed as a zone of clearance around the filter paper discs. Blank solvent was used as a negative control. For AM activity assessment of CEE and DBP, 1 mg/ml solutions of dry samples were used (CEE was dissolved in methanol, DBP - in 0.1 M NaOH), along with blank solvents as controls. To determine the range of activity of TDA, 100 |Lig/ml of purified TDA solution in methanol was applied to filter paper discs, with blank methanol as a negative control. 5.2.6. Gas Chromatography - Mass Spectrometry (GC-MS) of isolate D323 crude bioactive extract GC-MS analysis of CEE of isolate D323 was performed on the 5890 series II gas chromatograph (Hewlett Packard), using a DBS 30m x 0.32mm (internal diameter) x 250mm (film thickness) column, coupled to a 5972A mass-spectrometer (Hewlett Packard). The inlet temperature was set at 250°C; with a split ratio of 50:1. The GC oven was operated at an initial temperature of 50°C for 1 min and then increased to 200°C at a rate of 20°C/min. The transfer line temperature was set to 250°C. 5.2.7. Fractionation of isolate D323 CE and the assessment of the possible AM activity of phenol CEE obtained from isolate D323, was fractionated via solid phase extraction using Silica prepacked cartridge (Alltech) to obtain a phenol-free fraction. Six fractions (F1-F6) were eluted using an ethylacetate : hexane (5:1) mixture and collected into separate vials. The final fraction, F6, was obtained by eluting the cartridges with a 100 % methanol. All fractions were assessed by TLC, TLC-BOA (section 5.2.8), and the disc- diffusion assay (section 5.2.5). Various concentrations of phenol in ethylacetate (1 mg/ml, 0.1 mg/ml, and 0.01 mg/ml), blank solvents, as well as the ethylacetate extract of acidified MB medium were used as negative controls. 5.2.8. Thin Layer Chromatography (TLC) and TLC - Bioautography Overlay Assay (TLC-BOA) Extracts of interest were spotted on TLC plates (Merck silica gel 60 F254) in duplicate, along with a phenol standard (solutions of crystallized phenol (Sigma), in ethylacetate) and ethylacetate extract, obtained from the acidified blank MB medium as controls. The TLC plates were developed in ethylacetate : hexane (5:1), air-dried and stained in iodine vapour for 5 minutes to visualise the spots. The duplicate plates were used for the TLC Bioautography assay (TLC-BOA) (Hamburger and Cordell 1987). Briefly, the developed TLC plate was air-dried, sterilised under UV for 15 minutes and overlayed by soft LB 10 agar containing 0.01 % (w/v), 2,3,5-Triphenyl-tetrazolium chloride (TTC, Sigma) and inoculated with a 5 % (v/v) overnight culture of target indicator strain N. canis OH73. The overlayed plate was placed into a 37°C incubator overnight. AM activity was observed as a transparent halo on a red background, indicating bacterial growth. 5.2.9. Assessment for the production of phenol and TDA by D323 in marine minimal medium (MMM) in the presence and absence of L-Tyrosine Isolate D323 was grown in liquid MMM (Neidhard et al 1974), as well as in MMM supplemented with 0.9 mM L-tyrosine (Sigma) for 24 hours. The resulting supernatant was acidified, extracted with ethylacetate and analysed by TLC and TLC- BOA as described above (section 5.2.8). Extracts of P. inhibens T5 and T5-3, grown in liquid MMM, were also obtained and used for comparison in the TLC and TLC-BOA assays. 5.2.10. Purification and identification of the AM compound produced by isolate D323 and the related sponge associated bacteria CCSH21, CCSH24 and NWOOl 5.2.10.1. Purification Precipitated DBP obtained from the acidified spent medium of the isolate D323 overnight liquid culture was only soluble in highly alkaline water, such as a 0.1 M solution of NaOH, and not in any organic solvents. This made further purification of AM compound, present in the DBP, difficult, as the conventional chromatographic materials are incompatible with such alkaline conditions. Therefore, the focus was on the purification and characterisation of AM compound(s) present in the crude ethylacetate extracts (CEE). The CEEs, obtained as described in section 5.2.4.1, from the spent media of overnight liquid culture of isolate D323, related sponge-associated bacteria, as well as P. inhibens, were separately concentrated under vacuum, yielding an orange-red precipitate. Précipitants were washed with 15 %, 40 % and 60 % methanol, all containing 0.05 % trifluoroacetic acid (TFA) and dissolved in acetonitrile (ACN). Dissolved samples were analysed by reversed-phase high performance liquid chromatography (RP-HPLC) on an Alliance HPLC System comprised of a 2695 Separation Module and a 2998 Photodiode Array Detector (Waters). Operation of the HPLC equipment and data collection was controlled by Empower software (Waters). Samples dissolved in ACN were injected onto the column (Phenomenex, Prodigy ODS3, lOOA, 150 x 4.6 mm, 5 micron); at injection the column was equilibrated with 30 % methanol containing 0.05 % (v/v) TFA. The flow rate was set at 0.9 ml/min. After one minute, the concentration of methanol was increased to 80 % by a linear gradient between 1 and 15 minutes, then maintained at 80 % between 15-20 minutes and decreased to 30 % between 20 - 25 minutes. The UV spectrum of the dominant peak (6.957 minutes), was recorded. 5.2.10.2. Structural elucidation of antimicrobial compound present in CEEs The structure of the purified AM compound, present in CEE, was determined by Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS). 5.2.10.2.1, Liquid Chromatography - Mass Spectrometry (LC-MS) The bioactive fraction from CEE was purified as described above (section 5.2.10.1) and analysed by reinjection on a LCQ Deca XP plus LC-MS system combined with a photodiode array detector (LC-UV-MS) (Thermo Finnigan). Samples were separated on a Gemini CI8 column (50 x 4.6 mm, 5 micron) by using a water-ACN (containing 0.05 % TFA) gradient system (30% to 60% ACN in 10 min). The peak, corresponding to the bioactive compound, was analyzed in the positive electrospray ionization (ESI^) and at atmospheric pressure chemical ionization (APCI). Data were collected from m/z 100 to 220. 5.2.10.2.2, Nuclear Magnetic Resonance (NMR) For NMR analysis the purified dry samples obtained from CEE (as described in section 5.2.10.1) were dissolved in 1 ml of d6-DMS0 (for '^C NMR) and d6-C6H6 (for ^H NMR). Samples were analysed on a Bruker DFX 300 MHz system. The data was processed using TopSpin (Bruker) and SpinWorks (http://www.umanitoba.ca/chemistrv/nmr/spinworks) softwares. 5.3. Results 5.3.1. Phylogenetic identification of isolate D323 According to the 16S rRNA gene sequence, isolate D323 was phylogenetically related to the alpha-proteobacterium MBIC3368 with 98% identity in the 16S rRNA gene sequence (Figure 5-1). Moreover, its colony morphology, showing the formation of flat brown mucoid colonies when grown on MA, corresponded to that previously reported for sponge-associated MBIC3368 related bacteria (Webster and Hill 2001; Enticknap et al. 2006; Muscholl-Silberhom et al 2008). alpha proteobacterium MBIC3368, AF218241 tsolate_D323 sponge bacterium Isolates, AY948383 alpha proteobacterium CRA 8L. AY$625$4 alpha proteobacterium 1105, DQ888838 sponge isolate CCSH24 J sponge isolate CCSH21 ^ alpha proteobacterium NW001, AF29S099 f* alpha proteobacterium JE062,00097238 ^ alpha proteobacterium JE061, DQ097237 Roseobacter sp. 27-4, AJ536669 Phaeobacter galiaeciensis BS107, Y13244 Phaeobacter inhibens, AY177712 Phaeobacter sp. 2.10 JUIL Figure 5-1. Neighbour-joining phylogenetic tree showing the isolates used in this study (in red) and their close neighbours from GenBank related to isolate D323 (in black) and P. inhibens (in blue). 5.3.2. AM activity assessment of cell free spent medium of isolate D323 Cell free spent medium of isolate D323 showed an inhibitory activity against all the 16 target strains provided by WCPN and described as primary tooth colonisers (Table 5-1). Table 5-1. AM activity of cell free spent medium of isolate D323 liquid culture against primary tooth surface colonising bacteria provided by WCPN. Inhibition by D323 Target strains (WCPN) spent medium Streptococcus australis OH 116 4- Streptococcus gallinacous OH 19 -1- Streptococcus suis OH 78 + Pasteurella dagmatis OH 258 + Naisseria canis OH 73 + Neisseria canis OH 255 + Porphyromonas cangingivalis OH 87 + Porphyromonas canoris OH 64 + Porphyromonas macacae OH 77 + Porphyromonas gulae OH 67 + Porphyromonas gulae OH 168 + Porphyromonas endontalis OH 167 + Bacteroides steroris OH 186 + Prevotella heparinolyticum OH 188 + Actinomyces naeslundii OH 69 + Actinomyces hordeovulneris OH 273 + + - inhibitory activity observed - - no inhibitory activity observed I I - grown and tested aerobically I I - grown and tested anaerobically 5.3.3. AM activity assessment of CEE and DBF obtained from isolate D323 spent medium Both the crude ethylacetate extract (CEE) obtained by liquid-liquid extraction of acidified spent medium of isolate D323 and related bacteria, as well as the washed dark brown precipitant (DBF) formed in the same spent medium, showed inhibitory activity against indicator strain N. canis 73. The activity of CEE in the disc-diffusion assay was found to be stronger compared to that of DBP, as evident from the larger zone of inhibition around the filter paper disc containing the CEE (Figure 5-2). Assessment of the antibacterial activities of the CEE and DBP against various clinical strains revealed differences in their activities (Table 5-2) suggesting that the AM compound present in DBP differed from that present in CEE (later identified as tropodithietic acid or TDA). As opposed to the crude extract of acidified spent medium of isolate D323 (CEE, same as CI in Figure 5-9); no AM activity was found in the ethylacetate extract, when spent media were extracted at neutral (C2 in Figure 5-9) or alkaline conditions (C3 in Figure 5-9), which was indicative of the acidic properties of the AM compound present in CEE. Figure 5-2. Disc-diffusion assay of crude extract (CEE) and dark brown precipitate (DBP) obtained from isolate D323 spent medium against N. canis OH73. E A - blank ethylacetate control, NaOH - blank 0. IM solution of NaOH. Table 5-2. Antibacterial activities of the CEE extract and the DBP precipitated fraction against various clinical strains Growth Inhibition by Inhibition Clinical strains Growth media temperature TDA present by DBP (°C) in CEE Staphylococcus aureus 31 LBlOa/LBlOb 37 ++ + Staphylococcus aureus N135 LBlOa/LBlOb 37 + + Pseudomonas aeruginosa LBlOa/LBlOb 37 + - Neisseria canis OH73 CBA/LBlOb 37 +++ + Klebsiella pneumoniae UNSW 0045 LBlOa/LBlOb 37 + + Serratia liquefaciens MGl LBlOaVLBlOb 37 + + Serratia liquefaciens MG44 LBlOa/LBlOb 37 + - Xanthomonas sp. 9 LBlOa/LBlOb 37 ++ - Streptococcus sobrinus CBA/BHIb 37 ++ ++ Streptococcus salivarius CBA/BHIb 37 + + Corynebacterium xerosis CBA/BHIb 37 + + Corynebacterium GpC A48 CBA/BHIb 37 + + Corynebacterium jeikeium 96 CBA/BHIb 37 + - Salmonella typhimurium TA 100 NA/NB 37 +++ + Vibrio cholerae C7258 LBlOa/LBlOb 37 ++ + Vibrio cholerae 638 LBlOa/LBlOb 37 ++ + Vibrio vulnificus CI 184 (0) LBlOa/LBlOb 37 + + Vibrio vulnificus C7184 (T) LBlOa/LBlOb 37 +++ +++ + - zone of clearance in disc diffusion up to 3 mm from the edge of the disc ++ - 3-5 mm +++ more than 5 mm - -no inhibition observed LB 10b - LB 10 broth (liquid) LB 10a-LB 10 agar (solid) BHIb - BHI broth BHIa - BHI agar NB - Nutrient Broth (Oxoid) NA - Nutrient Agar (Oxoid) 5.3.4. Purification and RP-HPLC analysis of tropodithietic acid (TDA) in the CEE extracts of isolates D323, CCSH21, CCSH24, and NWOOl" The AM compound, purified from the bioactive CEE extracts, showed a dominant peak in the RP HPLC chromatogram at the retention time 6.957 minutes (Figure 5-3) under the chromatographic settings given above. The UV spectrum of the compound was in accordance with that reported for TDA (Liang 2003). Results, described in this section, were obtained in collaboration with Dr Tilmann Harder 133 6 957 Exiracted iJX 1/W \ \ 302.2 1J0 \\ / \ MCO a« \ / \ / \ i a»» \ / \ 0.70 \ / \ oeo 0.50 \ / \ 356.0 0/« V OJO \ 020 \ aio 210 00 moo ixco ? 10.00 1200 Minutes Figure 5-3. RP-HPLC chromatogram of the purified TDA obtained from isolate D323 showing a single dominant peak and its corresponding UV spectrum 5.3.5. Structure elucidation and identification of the antimicrobial compound present in isolate D323 CEE as tropodithietic acid (TDA)^^ 5.3.5.1. Liquid Chromatography - Mass Spectrometry (LC-MS) Liquid chromatography-electrospray ionisation tandem mass spectrometry (LC- ESf-MS) of purified TDA from GEE, performed in the positive ion mode, showed a molecular ion [M+H]^ of 212.88 Da and a dominant fragment ion [M+H-H20]'^ at 194.89 Da (Figure 5-4). Liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS) showed a dominant molecular ion [M+H]^ of 212.94 Da and a dominant fragment ion [M+H-C02]'" at 169.15 Da (Figure 5-5). Collision induced MS^ of the molecular ion in ESf did not result in further fragmentation. These data are in direct accordance with the LC-MS data of TDA reported by Bmhn et al (2005). ^^ Results, described in this section, were obtained in collaboration with Dr Tilmann Harder 134 RT: 0.00-10.01 4.04 NL: 5.35E6 lOQn 4.Qa 4J)8 TIC MS 323_ph3_eslpos_100ug r^ 6.47^4 7J6 8.69 9.60 NL: 5.35E6 TIC F:+ c ESI Full ms [190.00-220.00] MS ICIS 323_ph3 eslposJOOug NL: 7.40E5 TIC F: + c ESI Full m82 [email protected] [100.00-220.00] MS ICIS 323_ph3_esipo8_100u9 NL: 3.23E5 TIC F: + c ESI SRM m82 [email protected] [104.00-106.00. 168.00-170.00,195.00-197.00, 212.00-214.00] MS 323_ph3_e8ipos_100ug 4 5 6 Time (min) 323_i)h3_eslpo8_100ug #256-285 RT: 3.78-4.18 AV: 10 NL: 1.13E5 T: + c ESI SRM ins2 [email protected] [104.00-106.00,168.00-170.00,195.00-197.00,212.00-214.00] lOOq 195.15 95| 9 I ^ s. ^ 35^ 30£ 25 20z ^.91 lOl 5i 1^.79 196.80 T—1—I—I—I—I—I—I—I—I—I—I—r~1—I—I—I—i—I—rn— r —]—I—i—I—I—I—1—I—I—I—I—I—1—r "1—I—I—I—I—I—I—I—r I I I I I I I 110 120 130 140 150 160 170 180 190 200 210 m/z Figure 5-4. LC-ESf-MS spectra of TDA purified from isolate D323. NL: 1.68E8 TIC MS 323_apco lOOug 5 Time (min) 323_apoo lOOug #340-378 RT: 4.41-4.79 AV: 13 NL 2.24E7 T: + c APCI corona Full ms [100.00-214.00] 212.94 100^ 95| 90\ 80\ 7S\ 169.15 7(H 65^ 6(H 50\ 4S\ 40\ 35\ 30\ 16«£1 2S{ 21180 20\ 110.92 15^ 1U.91 195.02 144.89 17j.11 15J.09 200.18 11^.93 127.13 186.94 105.87 1^.91 176.83 ^.85 si 123J8 1.25 144^ 190.84 7 I . 111 iHiul LLTL L 100 110 120 130 140 160 160 170 180 190 200 210 Figure 5-5. LC-APCI-MS spectra of TDA purified from isolate D323 5.3.5.2. Nuclear Magnetic Resonance (NMR) Results of both ^H and '^C NMR of the AM compound present in CEE were consistent with the data previously reported TDA (Figure 5-6 a, b.) (Liang 2003), a compound comprising of a carboxylic tropone skeleton connected with a four-membered disulfide ring system. ^^C NMR showed the presence of eight carbon signals (Figure 5-6 a), including one carboxylic carbon, an unsaturated enolic carbon, and six aromatic carbons including two carbons connected with heteroatoms. 5.3.6. Analysis of CEEs by Gas Chromatography - Mass Spectrometry (GC- MS) and Thin Layer Chromatography (TLC)^^ GC-MS spectrum of isolate D323 bioactive crude ethylacetate extract indicated that phenol was one of the major components of isolate D323 CEE (Figure 5-7). TLC profiles of crude ethylacetate extracts of sponge associated isolates CCSH21, CCSH24 and NWOOl grown in MB were found to be identical to the TLC profile of algal associated isolate D323 (Figure 5-8), and also showed the presence of phenol. In contrast to isolate D323, no phenol was detected either in P. inhibens or its mutant T5-3 grown in the same medium, while the production of TDA was demonstrated by the zone of inhibition in TLC-BOA, in all the crude extracts of isolates grown in MB, except the TDA-deflcient mutant P. inhibens T5-3 (Figure 5-8), in accordance with the previous report {Brinkhoffet al. 2004). ^^ GC-MS results, described in this section, were obtained in collaboration with Dr Matthew Lee 137 •u o> WW Ua S.u, a H c 5c 300 MHz 6c 125 MHz (this study) (Liang, 2003) 1 182.9 182.4 2 120.3 120.0 3 167.9 168.1 4 150.9 150.2 5 134.2 133.4 6 138.0 137.3 7 138.1 137.5 8 170.9 170.6 PPM —I—'—I—'—I—'—I—'—I—'—I—'—I—'—I—^—I—'—I—'—I—'—1—'—I—'—I—'—ISO 176 172 16S 164 160 156 152 14S 144 140 136 132 i12—'—[4 —120 I w ^JUl ^ IAIlAaaA^ L PPM 6.70 6.60 6.50 6.40 6.30 6.20 6.10 6.00 5.90 5.80 5.70 5.60 5.50 5.40 Figure 5-6. ^^C (a) and ^H (b) NMR spectra of TDA obtained from CEE of isolate D323. IDA purified from isolate D323 was dissolved in d6-DMSO (for '^C NMR) and d6- benzene (for ^H) prior to measurements. The spectrum was obtained on Bruker DPX 300 MHz spectrometer. The chemical structure of TDA is given in (a). ^^C NMR spectroscopic data for TDA obtained from isolate D323 in this study, and the data previously reported for TDA by Liang (2003), are compared in the table included in (a). Abundance rrVZ"> Abundance m/z-> 100 10: Figure 5-7. GC-MS analysis of isolate D323 CEE (a) and corresponding MS spectrum of phenol (b). Identical masses are indicated by dashed arrows, the mass of the parental molecule (phenol) is indicated by the red arrow. PS P.i. P.i.-3 323 21 24 NW ?S a b Figure 5-8. TLC (a) and TLC-BOA (b) results of the crude extract obtained from isolate D323 (323); CCSH21 (21); CCSH24 (24); NWOOl (NW); P.inhibens T5 (P.i.) and its TDA-deficient mutant P. inhibens T5-3 (P.i.-3), all grown in MB. Purified TDA (TDA) and phenol (PS, 0.1 mg/ml, in ethylacetate) were used as standards 5.3.7. Fractionation of isolate D323 CEE and the assessment of the inhibitory activity of phenol TLC confirmed the presence of phenol in all three ethylacetate extracts obtained from isolate D323 at acidic (CI, same as CEE), neutral (C2) and alkaline (C3) conditions (Figure 5-9). For the six fractions, F1-F6, obtained from the acidified spent medium extract CI by solid phase extraction, no visible spots were found for fractions F1 - F3, phenol was found to be present in fractions F4 and F5, whereas fraction F6 did not contain phenol, but showed the presence of TDA as evident by inhibition observed in TLC-BOA. Phenol did not show any inhibitory activity in the TLC-BOA as indicated by the absence of inhibition in the spot, where phenol is located on the TLC plate (Figure 5-9). The AM activity of fraction F6 was confirmed in the disc-difftision assay, whereas no AM activity was observed for the ethylacetate solutions of various concentrations of pure commercially available phenol (Sigma) (Figure 5-10). J to ^^ / Figure 5-9. TLC and TLC-BOA of isolate D323 crude ethylacetate extracts obtained at acidic (CI), neutral (C2) and alkaline (C3) conditions, as well as the fractions (F1-F6) obtained via the fractionation of the C1 extract, indicated by a red arrow. The ethylacetate extract of acidified MB medium was used as a negative control (B). The pink arrow indicates the position of phenol on the TLC plate after developing it in ethylacetate : hexane (5:1) mixture. The red arrow indicates the crude CEE extract (CI in this figure). F6 pH2 crude extract pH2 crude extract Phenol (20ug) Pttend (2ug) F4 PtMnd (20ug> ptiz etude extract Figure 5-10. Disc-diffusion assays of isolate D323 crude ethylacetate extract of neutral (ph7 crude extract) and acidified (pH2 crude extract) spent medium, its fractions F1-F6, as well as ethylacetate solutions of commercially available pure phenol (Sigma). 5.3.8. Production of phenol and TDA by D323 in marine minimal medium (MMM) in the presence and absence of L-tyrosine To assess whether the production of phenol by isolate D323 correlateed with the presence of L-tyrosine in the growth medium, D323 was grown in marine minimal medium (MMM) with and without addition of L-tyrosine. Trace amounts of phenol were observed in the CEE of isolate D323 grown in MMM, whereas the production of phenol by D323 was greatly enhanced in the presence of L-tyrosine. TDA production was not observed in any of the extracts as shown by the absence of inhibition in TLC-BOA (Figure 5-11). The culture of isolate D323 grown in MMM supplemented with L-tyrosine acquired a characteristic brown colour; no pigment production was observed in the culture grown in normal MMM. 3M PS iL .w 333 CTvr) J Figure 5-11. TLC (a) and TLC-BOA (b) results of the crude extract obtained from isolate D323 grown in liquid MMM (323) as well as in MMM supplemented with L- Tyrosine (323 Tyr). Purified TDA (TDA) and phenol (PS, 0.1 mg/mL in ethylacetate), were used as standards. 3M - crude extract obtained from blank MMM medium. 5.3.9. Assessment of TDA, purified from isolate D323 cell free supernatant, for the AM activity against marine epibiotic strains In order to assess the inhibitory activity of TDA, purified from isolate D323, against a variety of environmental strains that are likely to be encountered by that bacterium in its natural habitat, the disc diffusion assay was performed against a range of marine epibiotic strains, including various D. pulchra associated bacteria. TDA was found to have a wide spectrum of activity against a range of strains tested (Table 5-3), including isolates belonging to all major phyla abundant on marine living surfaces, such as Alpha- and Gamma-protebacteria, Actinobacteria, Firmicutes, and Bacteroidetes with the exception of isolate D323 itself, as well as Phaeobacter gallaeciences strains 2.10, BS107, Phaeobacter inhibens T5 (Brinkhoff al. 2004) and its white mutant T5-3 . Table 5-3. Inhibitory activity of TDA produced by isolate D323 against various marine epibiotic bacteria Inhibition by Epibiotic strains Phylum TDA Alpha-proteobacterium D323*^ Proteobacteria alpha) - Phaeobacter inhibens T5^ Proteobacteria alpha) - Phaeobacter inhibens T5-3 Proteobacteria alpha) - Ruegeria sp. R11* Proteobacteria alpha) +++ Phaeobacter sp. 2.10'^ Proteobacteria alpha) - Roseobacter sp. 2516 Proteobacteria alpha) ++ Roseobacter sp. 2597 Proteobacteria alpha) +++ Roseobacter sp. 2601 Proteobacteria alpha) ++ Roseobacter sp. 2633 Proteobacteria alpha) ++ Roseobacter sp. 2654 Proteobacteria alpha) +++ Phaeobacter gallaeciensis BS 107^ Proteobacteria alpha) - Vibrio harveyi Proteobacteria gamma) +++ Pseudoalteromonas tunicata Proteobacteria gamma) +++ Pseudoalteromonas undina Proteobacteria gamma) -I-+ Pseudoalteromonas piscicida Proteobacteria gamma) -f Pseudoalteromonas nigrifaciens Proteobacteria gamma) ++ Pseudoalteromonas citrea Proteobacteria gamma) ++ Pseudoalteromonas haloplanktis Proteobacteria gamma) ++ Pseudoalteromonas ulvae Proteobacteria gamma) ++ Pseudoalteromonas aurantia Proteobacteria gamma) + Acinetobacter sp. ESS07* Proteobacteria gamma) +++ Marinomonas sp. ND73* Proteobacteria gamma) +++ Shewanella sp. ND51 * Proteobacteria gamma) ++ Thalassomonas sp. ND29* Proteobacteria gamma) -I-++ Thalassomonas sp. ND49* Proteobacteria gamma) ++ Aestuariibacter sp. ND16* Proteobacteria gamma) ++ Vibrio sp. ND23* Proteobacteria gamma) ++ Dokdonia sp. ESS 16* Bacteroidetes ++ Aquimarina sp. ND19* Bacteroidetes + Tenacibaculum sp. ND71* Bacteroidetes + Bacillus D203* Firmicutes ++ Bacillus sp. ESS03* Firmicutes +++ Micrococcus sp. ESS26* Actinobacteria ++ Agrococcus sp. LSS27* Actinobacteria ++ + - zone of clearance in disc diffusion up to 3 mm from the edge of the disc ++ - 3-5 mm +++ more than 5 mm - -no inhibition observed * - isolates obtained from the surface of D. pulchra ^ - TDA-producing bacteria 5.4. Discussion Phylogenetic analysis established that isolate D323 is closely related to the alpha- proteobacterium MBIC3368 (GenBank accession no. AB012864; Figure 5-1, Figure 2-1). Interestingly, MBIC3368 related bacteria are largely regarded as common sponge symbionts and have been reported to have AM activity (Muller et al 2004; Muscholl- Silberhom et al 2008), however, previous attempts to identify the bioactive compound were not successful (Muscholl-Silberhom et al. 2008). The fact that the alpha-protreobacterium MBIC3368 related strain D323 has been repeatedly obtained from D. pulchra (Longford 2008; Penesyan et al. 2009), suggests, that the symbiotic importance of these bacteria could possibly extend to a variety of eukaryotic hosts rather than being limited to sponges. The mechanisms of these possible symbiotic interactions are not clear. Taking into consideration the AM activity of isolate D323 and related bacteria, the mechanisms may be chemically mediated via the production of biologically active compounds, such as antimicrobials. However, despite the previously observed AM activities, to my knowledge, there are no reports of AM compound identified from MBIC3368 related bacteria. An AM compound - heptylprodigiosin was identified in a phylogenetically related organism - Pseudovibrio denitrifcans strain Z143-1 (Sertan-De Guzman et al. 2007). However, the colony morphology of strain Z143-1, which was reported to form red colonies while producing the bioactive compound, differs significantly from the colony morphology of isolate D323 and other antimicrobial producing MBIC3368 related bacteria that form brown, mucoid colonies (Webster and Hill 2001; Enticknap et al 2006; Muscholl-Silberhom et al 2008). The latter observation highlights the morphological and, possibly, physiological differences between strain Z143-1 and MBIC3368 related bacteria used in this study, including isolate D323, and confirms the importance of considering bacterial phylogeny in conjunction with its physiology and morphology in bacterial identification. Purification and the chemical identification of the ethylacetate extracts obtained from acidified spent media, established that the compound, responsible for the AM activity observed in CEE of isolate D323, is tropodithietic acid (TDA) or thiotropocin. This study also confirmed the presence of TDA in phylogenetically and phenotypically related sponge associated bacteria, including isolates CCSH21, and CCSH24, obtained from the surface of the marine temperate sponge C concentrica (Yung, P.-Y., unpublished data), and NWOOl found to be abundant among the culture collections from the tropical sponge R. odorabile (Webster and Hill 2001; Webster et al 2001). Until recently TDA and thiotropocin were regarded as separate compounds, however, Greer et al. (2008), using computer generated models, have demonstrated that there is no significant energetic barrier for the transformation of the molecule between the two structures, TDA and thiotropocin. Therefore, both those forms can be regarded as tautomers and are likely to coexist in a dynamic equilibrium in any given mixture. Hereafter, for simplicity, the name TDA is used, denoting both tropodithietic acid and thiotropocin. TDA is known as an AM compound produced by Pheaobacter inhibens and related strains within the Roseobacter clade (Brinkhoff et al 2004; Bruhn et al 2005; Martens et al 2006; Porsby et al 2008). In earlier studies, the presence of TDA was also reported in Pseudomonas sp. (Kintaka et al 1984) and Caulobacter sp. (Kawano et al 1997), however, as no 16S rRNA gene sequence information is available from these studies, no phylogenetic comparisons were possible. TDA, purified from isolate D323, was found to be active against the majority of marine epibiotic bacteria. The only strains found to be resistant to the inhibitory effect of TDA were P. inhibens, and the P. gallaceinces strains 2.10 and BS107, all of which also produce TDA (Brinkhoff et al 2004; T. Thomas, personal communications). It suggests that those bacteria, being TDA producers themselves, may possess a yet unknown, but efficient mechanism of resistance to prevent the autoinhibition by TDA. Notably, the TDA-deficient spontaneous mutant of T5 designated as T5-3 (Brinkhoff et al 2004), despite losing the TDA producing ability, still maintained this resistance mechanism (Table 5-3). The wide spectrum of activity of TDA against various marine epibiotic strains, demonstrated in the current study, suggests that production of TDA by these surface associated bacteria can serve as a defence against competitor bacteria, and could also be involved in the protection of the host against other surface colonisers. Notably, TDA was shown to be highly active against such a strain as Ruegeria sp. R11 (Table 5-3), which is linked to the aetiology of bleaching disease in D. pulchra (Case 2006). The observation. made by Webster et al (2008) that after losing the D323-related symbiotic strain NWOOl the sponge becomes heavily colonised by various microorganisms, subsequently leading to its death, supports this proposition. The importance of epibiotic bacteria in the chemical defence of the host has been previously highlighted for various marine eukaryotes, (Egan et al 2000; Holmstrom et al 2002; Thakur et al 2003; Harder 2009), however, to my knowledge, no such symbiotic relationship has previously been reported for the red alga D. pulchra. D. pulchra is well- equipped to perform its own chemical defence against surface colonisation via production of quorum-sensing inhibitory compounds - fliranones. Furanones, which are structurally similar to acylated homoserine lactones (AHLs) - common quorum signal molecules in gram-negative bacteria, act as AHL analogues by competitively binding to AHL binding sites, thus preventing the binding and subsequent action of the AHLs (Manefield et al 1999). The evidence presented here suggests that in addition to furanones, D. pulchra may complement its defence against colonisation by employing its surface associated bacteria, such as MBCI3368 related alpha-proteobacterium D323, to produce bioactives. This assumption is also supported by previous observations showing the presence of various AM producing epiphytic bacteria on the surface of D. pulchra (Chapter 2) (Penesyan et al 2009). Interestingly, isolate D323, as well as all the other related sponge isolates tested, in addition to TDA, were also found to produce and secrete phenol into the growth medium. Phenol production has been previously reported in enteric bacteria (Kawamoto 1986). Formation of phenol by those bacteria is carried out by the enzyme tyrosine- phenol-lyase (TPL) (Lloyd-George and Chang 1994; Katayama et al 2000; Lutke- Eversloh et al 2007) and is directly linked to the availability of L-tyrosine. The enhanced phenol production in the presence of L-tyrosine, suggests a similar pathway for the biosynthesis of phenol in isolate D323. No direct correlation was observed between the production of TDA and phenol, as shown by the absence of phenol in the corresponding extract of the Roseobacter related TDA producing bacterium P. inhibens T5 (Figure 5-8), which might have been expected, due to the involvement of various phenolic compounds in the biosynthetic pathway leading to TDA (Cane et al 1992). Therefore, production of phenol might be a unique feature of D323 related bacteria. Despite being a known disinfectant, phenol was not shown to cause significant AM activity (Figure 5-9, Figure 5-10), and the role of phenol production and secretion by isolate D323 related bacteria is not yet clear. It is possible that phenol, produced by these associated bacteria, could be further used by the eukaryotic host. Interestingly, in an investigation of the mechanism of symbiosis between the isolate D323 related bacterium SB2 and the sponge host, Muller et al. (2004) found a possible link between the tyrosinase activity of the sponge, involved in the formation of the diphenols from monophenolic compounds, and the abundance of those bacteria. In addition, Muller et al (2004) also described an operon within the bacterial genome, which was responsible for the synthesis of enzymes involved in the breakdown of phenolic compounds produced by the sponge host. The authors concluded that bacterium SB2 may use those sponge products to generate energy. Taking into account the probable intimate relationship between these bacteria and the host, which could also include the exchange of phenolic compounds, it is possible that phenol, produced by these bacteria, might be used by the host as a precursor for other phenolic compounds. However, this hypothesis needs to be tested in the future. In addition to CEE extract, from which TDA was obtained, AM activity was also observed in the precipitant formed in the acidified spent medium of isolate D323, designated DBP (dark brown precipitant). Whilst the exact nature of the compound responsible for the activity has not been determined, the colour of the precipitant, together with the enhanced production of the pigment in the presence of L-tyrosine (Slominski et al. 1988) (section 5.3.8) indicated the presence of a compound related to melanins. Futhermore the alkaline-solubility of DBP and its precipitation in acidic conditions were also in accordance with the previously reported properties characteristic for melanins (Bull 1970; Aurstad and Dahle 1972; Ivins and Holmes 1980). Melanins are pigments widespread in variety of organisms, from bacteria to humans. Their functions are well known and include photoprotection, photoconductivity, thermoregulation, and metal ion chelation (Liu et al. 1993; Plonka and Grabacka 2006; Wan et al. 2007). Besides these functions, recent studies have revealed the involvement of melanins in the virulence of many pathogenic microorganisms (Liu and Nizet 2009). For example, melanin deficient mutants of the pathogenic fungus Ctyptococcus neoformans are less invasive, and are less resistant to phagocytosis (Kwon-Chung et al 1982; Wang et al 1995). In addition, negatively charged melanin was also shown to neutralize the activity of some cationic antimicrobial peptides (Doering et al 1999). hi Vibrio cholerae, the production of melanins was observed under the conditions of stress, such as, for example, hyperosmotic shock or increased temperature (Denoya et al. 1994; Attridge et al 1996; Nag et al 2005; Chatfield and Cianciotto 2007), and led to te increased UV resistance and enhanced toxin production (Valeru et al 2009). Due to the insolubility of melanins in organic solvents, and difficulties with their purification and structure elucidation, knowledge about the structures of these pigments are limited (Piattelli et al 1965; Hamilton and Gomez 2002; Taborda et al 2008). Melanins are known to be polymers derived from phenolic compounds via the activity of the enzyme tyrosinase and, therefore, their production is directly linked with the availability of L-tyrosin (Claus and Decker 2006; Wang and Hebert 2006). Future studies would be needed to confirm whether the pigment present in DBF is related to melanins. If the presence of a melanin-like compound is confirmed, it would also be interesting to establish whether the biosynthesis of this compound is linked to the production of phenol by the same bacterium, or with the high tyrosinase activity previously reported for a sponge host of related bacteria (Muller et al 2004). The possibility that melanin related compound is responsible for the AM activity found in DBF obtained from isolate D323 spent medium is also worthy of further exploration. In summary, for the first time, this work has led to the characterisation of the antimicrobial compounds produced by epiphytic isolate D323 and related sponge- associated alpha-proteobacteria, as TDA or thiotropocine. The wide range of activity of TDA against various bacteria, including common marine epibionts, indicates that TDA could be used by D323 and related bacteria to defend themselves against other competitors. The frequent isolation of D323 related strains from marine sessile eukaryotic hosts also indicates a role for these bacteria in the defence of the eukaryotic host against heavy colonisation (Figure 5-12). In addition, the presence of specific resistance against the inhibitory activity of TDA in TDA-producing strains is suggested; however, the mechanisms require further investigation. oC phenolic compounds / Figure 5-12. Putative model of the interaction between isolate D323 and the eukaryotic host. Possible symbiotic relationship could include the protection of the host surface, as well as the producer bacterium, via secretion of AM compound TDA by D323 in return for nutrients available on the surface of the host. Phenol, also produced by D323, could possibly be used by the host organism as a precursor for various phenolic compounds. The presence of a second, potentially melanin related AM compound was also shown in isolate D323 spent medium in a fraction, described as a dark brown coloured precipitate. It indicated the production of multiple bioactive compounds by isolate D323. Interestingly, while production of phenol was also observed in D323 and related isolates, it was not shown to significantly contribute to the antimicrobial activity observed for these bacteria, consequently, its role remains unclear. Whether phenol, produced by these epibionts, might possibly serve as a precursor for the host organism in the biosynthesis of various other metabolites, is a topic for further investigation. CHAPTER SIX Construction, screening and analysis of a large insert DNA (fosmid) library using genomic DNA from cultured antimicrobial producing isolates 6.1. Introduction This chapter discusses the use of a large DNA insert library to characterize the antimicrobial (AM) activity of several AM producing isolates obtained during the course of this project. Metagenomics, defined as "the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms" (Handelsman 2004), has recently emerged primarily as a tool to gain access to the genetic machinery and biochemical potential of yet uncultured microorganisms. Consequently, frinctional metagenomics, that includes cloning of DNA obtained from the environment and screening of clones for desired activity, was mostly focused on obtaining bioactive compounds from these microorganisms. However, the latter can only be successful if the heterologous host has all the necessary machinery required, and, thus is capable of production of the foreign compound. As a result, the hit rates of obtaining clones with positive activity by using the environmental functional metagenomics approach have to date been were rather limited (Schloss and Handelsman 2003; Handelsman 2005; Pelaez 2006). Nevertheless, the use of metagenomics is currently not limited to production of bioactives. Genomic data obtained via a metagenomic approach may also provide invaluable information about the various ecosystems and the roles that microorganisms play in the environment (DeLong 2009). For example, recent metagenome analysis by 152 Konstandinidis et al. (2009) compared the abundance of various genes in surface waters and in the deep sea. That study demonstrated, for example, the high content of genes related to photosynthesis, UV-induced DNA damage repair and oxidative shock in the microorganisms obtained from the surface waters, where the organisms are constantly exposed to the effects of UV radiation and high oxygen content. In contrast, the deep sea metagenome revealed the abundance of transposases, phage integrases, plasmids and recombinases (Konstantinidis et al. 2009). Metagenomics methodology has greatly assisted in the understanding of the genomic bases of biosynthetic pathways underlying the production of bioactives. As an example. Burke et al (2007) used the genomic DNA of the cultured bacterium P. tunicata, known for its ability to produce various bioactives, to create a large insert fosmid library in an E. coli host followed by subsequent screening of the library for AM activity. As a result, a clone producing the antifungal compound, tambjamine, was identified and a biosynthetic pathway was proposed based on the function of the genes involved in its biosythesis and contained in the fosmids of the producer clone (Burke et al 2007). Likewise, two positive clones from a P. tunicata genomic library, with different modes of action against the nematode Caenorhabditis elegans, were successfully identified in another study (Ballestriero et al., unpublished data). Here I attempted to use a similar approach, however, expanding it to the use of not only one, but a collection of AM producing bacterial isolates. The overall objective of the work, presented in this chapter, was to fuse the state-of-the-art functional genomics with traditional culturing. It included cloning the large fosmid fragments of genomic DNA, obtained from a collection of isolates previously shown to produce AM compounds, into the E. coli host, screening the clones for AM activity, followed by the genetic analysis of the fosmids, responsible for the observed activity. Thus, by employing this mixed approach and by using the DNA known to contain genes encoding for biologically active compounds, I expected to maximally utilise the capabilities of the host strain to synthesize the bioactives and, therefore, to maximize the output of the AM expressing clones. Moreover, this approach could provide access to the genes involved in the biosynthesis of bioactives and, therefore, lead to a better understanding of the origin and the properties of these bioactive compounds. 6.2. Materials and methods 6.2.1. Extraction and size-selection of genomic DNA 6.2.1.1. Pulsed Field Gel Electrophoresis (PFGE) Six AM producing phylogenetically and phenotypically different isolates obtained from the surfaces of U. australis and D. pulchra during this project (Chapter 2) were used in the construction of a large insert DNA library. Briefly, the genomic DNA from isolates U95, U140, U156, D250, D295, and D323 was extracted separately as described in chapter 2 (section 2.2.3). To select for large fragments of ~ 35 kB, the extracted genomic DNA was sheared and subjected to PFGE on a 1 % agarose gel (Agarose, Low Melting, Axygen Biosciences) using the CHEF-DR II Pulsed Field Electrophoresis System (Bio- Rad) in TAE buffer (Appendix Ilf) under the following conditions: field strength - 6 V/cm; angle - 120°; switch time - linear ramp 1 - 6 seconds; run time - 14 hours; temperature - 14°C; voltage was maintained at 280-300 V. Genomic DNA from the isolates (4 |xg from each isolate) was run in separate lanes along with the small aliquots (0.4|ig) used as standards. 0.4|ig of each of MidRange I PFG Marker (New England BioLabs) and >.DNA/EcoRI+HindIII (Fermentas) were used as size and concentration markers. After electrophoresis, lanes containing DNA standards were excised from the gel and stained in ethidium bromide (EtBr) solution (1 mg/1) for 10 minutes, then visualised under UV light on the GelDoc transilluminator using the QuantityOne software (Bio-Rad). The regions corresponding to ~35 kB DNA size were excised from the unstained lanes containing large samples and used as inserts for the construction of the library. 6.2.1.2. Recovery of DNA fragments from agarose gel DNA was extracted from the agarose gel slice using the Gelase enzyme preparation included in the Copy Control Fosmid Library Production Kit (Epicentre Biotechnologies) according to the manufactureras instructions. Briefly, the gel slices were incubated at 70° C in water bath for 15 minutes to melt the agarose and then transferred to 45°C water bath. Gelase buffer was added to the tube to a 1 X final concentration 154 followed by Gelase enzyme preparation (1 U per 300 |LIL of molten agarose) and incubated at 45''C in a water bath for 1 hour. The enzymes were deactivated by incubating the mixtures at 70 ""C for 10 minutes. The digests were chilled on ice for 5 minutes and centrifuged at 21250 g for 10 minutes to remove the non-solubilised oligosaccharide material. The upper supernatant part containing the DNA was transferred to a fresh tube, the DNA precipitated according to the ethanol precipitation protocol and resuspended in EB buffer (Appendix lie). Recovered DNA was subjected to agarose gel electrophoresis in TBE buffer (Appendix He) to assess the quality and the quantity of the high molecular weight (HMW) DNA fragments obtained. After electrophoresis the gel was stained and the bands were visualized and photographed as described above (section 6.2.1.1). The HMW DNA samples from all six isolates, listed above, were mixed into a combined HMW DNA sample in equal proportions to ensure that each isolate's DNA was equally represented in the clone library. 6.2.2. Construction of the fosmid library The library was constructed using the CopyControl Fosmid Library Production kit (Epicentre Biotechnologies) and the manufacturer's protocol with modifications, as further described in this section. 6.2.2.1. End-repair of the size-selected genomic DNA After PFGE the size-selected DNA was end-repaired to form blunt ended 5' phosphorylated fragments. Briefly, size-selected DNA (2.5 }ig) was incubated with End- Repair enzyme mix (Epicentre) also containing End-Repair buffer, dNTPs (20 mM) and ATP (80 mM) for 45 minutes at RT. Enzymes were deactivated by adding EDTA (lOmM) and the mixture incubated at TO^'C for 10 minutes. To remove any residual kinase activity, DNA was purified from the mixture by isopropanol precipitation (Appendix Ild) and resuspended in EB buffer. 6.2.2.2. Ligation of insert DNA into the fosmid vector End-repaired DNA (0.25 ^g) and the CopyControl pCClFOS vector (Epicentre) (0.5 |ig) were incubated with 2 units of T4 DNA ligase in the Hgation buffer (Promega) for 16 hours at 16°C. The enzyme was deactivated by heating the mixture to 70°C for 10 minutes. 6.2.2.3. Packaging Twenty-five microliters of MaxPlax Lambda Packaging Extract (Epicentre) were added to 10 |il of ligation mix and incubated in a water bath at 30°C for 90 minutes. A further 25 |iL of extract was added and the mixture incubated for another 90 minutes at 30°C. Phage dilution buffer was added to a 1 ml final volume, followed by 25 |LIL of chloroform. Packaged phage was stored at -80°C. 6.2.2.4. Infection of the E, coli host The host strain E. coli EPBOO-Tl^ (Epicentre) was grown overnight in LB 10 broth and was reinoculated into fresh LB 10 supplemented with 10 mM MgS04 and 0.2 % maltose. The culture was grown on a horizontal shaker at 37 to ODeoo ^ 0.8-1.0. Ten microliters of packaged phage particles were added to 90 |iL oi E. coli EPI300-T1^ culture and incubated for 20 minutes at 37°C. hifected host cells were spread onto plates containing solid LB 10 (Appendix la), supplemented with 12.5 (xg/ml chloramphenicol (Cm), to select for fosmid clones. Plates were incubated at 37°C for 24 hours. 6.2.2.5. Determination of number of clones for complete genome coverage The number of fosmid clones required to ensure a 99.8 % probability that any given DNA sequence from isolates used to create the library is contained within the library was determined according to the following formula: N = ln(l-P)/ln(l-f) Where P is the desired probability; f is the proportion of the genome contained in a single clone; and N is the required number of fosmid clones. 6.2.2.6. Storage of library clones Single colonies each representing an individual fosmid clone were picked and inoculated into the individual wells of 96-well plates, each well containing 150 ^iml of LB 10 liquid medium supplemented with 12.5 ^g/ml Cm. Inoculated plates were incubated on a horizontal shaker at 100 rpm overnight. After incubation, sterile glycerol was added to each well to a final concentration 25 % and mixed. The library was stored at -80°C. Fosmids were maintained in the clones at a single copy per cell unless induced to high copy number (10-50 per cell) in the presence of L-arabinose. 6.2.2.7. Screening of the clones for antibacterial activity Clones were inoculated from the frozen stock solutions, using a 96 pin replicating tool, onto large square dishes (Nunc) containing LB 10 agar with Cm (12.5 |xg/mL) and L- arabinose (0.02 % w/v) followed by incubation for 48-72 hours at 25''C. After incubation, the trays containing colonies of clones were overlaid with soft LB 10 agar (0.7 % agar) and inoculated with the liquid culture of the indicator strain S. aureus 31 (CMB culture collection) or N. canis OH73 (WCPN culture collection). Overlaid plates were incubated overnight at 3>TC to facilitate the growth of indicator strains. All clones were screened against both indicator organisms. Positive antibacterial activity was observed as a zone of clearance around the bioactive producing clone. To further confirm the activities, positive clones were re-assessed by streaking them on separate petri dishes containing LB 10 agar with chloramphenicol (12.5 |Lig/ml) and L-arabinose (0.02 % w/v) and performing an overlay assay as described above. Ten positive clones, named 3-Gll, 9-E12, 10D3, 12-Al, 14-D9, 15-ElO, 16-B12, 20-G8, 19-FlO and 23-H6, were selected for further fosmid extraction and sequencing. Fosmids were named after the E. coli clones they were obtained from, preceded by "f. 6.2.3. Extraction and purification of fosmid DNA from bioactive producing clones Overnight liquid cultures of ten antibacterial producing E. coli fosmid clones, mentioned above, were used to inoculate 10 ml of LB 10 liquid media containing 12.5 ^g/ml Cm and 0.02 % L-arabinose; the latter was used to induce fosmids to a high copy number. Inoculated cultures were incubated on a horizontal shaker at ?>TC and 310 rpm overnight. After incubation, 3 ml of each culture were used for fosmid extraction and purification using the Illustra plasmidPrep Mini Spin Kit (GE Healthcare) according to the manufacturer's protocol. Extracted fosmid DNA was subjected to agarose gel electrophoresis to assess the quality and the quantity of the fosmid DNA obtained. After electrophoresis the gel was stained in the solution of EtBr. The bands were visualized and photographed under UV light using the GelDoc gel imaging apparatus and the QuantityOne software (BioRad). 6.2.4. Sequencing and analysis of bioactive producing fosmid sequences Purified fosmid DNA was random shotgun sequenced at the J. Craig Venter Institute using the ABI3730XL and the Roche Titanium FLX DNA sequencers. Each sequencing read was trimmed for vector contamination (i.e. pCClFOS) and low quality, using the Phred/Phrap/Consed software pipeline (Gordon et ai 1998). Reads from the shotgun library were assembled with Phrap and the assembly manually checked in Consed. Open reading frames (ORFs) were identified with the program MetaGene (http://metagene.cb.k.u-tokyo.ac.jp/metagene/), a gene-finding software that targets archaea and bacteria and is able to predict a range of prokaryotic genes from anonymous genomic sequences based on di-codon frequencies estimated by the GC content of the given sequences (Noguchi et al. 2006). All putative ORFs were searched (using an in- house pipeline) against Swiss-Prot database (http://expasy.org/sprot/), the Institute of Genome Research Family (TIGRFAM) database (http://www.tigr.org/TIGRFAMs/), the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/), and the Cluster of Orthologous Group of proteins (COG) database (http://www.ncbi.nlm.nih.gov/COG/) to obtain a ftmctional annotation. The output for each fosmid sequence was tabulated with the coordinates of each ORF and the results of each database search. The ORFs were visualised using Artemis software (http://www.sanger.ac.uk/Software/Artemis/). 6.2.5. Identification of the strains from which bioactive producing fosmids originated PCR amplification was used to identify to which parent genome the selected fosmids belonged. Specific primer pairs were designed based on the sequence of the fosmids. The product sizes varied between 432 - 772 bp to help their further identification. The primer sequences and the expected product lengths are given in Table 6-1. Genomic DNA of the six isolates, included in the construction of library, were used as templates for amplification. Table 6-1 Primer pairs used for fosmid identification and their expected product lengths. Fosmid Primer pairs Sequence (5' to 3') Product length 3G11 forward GGC TAG AGG CGT TGC GTA TTG TGC 3G-11 679 bp 3G11 reverse CTT TAA AGG CGC CGG GCT CCA TCT 9E12 forward TGC TGA AGC GGA AGT GGA GTA TGA 9E-12 388 bp 9E12 reverse CGG CAC GTT GAA GTC GAA GTA GTC 10D3 forwards CTA TGA TCA CGA CCA GCA CAC GAG 10-D3 571 bp 10D3 reverse ACC AGG TCC GAG CCA TCT ACA CAA 12A1 forward ACA GCG GTG GTC ATT ATT GGA ACG 12-Al 432 bp 12A1 reverse GGC GGT GTG AAA GCG GTG ATA GTC 14D9 forward GGC ACA CGG CTC TTC ATC TTC ACA 14-D9 532 bp 14D9 reverse GCC GCG TTC GTT CCC GTC AC 15-ElO 15E10 forward GCT AAA CTG CCT GAC TTC TAC ACG 20-G8 509 bp 23-H6 15E10 reverse CTG GAT ACT GCT GGT TTG ACT ACG 16B12 forward CTC TTT ACG CCC AGT GAT TCC 16-B12 613 bp 16B12 reverse TTA TTT GCG TGT TCC TCG TCT ATT 19F10 forward ACA TCA TCG CCG CTA AGG TA 19-FlO 772 bp 19F10 reverse TAT GGG ATT CTG TTG TTT CGT AA These fragments were PCR amplified in 20 fxL reaction mixes each containing 50 ng of genomic DNA of one of the isolates U95, U140, U156, D250, D295, D323; 2 ^L REDtaq buffer (Sigma), 2.5 mM each dNTP (Roche), 12.5 pmol of each of the forward and reverse primers (Table 6-1.) obtained from Sigma, 0.5 |il of 10 % BSA (w/v, New England Biolabs) and molecular grade water (Eppendorf). One unit of REDtaq Polymerase (Sigma) was added at the Hot Start, after the initial thermal ramp. The PGR conditions were 94°C for 3 min, then 25 cycles each of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. A final extension step of 72°C for 6 min was performed. Amplified DNA fragments were subjected to agarose gel electrophoresis to check for the presence of amplification products. 6.3. Results 6.3.1. Size-selection of genomic DNA Analytical PFGE was performed to evaluate the quality and quantity of the genomic DNA, as well as to find the optimal conditions for separation. The conditions outlined in section 6.2.1.1 were chosen as a result of several trial runs and produced the best separation (Figure 6-1). After preparative PFGE, fragments of genomic DNA from 6 bioactive producing isolates U95, U140, U156, D50, D295, and D323 were size separated. The DNA fragments of the size ~ 35 kB were successftilly excised and recovered from the gel (Figure 6-2). •iflWhlB l^plMfiril' ^ S^jji^w*'- ^llWWML " Ml 95 140 156 250 295 323 IV12 > 35 kB Figure 6-1 Analytical PFGE of genomic DNA obtained from AM producing isolates. Ml - MidRange I PPG Marker, M2 - EcolHindlll marker, 95 - genomic DNA of isolate U95, 140 - genomic DNA of isolate U140, 156 - genomic DNA of isolate U156, 250 - genomic DNA of isolate D250, 295 - genomic DNA of isolate D295, 323 - genomic DNA of isolate D323. feto--' - sy» ?» icrr it r56 250 295 323 Ml diluted bp 21226 d" 5148 2027 1584 947 Figure 6-2. Agarose gel electrophoresis of HMW DNA (35-40 kB) recovered from the gel slices after preparative PFGE. Ml - MidRange I PFG Marker, M2 - EcolHindlll marker, 95 - genomic DNA of isolate U95, 140 - genomic DNA of isolate U140, 156 - genomic DNA of isolate U156, 250 - genomic DNA of isolate D250, 295 - genomic DNA of isolate D295, 323 - genomic DNA of isolate D323. 6.3.2. Construction and screening of the fosmid library for the antibacterial activity 6.3.2.1. Determination of number of clones in the library necessary for a complete coverage of six bacterial genomes included in the library The E. coli fosmid library was grown on LB 10 containing Cm and yielded in excess of 100 000 clones. Since the exact genome sizes of the six isolates used for library construction are unknown, the average bacterial genome size of 3.5 Mb (Whitworth 2008; Whitworth and Cock 2009) was used to evaluate the number of clones necessary for complete coverage of all six bacterial genomes included in the library. Thus, by using the formula described in section 6.2.2.5, N - 6 X {In (1 - 0.99) / In (1 - [4 x 10"^ bases / 3.5 x 10^ bases])} = 6 (-4.61 / -0.01) = = 6 X 461 - 2766 clones. Hence, 2766 clones were considered as sufficient for the complete coverage of six bacterial genomes included in the fosmid library construction. 6.3.2.2. Screening of the library clones for antibacterial activity Some of the library clones, upon induction to fosmid high copy numbers, showed visible pigment production (Figure 6-3), demonstrating the successful expression of foreign genes. The clones 15-ElO and 20-G8 expressed dark purple pigmentation. Li addition clone 23-H6, expressed a pale purple pigmentation, and clone 19-FlO, was found to produce a dark green pigment (Figure 6-4). Clones 15-ElO and 20-G8 were morphologically identical, forming dark-purple colonies when induced to high copy fosmid number; the clone 23-H6 differed morphologically, consistently showing lighter grayish-purple pigmentation. The AM activity of clone 23-H6 was also significantly lower and hardly detectable compared to the potent inhibitory activities observed for clones 15-ElO and 20-G8 (Figure 6-5) Figure 6-3. E. coli clones carrying the fosmids grown in the presence of L-arabinose. The arrow indicates a clone 20-G8 expressing dark purple pigmentation. Figure 6-4. The pigment expressing E. coli clones 15-ElO (a), 23-H6 (b), 20-G8 (c), and 19-FlO (d) growing on petri dishes containing LB 10 agar supplemented with chloramphenicol (12.5 ixg/ml) and L-arabinose (0.02 % w/v). Figure 6-5 Antibacterial activities of violacein producing E. coli fosmid clones 23-H6 and 20-G8, originated from the genome of isolate D250, against S. aureus. A total of 2880 library clones were screened for antibacterial activity in an overlay assay against both S. aureus and N. canis. One hundred and forty seven clones, or 5.1 % of all clones screened, showed activity in at least one of the screens, even though, in many cases, the activities were either weak or inconsistent. Ten clones, namely 3-Gl 1, 9-E12, 10D3, 12-Al, 14-D9, 15-ElO, 16-B12, 20-G8, 19-FlO and 23-H6, which showed consistently high levels of antibacterial activity or pigment expression, were chosen for further analysis. 6.3.3. Sequencing and analysis of selected fosmids The sequencing of the fosmids revealed that the clones 15-ElO, 20-G8 and 23-H6 contain similar fosmids, and, therefore, their data were combined and annotated as one fosmid sequence, yielding overall 8 different fosmid sequences. Annotated data was analysed and predicted gene products were classified into COG functional groups according to the COG database (www.ncbi.nim.nih.gov/COG) (Tatusov et al 2000; Tatusov et al. 2001). To demonstrate that fosmids, sequenced in this study and linked with AM activity, are enriched in genes involved in secondary metabolism, the COG data from a whole genome of marine bioactive producing epibiotic bacterium P. tunicata was used for comparison (Thomas et al 2008). The COG annotated data, obtained from the fosmids in this study, showed the presence of genes belonging to the different functional groups with various abundance. Notably, it demonstrated a high abundance of genes (more than 10 % of all the genes contained in the fosmids) involved in secondary metabolism (Figure 6-6). The fosmid sequencing data were also noted for the high abundance (more than 15 %) of genes with as yet unknown function (Figure 6-6), which might also potentially be involved in the biosynthesis of novel biologically active compounds. 0 O) B 14 H c ifosmid data s I ^ 12 -! I P. tunicata D2 genome Q. 10 6 ^ 2 1 .iiliLliiiiiljtV i\ V V X X X \ \ V \ 4A \ Figure 6-6. Percentage of genes belonging to each COG functional groups present in P. tunicata whole genome and 8 different fosmids obtained in the current study, 6.3.4. Identification of the parental strains from which fosmids originated Identification of the fosmids was achieved based on the presence / absence of the products obtained by amplifying the specific fosmid sequences using the genomic DNA of each isolate previously used for fosmid library construction as a template. PGR amplification product sizes were compared with the estimated product sizes obtained during the primer design (Table 6-1) and only the correct size products were considered in the fosmid identification process. As evident in Figure 6-7, a 772 bp PGR product was obtained by using the primers specific for the fl9-F10 fosmid sequence and the genomic DNA of isolate U95 as template. Therefore, it may be concluded that the cloned DNA in fosmid fl9- FIO originated from isolate U95. Likewise, three amplification products, 679 bp, 432 bp, and 509 bp, were obtained using the isolate D250 genomic DNA, which ascribes the fosmids 3-Gl 1, 12-Al, as well as all the sister fosmids fl5-E10, f20-G8 and f23- H6, to isolate D250. Four amplification products, 388 bp, 571 bp, 532 bp, and 613 bp, observed by using the genomic DNA of isolate D323 as template attributed the fosmids f9-E12, flO-D3, fl4-D9, and fl6-B12 to isolate D323. No amplification products were observed when the genomic DNA of isolates U140, U156 and D295 were used as templates, consequently, it can be concluded that none of the 10 sequenced fosmids originated from any of those three isolates. A summary of the fosmid identification is given in Table 6-2. Table 6-2. Summary of the identification of the parental strains from which fosmids originated. Fosmid Parental organism f3-Gll Isolate D250 f9-E12 Isolate D323 flO-D3 Isolate D323 fl2-Al Isolate D250 fl4-D9 Isolate D323 fl 5-E10, f20-G8, f23-H6 Isolate D250 fl6-B12 Isolate D323 fl9.F10 Isolate U95 Ml 2 3 4 5 6 7 8 VI ^l(X)OKb — 800 Kb - 500 K b Ml 2 3 4 5 6 7 8 M —1000 Kb' — 700 Kb A — 500 Kb Ml 2 3 4 5 6 7 8 M —1000 Kb — 700 Kb — 500 Kb Figure 6-7. Agarose gel-electrophoresis showing the amphfication products obtained using the primers specific for the fosmids f3G-ll (1); f9-E10 (2); flO-D3 (3); fl2-Al (4); fl4-D9 (5); fl5-E10. G0-G8, f23-H6 (6); fl6-B12 (7); fl9-F10 (8); and the genomic DNA of the isolates U95 (a), U140 (b), U156 (c), D250 (d), D295 (e), and D323 (f). The GeneRuler 100 bp DNA Ladder Plus (Fermentas) was used as a size marker (M). Bands, containing amplification products, are highlighted with arrows. 6.3.5. Analysis of fosmid sequences 6.3.5.1. Fosmids originated from isolate D250 6.3.5.1.1, Fosmid J3-G11 The fosmid extracted from clone 3-Gl 1 was found to be 24296 bp in length. When searched using the protein BLAST and pblast algorithm (Altschul et al 1997), it featured several conserved hypothetical proteins (CHP) followed by multiple transposable elements. The sequence ended with a stretch of 12 NADH dehydrogenase components. The GC percentage plot revealed a lower than average GC content in the first half of the sequence, including CHPs and the transposable element sequences. In contrast, the last 12000 bp of sequence had a higher than average GC content for NADH-dehydrogenase components (Appendix Ilia). 6.3.5.1.2, Fosmid fl2-Al The fosmid sequence of fl2-Al was shown to consist of 32547 bp. Among the genes contained in the fosmid were several genes involved in signaling, a tonB- dependant receptor protein, and a cluster of genes iucA-D previously shown to be involved in the biosynthesis of the siderophore aerobactin (Williams 1979; Stuart et al 1980; Braun 1981; Loper et al. 1993; Yokes et al. 1999) (Appendix Illd). 6.3.5.1.3, Fosmids fl5-E10,f20-G8, andf23-H6. Fosmids extracted from the purple pigment producing clones 15-ElO, 20-G8, and 23-H6 were found to share a similar nucleotide sequence and were considered as sister fosmids. However, fosmid f23-H6 contained less DNA and, hence, lacked genes, such as those encoding CHP and a Ton-B dependant receptor, found at the beginning of the sequence of the fosmids fl5-E10 and f20-G8. The remaining sequences were similar among all three sister fosmids, and included transposable elements, such as a transposase and an integrase, as well as genes encoding VioA-E, previously shown to be responsible for the production of the purple pigment violacein in C. violaceum and P. tunicata (August et al. 2000; Brady et al 2001; Antonio and Creczynski-Pasa 2004; Balibar and Walsh 2006; Asamizu et al. 2007; Thomas et al 2008). The sequence ended with several CHPs preceded by a transcriptional regulator. The GC content plot showed a significantly lower GC content for the genes in the violacein cluster compared to the other half of the fosmid sequence (Appendix Illf). 6.3.5.2. Fosmids originated from isolate D323 63,5,2,1, Fosmid f9-E12 Consisting of 37944 bp, fosmid f9-E12 contained numerous genes, including those encoding two-component response regulators, proteases, chaperonins, CHPs, as well as various proteins involved in transport, such as permeases, a sugar transporter, and a protein translocase. The sequence ended with a gene encoding for bacteriocin found in Pseudovibrio sp. JE062 (Appendix Illb). 6.3.5.2.2, Fosmid flO-D3 Fosmid flO-D3 was found to contain 36314 bp of insert DNA. Sequence analysis revealed genes encoding enzymes involved in electron transport and energy generation, followed by a TonB receptor and CHPs. Interestingly, this included two very large CHPs, the first consisting of 1605 amino acids, and the second - 654 amino acids. The first of these two CHPs is predicted to consist of 1605 amino acids and has 57 % identity to its only hit in the NCBI database - hypothetical protein PJE062_2494 from Pseudovibrio sp. JE062. These two CHP encoding genes were followed downstream by several genes encoding transporter proteins and a two-component response regulator (Appendix IIIc). 6.3.5.2.3, Fosmid fl4-D9 Fosmid fl4-D9 consisted of 19858 bp insert DNA and was found to encode for genes involved in riboflavin biosynthesis, transporters, CHPs, as well as several coenzymeA-dependent dehydrogenases (Appendix Ille). 6.3.5.2.4, Fosmid fl6-B12 Fosmid fl6-B12 was comprised of 39325 bp of insert DNA. It included genes encoding a peptidase, various Fe-S proteins, chemotaxis sensory protein, translocases and several CHPs. Remarkably, the full 16S rRNA gene was located in the beginning of the fosmid, which perfectly matched the 16S rRNA gene of isolate D323, previously amplified and sequenced in the process of identification of this isolate (Chapter 2) (Appendix Illg). 6.3.5.3. Fosmids originated from isolate U95 The fosmid extracted from the dark-green pigment producing clone 19-FlO, was found to originate from isolate U95. 6.3,5.3 J. Fosmid fl9-F10 Sequencing of forsmid fl9-F10 showed that it contains 29696 bp of insert DNA. It included a cluster of genes encoding proteins involved in the type VI secretion system (T6SS), as well as the type VI secretion Vgr family protein. The sequence also contained genes encoding for various transporters, such as the ATP- binding cassette (ABC) transporters, as well as major a facilitator superfamily (MFS) permease. A large non-ribosomal peptide synthetase (NRPS) was also found in the sequence of fosmid fl9-F10. The adenylation (A), thiolation (T) and thioesterase (TE) domains were successfully located in the sequence of that NRPS using the NRPS- PKS database search tool found at http://www.nii.res.in/searchall.html. In the sequence of the fl9-F10 fosmid, the NRPS was downstream followed by CHP and, then, by a gene encoding a protein with 56 % identity to the indigoidine biosynthesis protein IndA, found in E. chrysanthemi. Fosmid fl9-F10 also contained a gene encoding a protein similar to the antifungal compound nikkomycin biosynthesis protein SanR (Zeng et al. 2002) (53 % identity in protein BLAST) (Appendix Illh). 6.4. Discussion The aim of this chapter was to use a functional genomics approach to identify genes involved in the biosyntheses of AM compounds produced by selected marine surface bacteria. This approach has been successfiilly used by others to identify genes involved in secondary metabolite production either from a single bacterial strain (Burke et al 2007) or genes from an environmental metagenomic DNA sample (Brady et al. 2001 ; Gillespie et al. 2002). Here, rather than using a single isolate or an environmental DNA sample, I have taken the genomic DNA from phylogenetically and phenotypically different bioactive producing isolates that were mixed and used to create a large insert fosmid library. Many AM producing clones were identified; however, most of the activities were rather weak. The weak antibacterial activity of fosmid clones might be due to the limited ability of the host organism to produce bioactives of foreign origin. Future studies could use techniques to improve the expression of foreign genes in E. coli host on transcriprional level, and may include the use of stong promoter systems, such as, for example, the T7 promoter system (Studier and Moffatt 1986). However, the functional screening of library clones for antibacterial activity introduces an additional limitation for expression of these bioactive compounds. Li such case, the expression of broad-spectrum antibiotics with high potency is very limited since it could be toxic to the host organism. Host toxicity was confirmed recently as a major obstacle for the successful transfer and expression of foreign genes to the host organism (Sorek et al 2007). In the current study a potent antimicrobial - violacein, was successfully expressed by the E. coli host, which could be explained by the limited activity of violacein against gram-negative bacteria, in contrast to the potent activity it exhibits against gram-positives, as reported in several studies (Lichstein and Van de Sand 1945; De Moss 1967; Nakamura et al 2002; Nakamura et al 2003). Furthermore, a limitation may also be introduced if the genes necessary for the biosynthesis of a bioactive compound are located in various parts of the genome which would make it impossible to encompass them within a single fosmid. To validate the selection of fosmids for the production of bioactive secondary metabolites, the sequence data obtained from 8 different fosmids (fosmids f20-G8 and f23-H6, sharing similar sequences with fl5-E10, were excluded from this calculation) were assigned to various COG functional groups to establish the ratio of the genes involved in various physiological processes in the organisms. To assess the richness of the selected fosmids in the genes related to secondary metabolism, the fosmid sequence data was compared to the functional annotation of the whole genome of P. tunicata- a surface-dwelling marine epibiotic bacterium best known for its ability to produce an array of biologically active compounds targeting various marine organisms, such as bacteria, fungi and larvae (James et al 1996; Holmstrom et al 1998; Egan et al 2001; Egan et al 2002; Franks et al 2006), and, subsequently, is known to contain a multitude of genes involved in the biosynthesis of secondary metabolites (Thomas et al 2008). Nevertheless, the content of genes involved in secondary metabolism was found to be almost 5 fold higher in the fosmids, compared 171 to the whole genome of P. tunicata (Figure 6-6), confirming that fosmids were enriched in genes involved in the transport and metabolism of secondary metabolites. Some of the isolates, which genomic DNA was used to create the fosmid library, had already been characterized for their AM activity in the course of the current project. For example, isolate U156 was shown to produce a potent AM compound in the presence of high concentrations of iron in the environment (Chapter 3), isolate D323 was shown to produce the broad-spectrum AM compound tropodithietic actid (TDA) (Chapter 5), and isolate D250 was found to produce the bioactive purple pigment violacein (Chapter 4). It should be noted that in both cases, where the AM compound was chemically characterized (TDA from isolate D323 and violacein from isolate D250), these compounds were found not to be the sole AM compound produced by the particular bacterium suggesting the presence of yet unknown bioactive compounds. The presence of multiple bioactive compounds produced by isolate D250 and D323 was further evident by multiple and different fosmid sequences encoding for AM compounds, that were assigned to those two isolates: fosmids fl5-E10, 20-G8, f23-H6, D-Gll and fl2-Al were assigned to isolate D250, and fosmids f9-E12, flO-D3, fl4-D9, fl6-B12 were assigned to isolate D323. For the sister fosmids fl5-E10, f20-G8, and f23-H6, the AM activity is likely to be due to violacein for which genetic machinery was found to be present in those sequences. However, no violacein related genes were found in the bioactive encoding fosmids O-Gl 1 and fl2-Al also belonging to isolate D250. Similarly, no TDA related genes were found in any of the fosmids ascribed to isolate D323. The violacein biosynthetic cluster of isolate D250, found in the sequence of fosmids fl5-E10, f20-G8 and f23-H6, resembled similar clusters found in other violacein producer bacteria, such as C violaceum and P. tunicata, and consisted of genes vioA-E (August et al. 2000; Brady et al 2001; Antonio and Creczynski-Pasa 2004; Balibar and Walsh 2006; Asamizu et al. 2007; Thomas et al. 2008). This strong resemblance and the presence of mobile elements, such as transposases and integrases, adjacent to the violacein operon in strain D250, suggests horizontal transfer of the violacein biosynthesis genes between violacein producing strains. This conclusion is further supported by analysis of the GC content that demonstrated a lower than average GC content for the predicted violacien cluster and the surrounding ORFs, compared to the rest of the fosmid insert sequence (Appendix Illf). Notably, the insert sequences of the fosmids fl5-E10 and f20-G8 were identical, whereas f23-H6 lacked two genes: a CHP and a short chain dehydrogenase/reductase. Similar genes were also found in the sequence of other violacein producing bacteria, such as P. tunicata and C violaceum, however, they were located in different parts of the chromosome. Further investigation of the functions of these genes would help to identify the mechanism by which the absence of these genes could affect the expression of violacein in isolate D250, which is evident by the altered violacein production in the clone 23-H6. Such research could include creation of knock-out mutants in those two genes of isolate D250, as well as other violacein producer bacteria, and screening of the mutants for the altered violacein production, similar to the phenotype expressed by the clone 23-H6. Other than violacein producing clones, fosmids extracted from clones 3-Gll and 12-Al, were also assigned to isolate D250. Given that almost half of fosmid f3- Gll was comprised of the genes responsible for the biosynthesis of various NADH dehydrogenase components, which would not be expected to be involved in any other function then energy generation, the AM encoding genes are likely to be located in the other half of the fosmid sequence, containing multiple mobile elements and CHPs. Notably, the GC content plot showed significant differences between those two parts of fosmid OG-11. Taking into consideration also the presence of mobile elements, it is suggestive of a recent HGT, which could be investigated in the future. Fosmid fl2-Al was shown to contain a gene cluster similar to the genes encoding for the production of the siderophore aerobactin. Aerobactin was first characterized in 1969 in Aerobacter aerogenes (Gibson and Magrath 1969) and was long considered as one of the virulence factors exclusive to enteric bacteria (Davis et al 1981; Shandera et al. 1983; De Lorenzo and Martinez 1988). However, aerobactin production was later also demonstrated in various marine bacteria (Buyer et al. 1991; Haygood^/fl/. 1993). The genes required for transport and biosynthesis of aerobactin can be either located on a plasmid or in the chromosome (Williams 1979; Stuart et al. 1980; Braun 1981; Loper et al. 1993; Yokes et al. 1999). These genes usually include conserved genes /wcABCD and /w/A, and are surrounded by insertion elements (McDougall and Neilands 1984; Yokes et al. 1999), suggesting the possibility of their transfer via transposition. However, no such elements were found in the sequence of 12-Al fosmid. Notably, among other transporters, a TonB receptor protein known to mediate the transport of siderophores, was also found in the sequence of this fosmid. Despite being a well-known virulence factor, aerobactin has not been previously reported to have AM activity. Moreover, while not directíy confirmed for aerobactin, the iron-binding activity of siderophores may not be sufficient to cause a zone of inhibition as observed in the AM tests used in this study (Chapter 3, section 3.3.4.1). Therefore, the antibacterial activity, conferred by fosmid 12-Al, is likely to be caused by non-aerobactin related genes. Otherwise, aerobactin may have another, as yet-unknown, likely "non-siderophoric" mechanism of action against bacteria, which might be revealed by further mutagenesis of the aerobactin biosynthesis genes contained within the fosmid fl2-Al and screening of the mutants for the loss of AM activity. The only sequence derived from isolate U95 - a representative of possibly a novel genus belonging to the Roseobacter clade, was fosmid fl9-F10 extracted from the corresponding E. coli clone. Clone 19-FlO was shown to produce a dark-green pigment when the fosmid was induced to a high-copy number (Figure 6-4 d). Sequence analysis revealed the presence of a large NRPS within the fosmid fl9-F10, which predicted product had 45 % identity to both the blue pigment indigoidine synthetase BpsA of Streptomyces lavendulae (Takahashi et al. 2007) and indigoidine synthase IndC from Erwinia chrysanthemi (Reverchon et al 2002). There is strong evidence in the literature which indicates that NRPS are often responsible for the production of molecules that possess various biological activities (Schwarzer et al. 2003; Salomon et al 2004; Bergmann et al 2007; Dunlap et al. 2007; Zhu et al. 2007; Caboche et al. 2008; Collemare et al. 2008; Jirakkakul et al. 2008; Pearson et al. 2008; Caboche et al. 2009). Therefore, this gene could be the primary candidate responsible for the antibacterial activity of the clone 19-FlO, and, subsequently, of isolate U95. In the sequence of the fl9-F10 fosmid, the NRPS was followed by a CHP and then by a gene encoding a protein with 56 % identity to the indigoidine biosynthesis protein IndA, also found in E. chrysanthemi. However, no indB related gene, reportedly required for the biosynthesis of indigoidine (Reverchon et al. 2002), was found in the sequence of fl9-F10, which may indicate that the NRPS, derived from isolate U95, is likely to encode for a peptide different from indigoidin, or that the indB related gene could be present on a separate part of the chromosome. 174 Notably, the fosmid fl9-F10 was found to be rich in various transporters, such as multiple ABC transporters and MFS permease, which are usually linked with resistance to antimicrobials (Jacquot et al 1997; Nourani et al. 1997; Martin et al 2005). It is possible that these transporters are involved in autoprotection of the producer organism U95, against the high concentrations of the bioactive compound. The sequence of fl9-F10 also contained genes encoding for a type VI secretion system (T6SS), for example, proteins related to ImpA and ImpB, found in other organisms, such as, Rhizobium leguminosarum, Vibrio cholerae and Pseudomonas aeruginosa (Das and Chaudhuri 2003; Pukatzki et al. 2006; Filloux et al. 2008) as part of a T6SS gene cluster. T6SS has been recently described as a complex multicomponent secretion machinery, which has a major role in the interaction of bacteria with eukaryotic hosts, both in pathogenic and symbiotic relationships (Filloux et al. 2008). For example, in Rhizobium leguminosarum the T6SS was shown to prevent pea nodulation and nitrogen fixation (Bladergroen et al. 2003). In V. cholerae and P. aeruginosa it was found to contribute to pathogenesis (Mougous et al. 2006; Pukatzki et al. 2007). In fact, there is evidence that the T6SS clusters are usually located within pathogenicity islands (Cascales 2008). Previous studies have suggested that the T6SS may employ a bacteriophage- like strategy to puncture host cell membranes and, thus, making a route for the passage of substrates across the bacterial cell envelope into the host cytosol. (Filloux 2009; Pukatzki et al. 2009). Interestingly, the T6SS Vgr family protein was also located in the sequence of the fosmid 19-FlO, upstream of an ImpA encoding gene, several genes apart. As previously shown for other bacteria, members of this protein family are relatively conserved and may often be encoded by genes located far from the type VI secretion locus (Filloux et al. 2008). VgrG components share similarities with gp5/gp27 proteins of the bacteriophage T4 tail spike used to puncture the bacterial membrane to allow bacteriophage DNA injection. Despite rapid progress in understanding of the T6SS in recent years, there is still a lack of information regarding the substrates or proteins which are being translocated. Several proteins which require T6SS for secretion have been recently reported (Bladergroen et al. 2003; Schell et al. 2007; Zheng and Leung 2007), moreover, their encoding genes were found to be located in close proximity to T6SS clusters (Pukatzki et al. 2009). Therefore, based on these data, future studies could 175 establish whether the NRPS encoded bioactive compound located on the same fosmid, is the substrate for T6SS in isolate U95. Analysis of the genes contained within the fosmids obtained from marine epibiotic bacteria in this study, besides providing information in relation to the production of bioactive compounds, also revealed the presence of genes that could potentially be involved in symbiosis with their eukaryotic host. For example, several genes involved in the biosynthesis of riboflavin (vitamin B2) were found in fosmid fl4-D9, originated from isolate D323. Taking into account the recent literature data suggesting that symbiotic bacteria can synthesise and provide vitamins to host (Croft et al. 2005; Wu et al. 2006; Wagner-Dobler et al 2010), a similar relationship might also exist between the isolate D323 and its eukaryotic host. Overall, the fosmid library created using the genomic DNA of several bioactive producing isolates has given an invaluable insight into the genetics behind the production of biologically active compounds, and, possibly, the role that these bacteria may play in the environment, such as being possible pathogens, or their likely importance in the symbiosis with the eukaryotic host. Sequencing confirmed the high content of genes involved in secondary metabolism in the fosmids contained within AM producing E. coli clones. Moreover, the biosynthetic cluster of violacein, a compound successfully isolated and chemically characterized in the current project from isolate D250, was successfiilly cloned and the genes were identified in the fosmid originated from isolate D250. This study also clearly illustrated the production of multiple bioactive compounds by isolates D250 and D323 on a genetic level, confirming previously obtained data (Chapters 4 and 5 respectively). Furthermore, besides the biosynthesis of bioactive compounds, this study has provided suggestions for the presence of genes that may be involved in various processes linked with the antibiotic production, such as transport and antibiotic resistance, which, could be investigated in the future. CHAPTER SEVEN General Discussion The work presented in this thesis has demonstrated the vast biotechnological potential of marine epibiotic bacteria, particularly those living on the surfaces of marine algae D. pulchra and U. australis. During this project 325 bacterial strains were isolated from these algae, 12 % of which demonstrated the capacity to produce antimicrobial compounds. Among these, two different isolates designated D250 and D323 were found to produce violacein and tropodithietic acid respectively. This study also attempted to understand the role of the antimicrobials for the producer organisms - a long neglected avenue of antibiotic research. Consequently, as an example, the role of violacein in the biofllm formation has been proposed. In addition, TDA obtained from isolate D323 was shown to have a strong inhibitory effect on various marine epibionts likely to exist in the same natural environment as the isolate D323, suggesting the role of TDA in the defence of both the producer microorganism, as well as the eukaryotic host. Construction of a large DNA insert (fosmid) library using the DNA of AM producing isolates proved to be successful and allowed for a significant increase in the hit rates in obtaining bioactive producing clones compared to the environmental metagenomic libraries. Moreover, analysis of the insert sequences from AM producing clones identified not only the genes that could be responsible in the biosynthesis of the AM compounds, but also genes potentially involved in the associated processes, such as the transport of the AM compounds and the resistance towards its inhibitory effects. This final chapter outlines the major findings and conclusions of this PhD research project and gives directions for future research. 7.1. The potential of marine epibiotic bacteria as a source for future drug discovery The work carried out in this project demonstrated that marine epiphytic bacteria represent a great potential as a source of biologically active compounds such as antimicrobials by showing that 12% of all epiphytic isolates possessed an antimicrobial (AM) activity. Moreover, many of the AM producing isolates were preliminarily identified as new species or genera. For example, isolates U82 and U95 presumably represent a novel genus within the Roseobacter clade. The abundance of bioactive producers among marine epibionts was also observed in several other studies (Lemos et al 1985; Burgess et al 1999; Hentschel et al 2001; Muscholl- Silberhom et al 2008; Wilson et al. 2009). This cleariy illustrates that the targeted isolation of marine epibiotic bacteria could provide a rich source for bioactive compounds in future drug discovery. During the course of this project, the antimicrobial compounds violacein and tropodithietic acid were chemically purified and identified from isolates D250 and D323 respectively. However, both these isolates were also found to synthesize other AM compounds as deomonstrated by both chemical and clone library analyses. It has been suggested that bioactive producing bacteria tend to synthesize not only one, but a variety of different bioactive compounds. Such strains that are rich in secondary metabolites have been termed "metabolically talented" (Trujillo et al. 1997; Knight et al. 2003). Examples include Streptomyces sp. strain Go.40/10, P. tunicata, and the cyanobacterium Lyngbya majuscule. Streptomyces sp. strain Go.40/10 was found to synthesize at least 30 different secondary metabolites, many of which were structurally novel and included antifungal and antibacterial compounds (Schiewe and Zeeck 1999). The marine surface associated bacterium P. tunicata and its secondary metabolites have been extensively studied (Bowman 2007). The bioactive compounds identified include the antibacterial compounds violacein and the protein AlpP (James et al. 1996), and the antifungal compound tambjamine (Franks et al. 2005). Lyngbya majuscule, in turn, has been shown to produce several unique bioactive compounds including hectochlorin and the jamaicamides A-C (Márquez et al. 2002; Edwards et al. 2004; Ramaswamy et al. 2007). Taking into consideration the production of multiple AM compounds by the isolates used in this study, further research would be necessary to fully investigate the metabolic potential of these bacteria and could also identify compounds with biological activities other than antimicrobial, such as, for example, those of antiprotozoan, antinematode, antiviral and cytotoxic activity. 7.2. Importance of environmental conditions in the expression of bioactive compounds The fact that the conditions of growth can influence the formation of various bioactive compounds is well known and was discussed in Chapter 1 (section 1.5.1). Likewise, this study also demonstrated the effect of various parameters, such as the growth media composition, use of solid or liquid media, and the length of cultivation, on the presence of AM compound in isolates U156 and D245. Moreover, in all cases "incorrectly" chosen parameters could lead to the complete loss of the production of the bioactive compound. These observations highlight once more the importance of choosing the right cultivation conditions that would promote the formation of desired natural products, while slight differences could create an illusion of inability of the strain to produce these compounds. The sequencing of whole microbial genomes as well as environmental metagenomes can indicate the presence of genes involved in biosynthetic pathways leading to the production of bioactive metabolites in strains not previously known for their biosynthesis (Jensen 2010). For example, recent analysis of the genome of P. tunicata revealed the presence of nine NRPS genes (Thomas et al 2008). Two products of these NRPS, with predicted biological activity have been recently confirmed in the original strain of P. tunicata by varying its conditions of growth (Blasiak and Clardy 2010). Therefore, variations in the growth conditions appears to be a valuable strategy both to improve the production of natural products, as well as to promote their expression in the non-producing strains, especially, when their presence is suggested by the genomic data. 7.5. Intracellular localization of bioactive compounds: A possible adaptation to the marine environment Data obtained in this study have shown, that, in many cases, marine bacteria do not readily secrete bioactive compounds. For example, in the case of isolate U156 and all the other LGM bacteria, as well as isolates D245 and D250, the AM activity was localized in the cell extract but not in the growth medium. In order for the toxic compounds to have an effect, one of the possible strategies for these producer bacteria might be to be consumed by the predators, such as protozoa, so that the sacrifice of few members would generally benefit the overall population. Such an effect has been suggested for the broad-spectrum inhibitory compound violacein, active against both bacteria and eukaryotes (Matz et al. 2008). It is also possible that these cell-bound bioactives, in order to have an effect on antagonistic organisms, can be transported and released only when the producer is in direct contact with the antagonistic organism. In that case, the effector molecules could be directly "injected" into the target organism via secretion systems, such as Type III, Type IV and the recently described Type VI system widely present in gram- negative bacteria (Filloux et al. 2008; Pukatzki et al. 2009). Substrates (transported molecules) of these systems often include bioactive toxins, such as, for example, BteA protein and pertussis toxin of Bordatella (Shrivastava and Miller 2009), and CagA protein of Helicobacter pylori (Stein et al. 2000). These transport systems resemble the delivery systems employed by bacteriophages and are believed to form syringe-like structures that allow them to transport molecules directly from the producer cell to the cell cytosol of the target organism. As a result of this particular feature, the transported molecules do not come into contact with the environment and, thus the successful delivery of secreted molecules into the target organism is ensured. In the aquatic environment, secreted compounds can rapidly be diluted and carried away by the flow of water upon their release, before having an effect onto a target organism. Therefore, for marine bacteria it is of highest importance to prevent such a waste of valuable products. Consequently, the strategies outlined above, could prevent the waste of these metabolites, and, therefore, could be regarded as adaptations to life in the marine environment. 7.4. Antimicrobial compounds produced by bacteria may have multiple ecological functions Historically, research on AM compounds mostly focused on their antibiotic effects on target organisms, such as, for example, the assessment of the spectrum of activity against various organisms, minimum inhibitory concentrations, the mechanisms of action and the development of resistance by the target organisms. As a result, after obtaining the bioactive compound from the producer organism, the foci of further research shifted to target organisms. Thus, the purpose of the production of these bioactives and their role for the producer organisms was overlooked for many decades. It is widely accepted that nature acts in a very economical way and that traits that are not necessary for the survival in a particular environment are quickly eliminated during the course of evolution. In that respect, the production of natural products is often costly, and therefore, it can be assumed that the costs associated with their biosynthesis must be justified by the importance of their role in the survival of the producer organism. As mentioned above, it has long been presumed that the role of AM compounds is limited to their growth inhibitory activities, and, therefore, often the only ftjnction that was ascribed to these compounds was an antibiotic activity leading to the elimination of predators and competitors by the producer organism. However, recent literature suggests, that some bioactive compounds could also perform a different role, often acting at low, subinhibitory, concentrations. These effects can include the regulation of bacterial behavior via modulation of various gene expression levels, challenging the very understanding of what is an antibiotic (Linares et al 2006). The recognition of such effects, besides shedding light on the ecology of the producer organism, could be crucial for the potential applications of these bioactive compounds, for example, in clinical practice. For instance, the dual effect of well- known and widely used antibiotics such as tobramycin, tetracycline and norfloxacin has been demonstrated showing that at subinhibitory concentrations they can promote biofilm formation in Pseudomonas aeruginosa (Hoffman et al 2005; Linares et al. 2006), thus making these bacteria even more resilient to antibiotics. In addfition, tobramycin was also shown to increase bacterial motility, and tetracycline triggered the expression of type III secretion system and, consequently, increased the cytotoxicity of P.aeruginosa (Linares et al. 2006). Hence, these compounds can have opposite effects and could be harmful rather than beneficial if administered in concentrations below the inhibitory level. Violacein is a compound that has been known for more than a century and was extensively studies in relation to its various growth inhibitory activities (Lichstein and Van De Sand 1946; May et al 1991; Duran et al 1994; Leon et al 2001; Andrighetti- Frohner et al 2003; Duran et al 2003; Melo et al 2003; Nakamura et al 2003; Matz et al 2004; Bromberg et al 2005; Kodach et al 2006; Becker et al 2009). However, for the fitst time, the current study has also demonstrated the involvement of violacein in surface colonization and biofilm formation of isolate D250, as evident by the lack of biofilm formation observed in violacein-deficient mutants (Chapter 4). Given the surface-attached biofilm lifestyle of this epibiotic bacterium, such an activity of violacein, could provide a significant ecological advantage for isolate D250, allowing it to rapidly colonize the surface of the eukaryote. Future research into the subinhibitory concentrations of various AM compounds could reveal yet unknown functions of these compounds, as well as provide additional insight into the chemical ecology of producer organisms. 7.5. Identification of microorganisms: 16S rRNA based phytogeny andphenotypic characterization Li the past, microorganisms were classified based on resemblances in their morphological, physiological and biochemical characteristics (Buchanan and Gibbons 1974). However, these classification systems often did not take into consideration phylogeny and they barely reflected the true evolutionary relationships between microorganisms (Van Belkum et al 2001; Gupta and Griffiths 2002). In contrast, determination of phylogeny provides an insight into the course of bacterial evolution. 16S rRNA gene sequencing is currently accepted as a universal tool for the initial determination of bacterial phylogeny and is indispensable for the quick identification of bacteria (Woese 1987; Woese 1991; Olsen and Woese 1993; Ludwige/a/. 1998). Merging bacterial phylogeny and phenotypic characteristics represents the ultimate goal for bacterial identification; however, it still remains a challenge. Thus, for 16S rRNA gene phylogeny, 97% similarity is often used as a cut-off value for species differentiation (Stackebrandt and Goebel 1994). This may be highly predictive for many bacteria. For example, the epiphytic isolate D323 and related sponge- associated bacteria used in this study (Chapter 5), which, sharing only 98% 16S rRNA gene similarity, show many similarities in phenotypes (Chapter 5). However, there is now a growing acceptance in the scientific community that even a 16S rRNA gene similarity> 99 %, does not always translate into phenotypic similarities, especially when it comes to secondary metabolites. In fact, recent studies have suggested that such organisms could be assigned to separate species despite their close phylogenetic relationship (Maldonado et al 2005; Jensen et ai 2007; Jensen 2010). For example, the recently discovered species Salinispora arenicola and Salinispora tropica share 99.5 % 16S rRNA gene similarity (Jensen 2010), however, differ phenotypically, particularly in their secondary metabolite profiles (Jensen et al. 2007), which was fiirther supported in whole genome comparative analysis (Penn et al. 2009). Moreover, the spectrum of secondary metabolites was largely consistent within each species, leading to the proposal, that in Salinispora, the production of secondary metabolites occurs in a species-specific manner (Jensen et al. 2007; Jensen 2010). The phenotypically dissimilar isolates U156 (representative of bacteria assigned to the Hght green morphotype, LGM) and D250, used in this study, are clear examples of phylogenetically closely related bacteria with 16S rRNA gene sequence pairwise comparison revealing as little as 6 single nucleotide differences between the two. Consequently, such fine-scale differences may not be easily resolved by 16S rRNA gene based phylogeny studies, especially in the presence of other bacteria likely to exist in an environmental sample. For example, in a hypothetical large culture-independent community analysis study, 16S rRNA gene based phylogeny would, arguably, place D250 among other related bacteria, such as the LGM (Chapter 3), without a clear distinction between the two groups, thus, further highlighting the importance of considering phenotypic characteristics in bacterial identification. Future studies could possibly reveal other differences or similarities between LGM and D250 isolates, particularly in relation to metrics commonly used for characterization of bacterial species, which could further clarify the possible delineation of these bacteria into separate species. The absence of a universally accepted species concept in bacterial taxonomy makes bacterial speciation even more challenging (Gevers et al. 2005). One of the 183 difficulties that arises in defining the bacterial species is due to the high rates of recombination in bacteria, e.g. via horizontal gene transfer (HGT), which happens frequently for genes involved in natural product biosynthesis (Walton 2000; Egan et al. 2001; Koonin et al 2001; Ginolhac et ai 2005; Khaldi et ai 2008; Fitzpatrick 2009). The present study also demonstrated that, despite the great number and variety of microbially derived bioactive compounds, the same compound can be produced by phylogenetically distant organisms. For example, the production of TDA, a common metabolite of various strains related to P. inhibens, was confirmed as a metabolite of the phylogenetically distant isolate D323 and related bacteria. Likewise the isolate D250 investigated in this study, was the first representative of the genus Microbulbifer that produces violacein - a bioactive compound found in many other bacteria. Moreover, the similarities in the violacein biosynthetic cluster of isolate D250 with those of the distantly related bacteria, such as C. violaceum and P. tunicata, and the presence of mobile elements in close proximity with the violacein cluster, observed for the first time in this study, could support the possibility of recent horizontal transfer of violacein genes (Chapter 4). In order to avoid the difficulties introduced by HGT in defining bacteria species, Lan and Reeves (1996) have proposed the Core Genome Hypothesis which suggested, that there is a "core" genome, mostly comprised of housekeeping genes, that are shared within a species, whereas mobile "auxiliary" genes, such as those encoding for the production of bioactives, are only specific to a subpopulation of bacteria within a given species. It has also been suggested, that these core genes would be mostly vertically transferred and the divergence of the core genome during the course of evolution would lead to the divergence of species (Riley and Lizotte- Waniewski 2009). Besides 16S rRNA gene based phylogeny, the sequence information from a collection of housekeeping genes (from the "core genome") is widely used in the identification of various pathogenic bacteria in epidemiological studies in an approach called multilocus sequence typing (MLST) (Maiden et al 1998). MLST provides portability and easy exchange of sequence information between research groups. However, due to the sequence conservation in housekeeping genes, MLST sometimes lacks the discriminatory power to differentiate bacterial isolates (Fakhr et al 2005; Johnson et al. 2007). In contrast to the notion to only include housekeeping genes for species identification, Jensen et al (2007) have proposed that "auxiliary" genes are important 184 for niche adaptation and, consequently, for species delineation. Specifically, they argue that through HGT, microorganisms may "sample" various genes and gene clusters from the "genetic pool" and the most beneficial genes, such as the ones encoding for the production of bioactive compounds that are necessary for survival and niche adaptation, could become fixed in the population and later vertically inherited by subsequent generations (Jensen et al 2007). Likewise, Hunt et ai (2008), by studying marine Vibrionaceae, provided evidence, that environmental adaptation could be a trigger for speciation in closely related bacteria, and also indicated that in community-wide assessments such ecological adaptations may not be reflected by the use of universal gene markers (Hunt et al. 2008). Consequently, it has been suggested that acquisition and fixation of genes involved in functional adaptation, such as those encoding for natural products, act as a previously unrecognized force driving the functional diversification of bacteria (Hunt et al 2008.; Penn et al. 2009). Production of secondary metabolites represents one of the major means by which microorganisms interact with the environment, and, therefore, the production of natural products can have implications in the ecological role of the bacterium in the environment as a member of a multispecies community, as well as in the interaction with higher organisms. Therefore, phenotypic characteristics, such as production of secondary metabolites, should not be overlooked in the functional assessment of microbial communities and should be regarded as an integral part of bacterial identification. 7.6. Large DNA insert libraries based on the genomic DNA from cultured isolates^ known for the production of bioactive compounds^ enhances the hit rate of positive clones and provides an insights into the genes involved in the their production This study has described the successful cloning of the genomic DNA from the collection of cultured isolates, the functional screen of the resulting clone library, and the analysis of the fosmid sequences that led to the production of antimicrobials. Functional genomics is currently a widely used metagenomics tool. It includes the cloning of total DNA obtained from an environment into the host bacterium and screening of the clones for desired activity. However, as was previously mentioned, the hit rate of obtaining bioactive producing clonbes can be as low as 1 in 730000 (Henne et al 2000) clones screened. The main reasons behind this are thought to be the absence of the machinery necessary for the production of bioactives in the host bacterium as well as the toxicity of the bioactive product (Schloss and Handelsman 2003; Handelsman 2005; Pelaez 2006; Sorek et al 2007). However, the insufficient hit rates could also be a direct consequence of a scarcity of bioactive producing bacteria in a particular environment compared to the abundance of DNA from non-bioactive bacteria. The use of genomic DNA from isolates, already known for the production of biologically active compounds, such as antimicrobials, could minimize that "dilution" effect and allow the maximum utilization of the capabilities of the host strain to produce these antimicrobials. It can also give an estimate of the contribution of "dilution" in the low hit rates often observed in clone libraries. The approach used in this study has proven to be successfiil and led to the increase in positive hit rate by several orders of magnitude. In indicates that the underrepresentation of DNA from bioactive producing bacteria within the environmental DNA has serious implications and significantly contributes to the low hit rate observed in metagenomic libraries. Construction of large DNA insert libraries can also provide valuable information concerning the genes involved in the production of bioactive compounds, both directly involved in the biosynthesis, as well as in the processes associated with the production of these compounds, such as, for example, the transport of these natural products and the resistance mechanism that producer bacteria may employ to prevent the autoinhibitory effects. For example, close to the NRPS, presumably responsible for the biosynthesis of the bioactive compound, the presence of genes encoding the components of T6SS was revealed on the fosmid fl9-F10. This system could possibly act as the main transporter system for the bioactive compound, whereas various ABC transporters and MPS permease, also found on the same fosmid, could be associated with the resistance. Consequently, analysis of fosmids provided a new insight both into the nature of the bioactive compound, as well as the processes it undergoes after its biosynthesis, and, thus, offers great opportunities for fiiture research designed to reveal the roles of separate genes within those fosmids. Future studies may include transposon mutagenesis of the fosmids and screening of the mutant clones for the loss of activity; 186 the latter would indicate the essential role of the altered gene in the production of bioactive compound. The fosmid libraries could also be used to simplify the natural product purification process and allow for the rapid identification of the additional metabolite when compared to a reference extract of a non-bioactive producing host culture. In addition, this facilitates the identification and separation of multiple bioactive compounds produced by the same bacterium, as demonstrated in the current study where several different bioactive-encoding fosmid sequences were obtained from the genome of the same bacteria (Chapter 6). Furthermore, sequencing of the insert DNA could provide information regarding the biosynthetic pathway of the natural product, and, consequently, on its nature, and, would, therefore, help in designing further purification strategies. 7.7. Concluding remarks Even though this study was initially focused on obtaining novel antimicrobials, it had broader implications and significantly contributed to a better understanding of the chemical ecology and functioning of marine epibiotic bacteria, as well as their relationship with host organisms. The study has provided new insights into the production of antimicrobial compounds by marine epibiotic microorganisms and indicated novel avenues for future research. 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APPENDIX I a) Luria-Bertani medium (LBIO) (per litre of distilled water)* NaCl: 10 g, Tryptone: 10 g, Yeast Extract: 5 g b) Nine Salts Solution (NSS, per litre of distilled water)* NaCl: 17.6 g, Na^SO.: 1.47 g, NaHCOa: 0.08 g, KCl: 0.25 g, KBr: 0.04 g, MgCl2.6H20: 1.87 g, gCaCl2.2H20: 0.41 g, SrC1.6H20: 0.008 g, H3BO3: 0.008 g, - Adjusted to pH 7 c) Vaatanen Nine Salt Solution (VNSS, per litre of NSS)* peptone: 1.0 g, yeast extract: 0.5 g, glucose: 0.5 g, Fe SO4.7H2O: 0.01 g Na2HP04: 0.01 g d) Columbia Blood Agar (CBA, per litre of distilled water)* Columbia Blood Agar Base (Oxoid): 39 g, cysteine hydrochloride: 0.75 g The following components were filter sterilized before being added to sterile autoclaved media at 50°C: hemin: 5 mg, menadione: 0.5 mg, defibrinated horse blood: 50 ml •supplemented with 1.5 % agar (Agar-Agar, Research Organics) to make solid media e) Phosphate Buffered Saline (PBS, per litre of distilled water): NaCl: 8 g KCl: 0.2 g Na2HP04: 1.44g KH2PO4: 0.24g pH 7.2 - 7.4. APPENDIX II a) RNase treatment of DNA To prepare a of stock solution of RNase dissolve RNase A at a concentration of 10 mg/ml in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCL Boil for 15 min and cool slowly to room temperature. Store at -20 oC Prior to use boil stock solution for 5 min and cool to room temperature. Add 1 il of the stock solution for every 100 fl of DNA solution to be treated. Incubate at room temperature for 30 min. Follow with phenol : chloroform : isolamylalcohol extraction and ethanol precipitation b) Phenol : chloroform : isolamylalcohol extraction of DNA Add an equal volume of phenol : chloroform : isolamylalcohol (25:24:1 (v/v/v)) to the DNA solution, mixthoroughly. Separate the phenol and aqueous phases by centriftigation at 14 000 x g for 5 min at room temperature. Transfer the upper aqueous phase into a fresh tube. Repeat extraction until the interface is no longer visible c) Ethanol precipitation of DNA Add 1/10 th volume of a 3 M sodium acetate (pH 5.2) solution. Mix well. Add 2.5 volumes of ice-cold absolute ethanol. Mix well. Chill at -20 oC for 60 min (longer times for small concentrations or small fragments of DNA). Pellet the DNA by centrifugation at 14 000 x g at 4 oC for 15 min (longer times for smaller fragments). Discard supernatant and wash pellet in 70 % ethanol to remove salts. Invert tube and dry the pellet or use a vacuum desiccator. Resuspend DNA in appropriate volume of milli-Q water or EB Buffer (10 mM Tris-Cl, pH 8.5, Qiagen). d) Isopropanol precipitation of DNA Add 120 |il of molecular grade water, 20 |il of 3M sodium acetate (pH 5.0) and 140 ^il of isopropanol to 80 ^il of DNA solution Gently mix by inversion and incubate for 30 minutes at RT Centrifuge at top speed for 30 minutes at 4°C Remove the supernatant and add 500 |il of 70 % ethanol to the pellet Centrifuge at top speed for 5 minutes Remove the supernatant and air-dry the pellet at RT for 20 -30 minutes Resuspend the pellet in 25 |LI1 of EB buffer (10 mM Tris-Cl, pH 8.5) e) 1 X TBE buffer (per liter) Tris base: 10.8 g, boric acid: 5.5 g, 0.5M EDTA (pH 8.0): 4 ml f) 1 X TAE buffer (per liter) Tris base: 4:84 g, 0.5M EDTA pH 8.0: 2 ml, glacial acetic acid: 1.14 ml. APPENDIX III Fosmid sequences Dashed lines represent the GC content plot, in blue -the GC content is below the average, in red - GC content is above the average. a) f3-Gll 11 ' ' ' ' ' • ( I I I t I I ^ 2000 4000 6000 8000 CHP CHP CHP CHP CHP transposase integrase integrase transposase II IIIIMH" 10000 12000 14000 16000 transposase NADH dehydrogenase components I I I I I M M M I I I I I 11 I M I I I I I I I M I M I It I I I I I I I I , , . 18000 20000 22000 24000 NADH dehydrogenase components (continued) b) f9-E12 * I M I M I M I M I I M I M M I M t * I I H I I I M I I I I I I I 0 3000 6000 9000 CheZ MaoC like . Chemotaxis dehydratase permease isoleucyl-tRNA signal peptidase II protein CheY kinase synthetase Chemotaxis response regulator receiver I 11 M M 11 I 11 I. II III I M I III M I I M M M I I I I I 12000 15000 18000 CHP protease protease diguanylate LuxO repressor, CHP Gro S Gro L phosphodiesterase two component chaperonin chaperonin system response (continued) regulator II I I II I I f N M I II |l IN ' ' I t I M MM 21000 24000 27000 \ CHP GroL CHP putative CHP sugar methionine- (^fj^g CHP sec- YciA chaperonin NAD- transporter S-sulfoxide giucose/sorboson independent acyl- (continued) dependent putatiye e dehydrogenase P'"«^^ ^^ epimerase/ acetyl translocase thioester transferase hydrolas dehydratase I I t I M I M M M IM II II I 30000 33000 36000 ATP bacteriocin CHP CHP elvcosvl dolichyl-phosphate histidyl-tRNA , . fransfLe beta-D- synthetase P^osphon mannosyltransferase transterase ^^^^^ regulatory subunit c) flO-D3 I I I I I I I . . II I I I I I M ' I I I I I 0 3000 6000 9000 periplasmic quinol quinol periplasmic NirT j^^^g CHP short-chain CHP nitrate reductase dehydrogenase dehydrogenase nitrate denitrificati receptor dehydrogenase periplasmic „lembrane reductase plug ' component component ^ ® /reductase ^ component ' ' 12000 15000 18000 CHP CHP ABC transporter I I M ) . I I I I II II I I I I II II t I I ( Il I I I I I I I I • I . . "'Ill IIIIIM"' 21000 24000 27000 ABC type I response regulator transporter secretion RpfC sensory / regulatory enoyl-CoA receiver (contains (continued) membrane protein (two-component hydratase/isomerase fusion protem CheY-like receiver VirA-like sensor kinase) domain) I I I I II I I I M I I I I I I I I I I I I I I I I I I I I I i I I I I I I I I I I I I I ( 30000 33000 36000 photolyase short-chain CHP cyclopropane- amine oxydase RNA ChrR dehydrogenase fatty-acyl- polymerase ,transcriptional phospholipid sigma-70 synthase factor d) fl2-Al , MIIIM*' IIIIIMMI|||||||||||||||I I 3000 6000 9000 acyl-CoA cell-cell argynine NUDIX phospholipase CHP lucD - lysine dehydrogenase signaling decarboxylase hydrolase protein monooxygenase illlllllllllhli. Hill I I II I n . I I I II II I illllllliiillMlllll"" 12000 15000 18000 21000 lucD - lysine lucC - lucB - lucA - permease rubredoxin / flavin (possibly carbonic rubredoxin reductase monooxygenase aerobactin acetyl aerobactin MatB) transferase anhydrase reductase domain (continued) synthetase transferase synthetase protein llllllllll , ,,.. • IIIIIII11II iiiiih iiiiiiiiiiiiiiin 4 24000 27000 30000 glycerophosphoryl CHP diester tonB receptor aminopeptidase putative peptidyl- outer membrane phosphodiesterase tRNA hydrolase autotransporter domain protein e) n4-D9 Il II I II II I , Milli I I I I I I I 0 1000 2000 3000 4000 5000 6000 » riboflavin MFS permease thiamine- synthase beta RibD riboflavin NrdR senne monophosphate NusB ^hain riboflavin biosynthesis transcriptional hydroxymethyl- kinase transcription synthase nrotein reeulator antitermination alnha-chain . I t I I I I I I • , • ill.., iiiMIM Mill I I I I I M ' ' ^ i 7000 8000 9000 10000 11000 12000 13000 CHP twin-arginine MarR family 3-hydroxyisobutyrate CHP enoyl-CoA acyl-CoA translocation ranscriptional dehydrogenase hydratase/camithine dehydrogenase nathwav siptial repiilator racemase . I M I M I M I I I I I I M I I I I I I I I II I II 'Milli! 'I I I 14000 15000 16000 17000 18000 19000 acyl-CoA acyl-CoA threonine CHP TrkA-C domain arginyl-tRNA dehydrogenase dehydrogenase dehydratase transporter protein transferase icontinned^ icnntinned^ f) flS-ElO, f20-G8, and f23-H6 (combined) I MM INI " 0 CHP 3000 6000 9000 short-chain dehydrogenase/ ^ ; transposase CHP integrase indolepyruvate Y reductase ferredoxin sigma factors oxidoreductase III Mil Mil I I I M I I M I I M i I I t I I I I I II I I I I I I I I M II I I II M II I I I I I II II ( ( 1 12000 15000 18000 indolepyruvate transcriptional peptidase VioE - VioC - ferredoxin regulator prodeoxi- VioD - monooxygenase ^ioB - oxidoreductase violacein hydroxylase poliketyde synthase synthase (continued) V ^ violacein biosynthetic cluster llljiilll ll|| nil nil Mil IIINIH nil I nil Mil Mill 21000 24000 27000 30000 VioB - poliketyde VioA - triptophan transcriptional CHP CHP CHP synthase monooxigenase regulator (continued) violacein biosynthetic cluster (continued) g) n6-B12 I I I • I M ' ' I I I 11 11 I Ml. I .. * 11 11 I I 11 I 11 I I 16SrRNA 3000 6000 9000 mosc domain peptidase M23B ClpB ATP-related chaperone containing Fe-S protein <• t • I I I I I M t t M I I I I I I I I I I I I I I I I I I I I I I I M I It I I I I I M . I . . , , M I I 11 12000 15000 18000 MOSC domain protem- peptide phosphoenolpyruvate- 3-demethyl alpha^eta CHP containing Fe-S (glutamine-N5) ^hain release protein ubiquinone-9 hydrolase protein (continued) methyltransferase ^ , , , 3-methyl release factor- ' phosphotransferase f. protem asparate kinase, iransterase specific monofiinctional class I H • I • I I I "IIIIIIIIIIIIIIIIIIIMIIIIIt I I I I I M I 21000 24000 27000 putative GNAT ArgJ arginine peptidyl prolyl preprotein CHP CHP putative Ph^P^'" pilin acetyl biosynthesis cis-trans translocase, SecA ^..nfH^ nitrilase/ gluta biflinctional isomerase D subunit cyanide fedoxm BioC biotin m^j protein signal peptide hydratase biosynthesis mutator protein protein t I I 11 11 t • •. t )M||||||| 30000 33000 36000 39000 preprotein CHP transcript CHP CHP Ybis putative acetyl ferredoxin Chemotaxis sensory transducer translocase, conserved transferase reductase protein SecA subunit regulator iront-d't h) fl9-F10 I I I I I I I I I I I . I I I lilllllllllllllllllll 0 2000 4000 6000 8000 type VI CHP type VI putative ABC major facilitator CHP putative type VI secretion secretion transporter superfamily (MFS) secreted secretion protein protein oermease orotein system Vgr family protein I I ' >" Ml I I II M II I I (III,,! 10000 12000 14000 16000 glyoxalase/ transcriptional type VI secretion CHP CHP system Vgr family ^ bleomycin regulator 4-hydroxythreonine- protein (cont-d) amidohydrolase resistance transcriptional 4-phosphate protein/dio regulator dehydrogenase xygenase) ,, 11II11 III IUI II11II11II11II11 II 11II11 I 1111 M M 11 * ' ' 18000 20000 22000 24000 CHP putative ABC putative putative IndA CHP putative IndC ABC putative transporter oxidoreductase - indigoidine indigoidine synthase transporters transcriptional biosvnthesis NRPS susbtrate- transporter regulator binding ATP binding """""uiiiiiii Mimi 4 26000 28000 putative IndC putative putative N-(5'- transcriptional indigoidine synthase transmembrane phosphoribosyl) regulator NRPS icont-d) transporter mkkomycm anthranilate biosynthesis protein SanR n/O Uß