Antibiotic Production and Resistance in the Microbial Community of Ulva Australis

Antibiotic production and resistance in the microbial community of Ulva australis

Melani Sutrisno

A thesis submitted for the degree of Doctor of Philosophy

School of Biotechnology and Biomolecular Sciences Faculty of Science September 2012

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Sutrisno

First name: Melani Other name/s:-

Abbreviation for degree as given in the University calendar: PhD

School: Biotechnology and Biomolecular Sciences Faculty: Science

Title: Antibiotic production and resistance in the microbial community of Ulva australis

Abstract 350 words maximum: (PLEASE TYPE) Shigella flexnery and Shigella sonnei isolated from several countries have shown resistance to most antibiotics used for shigellosis treatment and therefore new antibiotics against the two bacteria are urgently needed. Although most antibiotics were discovered from soil microorganisms, the marine environment, such as the microbial communities of the macro-alga Ulva australis,is a potential source for finding new antibiotics. Since most of the bacteria living in the alga are not readily cultured or “not-yet” cultured, metagenomics was applied to access the uncultured properties for antibacterial activity against the two Shigella species. To optimally capture antibiotic synthesis genes, two host systems (Escherichia coli and Streptomyces lividans) were used. Moreover, to obtain a link of antibiotic function and phylogenetic origin, Homing Endonuclease Restriction and Marker Insertion (HERMI) was applied. The functional screening of the metagenomic library expressed in the two host cells resulted in the antibacterial activity against Shigella and it is thought that the activity was due to production by a microcin-like compound by the host cell and microcin maturation through genes encoded by the cosmid. HERMI, a v aluable technique for the phylogenetic analysis of metagenomic libraries, has produced a cl one for phylogenetic analysis and identification of gene properties. This HERMI clone was identified as belonging to a new, uncultured bacterium with only 79% 16S rRNA gene identity to Hyphomonas oceanitis. Five antibiotic resistant clones were obtained from the HERMI application and five genes encoding for beta-lactamases were characterized. It was revealed that both clinical and novel beta-lactamases were present in the microbial communities. The gene encoding for putative cell wall-associated hydrolase, which are distantly related to the existing sequences were also identified. Furthermore, genes for virulence (Toll/ interleukin-1 receptor protein and leucine-rich repeat protein-like protein) were identified, indicating the presence of pathogens on the alga. Overall, the microbial communities of U. australis have possibly important properties for pharmaceutical applications and further work will be needed to uncover its full potential.

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 make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property 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 Abstracts International (this is applicable to doctoral theses only).

………MELANI SUTRISNO LIMAR SUTRISNO 19 SEPT 2012 …………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date

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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.’

Signed ……………………………………………......

Date ……21 SEPT 2012……………………………………......

‘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. I retain all proprietary rights, such as patent rights. I also retain to the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorize 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…………………………………………………………………………

Date………21 September 2012………………………………………………

AUTHENTICITY STATEMENT

‘I certify that the Library deposit 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…………21 September 2012……………………………………………………………………

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TABLE OF CONTENTS

Cover……………………………………………………………………………………….…..i Originality Statement……………………………………………………………………...…..ii Copyright statement…………………………………………………………………………..iii Table of contents……………………………………………………………………...... iv-vii List of figures……………………………………………………………………………...viii-x List of tables………………………………………………………………………………xi-xii Abbreviations………………………………………………………………………………..xiii Dedications…………………………………………………………………………………..xiv Abstract………………………………………………………………………………...... xv Acknowledgements…………………………………………………………………….xvi-xvii

Chapter I: Literature Review: Antibiotic Production and Resistance in the Microbial Community of Ulva australis 1.1 Antibiotic resistance………………………….……………………………………...... 1-3

1.2 Marine macro- and microorganisms as a reservoir for bioactive compounds………... 3-4

1.2.1 Marine algae as macro-organisms that are a potential source of bioactive compounds and metabolites………………...……………………………………………………………4-5 1.2.2 Antimicrobial compounds produced by bacteria associated with marine algae……....5-7 1.3 Shigella: the organism, the disease, its antibiotic resistance and the need for new antibacterial compounds….………………...... 7 1.4 Metagenomics as a method to access unculturable bacteria in a sample….…………...8-9 1.5 Host cell for metagenomics…………………………………………………………...9-10 1.6 The aims of the research……………………………………………………………...... 11

Chapter II: Construction of a Metagenomic Library fromMicrobial Communities of Ulva australis and Screening for Antibacterial Clones 2.1. Introduction……………...……………………………….……………………………...13 2.2. Methods 2.2.1. Bacterial strains, media, buffer and vector used in this project…...…………...... 14-15 2.2.2. Extraction of microbial genomic DNA of the microbial community of Ulva australis……………...………………………………………………………………….....15-16

2.2.3. End-repairing and size selection of genomic DNA of the of theUlva australismicrobial community………………….……………………………………….………………………17 2.2.4. Cosmid preparation before ligation……………………………………………….17-18 2.2.5. Ligation and transformation to generate metabolic libraries…...…………….……….18 2.2.6. Screening for antimicrobial activities from cosmid library clones…...……………….18 2.2.7. Cosmid retransformation and the antibacterial assay………………….……...…...... 19 2.2.8. Cosmid characterization……………………………………………………………19-20 2.2.9. Genetic characterization of antibacterial activities via transposon mutagenesis………………………………………………………………………..……...20-22 2.2.10. Analysis of whole cosmid sequences…………...……………….…………………...22 2.3. Results 2.3.1. Metagenomic DNA from Ulva australis……….…………………………...……..23-25 2.3.2 Library construction in Streptomyces lividans TK24 and Escherichia coli EPI300…………………………………………………………………………………….25-26 2.3.3. Screening of the antimicrobial activity of the library of the Ulva australis microbial community………………………….…………………………………………………….26-27 2.3.4. Retransformation of the cosmid with inhibitory activity against Shigella using the paper disc assay …………………….…………………………………………...... 27-28 2.3.5. End-sequencing and restriction digestion of the cosmids of clones with inhibitory activity against Shigella……………………...……………………………………..…….29-30 2.3.6. Sequence analysis of the SE9 cosmid………...……………...…….…………...... 30-31 2.3.7. Identification of the gene responsible for the inhibitory activity against Shigella………………………………………………………………………………...…32-33 2.3.8. Sequence analysis of the entire SE9 cosmid insert DNA…………...…………… 33-35 2.4. Discussion 2.4.1. The quantity and quality of metagenomic DNA of the microbial communities of U. australis.………………………………………………………………………….…………..36 2.4.2. Metagenomic DNA coverage of the library expressed in two host cells………..…….37 2.4.3. The U. australis microbial community as a source for antibiotics…...... 37-39 2.4.4. The presence of a plasmid containing the antibacterial genes in the microbial community of U. australis……………...………………………………………………...39-40 2.4.5. Phylogenetic analysis of SE9 clone …………………………………………………..40 2.4.6. Other genes identified in SE9 clone………………………………………………40-41

Chapter III: Phylogenetic Study of Metagenomic Libraries of the Bacterial Community of Ulva australis, Using Homing Endonuclease Restriction and Marker Insertion 3.1. Introduction……………………………………………………………...…………...43-44 3.2. Methods 3.2.1. Bacterial strains and media and culture conditions…...………………………...... 45 3.2.2. Extraction of fosmid DNAs from the pooled library of an U. australis microbial community, and an illustration of steps in performing HERMI…………………………...…46 3.2.3. Homing Endonuclease Restriction and Marker Insertion (HERMI)……...…………...47 3.2.4. Phylogenetic analysis of the HERMI clones …………………………………..…..47-48

3.2.5. Analysis of the sequence of the flanking region of the kanamycin cassette...... ….…48 3.2.6. Amplification of the kanamycin cassette and analysis of the sequence…………...... 49 3.2.7. Whole fosmid sequencing and analysis of genomic DNA of the HERMI clone……...49 3.3. Results 3.3.1. Construction of transformants via HERMI technique and clone characterization...... 50 3.3.2.1. Clone B1………………………………………………………………………....50-53 3.3.2.2. Clone B2-B11……………………………………………………………………54-58 3.3.3. Whole genome sequencing of fosmid B1…………………………………………58-63 3.4. Discussion 3.4.1. The specificity of the recognition site of I-CeuI………………………………………64 3.4.2. The coverage of HERMI clones in this project…………………...………………..64-65 3.4.3. HERMI as a tool to study the phylogenetic and potential functions of uncultured microorganisms………………………………………………………………………...... 65-67

CHAPTER IV: Natural antibiotic resistance genes in metagenomic library of Ulva australis 4.1. Introduction…………………………………………………………………...... 69-70 4.2. Methods 4.2.1. Bacterial strains and media and culture conditions……………………………………………………….……………………………71 4.2.2. Resistance analysis and the minimum inhibitory concentration (MIC) determination of the clones in media containing antibiotics………………………………………………..71-72 4.2.3. Genetic characterization and whole sequence analysis of the antibiotic resistance gene via transposon mutagenesis……………………………………………………………….72-73

4.3. Results 4.3.1. Analysis of growth and minimum inhibitory concentration (MIC) of the antibiotic resistance clones………………………………………………………………………...... 74 4.3.2. Identification of the gene responsible for the antibiotic resistance activity……….74-81 4.3.2.1. Clone B3…………………………………………………………………………75-76 4.3.2.2. Clone B5………………………………………………………………………... 76-77 4.3.2.3. Clone B6……………………………………………………………………...... 78-79 4.3.2.4. Clone B7……………………………………………………………………………..80 4.3.2.5. Clone B10……………………………………………………………………………81 4.3.3. Comparison of beta-lactamase sequences………………………………………….82-84 4.3.4. Comparison of resolvase sequences………………………………………………..84-86 4.3.5. Genomic context of resistance clones……………………………………………...86-92 4.4. Discussion 4.4.1. The presence of beta-lactamase genes in the microbial community of U. australis …………………………………………………………………………………………….93-94 4.4.2. Predicted proteins identified from the uncultured Alphaproteobacteria of the microbial community of U. australis………………………………………………………………..94-97

CHAPTER V: General discussion, concluding remarks and future direction 5.1. The presence of antibiotic biosynthesis and resistance elements in bacterial communities of U. australis…………………………………………………………………………99-101 5.2. The presence of virulence genes in the antibiotic resistant clones……………………..101 5.3. A management system to avoid the spread of antibiotic resistance genes………...101-102 5.4. Concluding remarks and future direction………………………………………………102

REFERENCES ……………………………………………………………………….103-133 APPENDIX……………………………………………………………………………134-164

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LIST OF FIGURES

Figure 1-1: The diagram illustrating the steps in metagenomics technique.The technique begins with the extraction of DNA genomic from a sample, ligation into a vector cloning, transformation into a host cell to produce metagenomic library for screening (functional- driven analysis or sequence-driven analysis). The picture was taken from Handelsman, 2004.

Figure 2-1.Vector map of the pFD666 cosmid.Picture was taken from Kieser et al, 2000.

Figure 2-2.The schematic protocol of EZ-Tn5Transposon insertion. Picture was taken from the instruction manual of Epicentre, Madison, WI, USA.

Figure 2-4. PCR amplification of 16S and 18S rRNA. A. Lane 1: Lambda DNA EcoRI+ HindIII (Fermentas Inc., Glen Burnie, MD, USA) (0.2 µg); Lane 2: 100 bp pl us DNA marker; Lane 3-7: amplicon resulted from ~9000 pg, 900 pg, 90 pg, 9 pg, 0.9 pg of genomic DNA isolated from U. australis, respectively; Lane 8-10: positive controls (genomic DNAof E. coli); Lane 11: negative control (sterile ultra-pure water). B. Lane 1: 100 bp plus DNA marker (Fermentas Inc., Glen Burnie, MD, USA); Lane 2-7: products resulted from ~9000 pg, 900 pg, 90 pg, 9 pg, 0.9 pg, 0.09 pg of the isolated genomic DNA, respectively; Lane 10: positive control (Saccharomyces cerevisiae genomic DNA); Lane 11: negative control (sterile water).

Figure 2-5. Size selection of metagenomic DNA of microbial communityof U. australis. Lane 1: 10 µl Mid- range PFG marker I (New England Biolabs, Beverley, MA, USA); Lane 2: Marker (Lambda EcoRI+Hind III, Fermentas Inc., Glen Burnie, MD, USA): 1 µg; Lane 3 and 5: 10 µl (~350 ng) of the genomic DNA.

Figure 2-6.The functional assay of the clone against S. sonnei. Black box: zone of inhibition against the target bacteria was observed. ZOI-: there was no inhibition zone observed for the kanamycin 12.5 µg/ml used for the cosmid induction.

Figure 2-7. Digestion of cosmids with NcoI-StuI. Lane1: the gene ruler 1 Kb DNA marker (Fermentas Inc., Glen Burnie, MD, USA). Lane 2: pFD666 with no insert (before the digestion); Lane 3: pFD666 with no i nsert (after the digestion); Lane 4: cosmid of SE11 (before the digestion); Lane 5: cosmid of SE11 (after the digestion). Blue box: upper bands >10 Kb; lower band: ~8KB; Red box: the 3.9 Kb cosmid fragment. Green box (with arrows: a band of 2 Kb and 2.5 Kb (the bands were not sharp).

Figure 2-8. Maximum likelihood trees of the 16S rRNA gene of SE9 and the closest representatives of culturable and uncultured from the 16S rRNA gene database (NCBI).Red box: 16S rRNA gene sequence of SE9. The scale bar indicates 0.02 divergencesof the sequences. The triangle is the collapsed branch for more than 40 species of Pseudomonas.

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Figure 2-9. Phylogenetic tree of the aligned protein sequences related to PmbA of SE9; and the selected sequences from the non-redundant protein from NCBI. The scale bar corresponds to a genetic distance of 0.4 substitutions per position.

Figure 3-1.The map of pCC1FOS (Epicentre, Madison, WI, USA). The fosmid contains Ori2 and OriV for single copy and multicopy replication in E.coli host cell, respectively. The presence of Cos enables the construct of DNA insert and the fosmid to be packaged in lambda bacteriophage system. Antibiotic selection marker (Choramphenicol resistance gene) is present in the fosmid for clone selection. The picture was taken from the manufacturers manual (Epicentre, Madison, WI, USA).

Figure 3-2. The diagram illustrating the steps in HERMI technique.The technique begins with the digestion of the pooled fosmids (pCC1FOS + genomics insert) with I-CeuI. Then, kanamycin cassette was inserted into the I-CeuI digested fosmid continued by transformants selection on plates containing both kanamycin (the inserted kanamycin cassette) and chloramphenicol (the antibiotic resistance gene contained in pCC1FOS). The picture was taken from Yung et al, 2009.

Figure 3-3: The PCR amplification of 23S rRNA gene using universal primers. Lane 1 and 11: 1 K b DNA ladder (Fermentas Inc., Glen Burnie, MD, USA); lane 2: B1 clone, it produced around 3.1 Kb of amplicon; lane 3 to 10: B2 to B9 clone; lane 12 and 13: B10 and B11, respectively. B2-B11 produced ~2.1 Kb PCR product.

Figure 3-4. Maximum likelihood tree of the 23S rRNA gene of B1and the closest representatives of micro-organisms from the nucleotide database (NCBI). Red box: 23S rRNA sequence of B1. The scale bar indicates 0.07 divergences of the sequences.

Figure 3-5. Maximum likelihood trees of the 16S rRNA gene of B1and the closest representatives of micro-organisms from the 16S rRNA database (NCBI). Red box: 16S rRNA sequence of B1. The scale bar indicates 0.04 divergences of the sequences.

Figure 3-6.The PCR amplification of kanamycin cassette.Lane 1: 1 Kb DNA ladder (Fermentas Inc., Glen Burnie, MD, USA). Lane 3-12: Kanamycin amplicon of fosmid B2- B11. All fosmid amplified KanFCeu and KanRCeu to result in a 1 Kb amplicon.

Figure 3.7.Schematic representation of the 5S-23S rRNA operon in fosmid B1. I-CeuI rec site: I-CeuI recognition site. Kan: the kanamycin resistance cassette (pBHS-Kan). Sequences in the box: A: Last 100 bp of the 16S rRNA sequence (position in fosmid: 13, 946-14,046); B: Last 100 bp of the 23S rRNA sequence (position in fosmid: 18,828-18,928); C: First 100 bp of the 5S rRNA sequence (position in fosmid: 19,036-19,136).

Figure 3-8. Multiple protein sequence alignments of the histidine kinase identified from B1with its closest representatives according to the NCBI database.M. maris: Maricaulis maris, O. alexandrii:Oceranicaulus alexandrii, P. bermudensis: Parvularcula bermudensis and H. neptunium; Hyphomonas neptunium.S7.5: the histidine kinase of B1. Accession numbers of the protein sequences are shown next to the organism to which the protein sequence belongs. Red letter was the conserved amino acid for each domain.Code for the column: (*): column of the alignment contains identical amino acid residues in all sequences. (: ): column of the alignment contains conserved substitution of amino acids. (.): column of the alignment contains semi conserved amino acids.

Figure 4.1.Schematic diagrams of the ORFs of B3 mutant (namely B3C12). The direction of the primers is shown by arrows. ORF2: unknown function (Unk), 50 a mino acids. ORF1: beta-lactamase (Bel1), 266 amino acids. T: transposon insertion site.

Figure 4-2.Schematic diagrams of the ORFs of B5 mutant. The direction of the primers is shown by arrows: ORF3: beta-lactamase (Bel2), 228 amino acids. ORF4: resolvase (Rvs1), 185 amino acids. T: transpososn insertion site.

Figure 4-3.Schematic diagrams of the ORFs of B6 mutant. The direction of the primers is shown by arrows. ORF5a: beta-lactamase (Bel3), 120 amino acids. ORF5b: beta-lactamase (Bel3), 160 amino acids. ORF6: resolvase (Rvs2), 133 amino acids. ORF7: predicted protein (Ppr1), 59 amino acids. T: transposon insertion site.

Figure 4-4.Schematic diagrams of the ORFs of B7 mutant. The direction of the primers is shown by arrows. ORF8a: beta-lactamase (Bel4), 164 amino acids. ORF8b: beta-lactamase (Bel4), 212 amino acids. ORF9: unknown protein (Unk2), 54 amino acids. ORF10: unknown protein (Unk3), 57 amino acids T: transposon insertion site.

Figure 4-5. Schematic diagrams of the two ORFs of B10 mutant. The direction of primers is shown by arrows. ORF11a: beta-lactamase (Bel5), 160 amino acids. ORF11b: beta-lactamase (Bel5), 139 amino acids. T: transposon insertion site.

Figure 4-6. Protein sequence alignment of the Bel1with sequences of Bel2, Bel3, Bel4 and Bel5. Symbols:”*”= column of the alignment contains identical amino acids residue in all sequences. “:”= column of alignment contains conserved substitutions of amino acids. “.”= column of alignment contains semi-conserved substitutions. “-“= gaps in the amino acid sequence.Code (1) indicates the active site (S*XXK motif), red letters. Code (2) indicates the S(Y) XN motif, red letters. Code (3) indicates the K (H) T(S) G motif, red letters. Blue letters in (2): deviation in the S (Y) XN motif.

Figure 4-7. Phylogenetic tree of the aligned protein sequences that were related to Bel1, Bel2, Bel3, Bel4 and Bel5; and the selected sequences from the Swiss protein from NCBI ftp site (2012-07-06) datasets. Klebsiella_oxytoca_75428812: TEM beta-lactamase Klebsiella oxytoca (BLAT_KLEOX). Escherichia_coli_50401825: TEM beta-lactamase E. coli (BLAT_ECOLX). The scale bar corresponds to a genetic distance of 0.3 substitutions per position.

Figure 4-8. Protein sequence alignment of the Rvs1, Rvs2 with Tn3 resolvases sequences of E. coli (TNR3_ECOLX) and Klebsiella pneumoniae (Accession no. T NR4_KLEPN). K.: Klebsiella pneumoniae. E.: Escherichia coli. Symbols:”*”= column of the alignment contains identical amino acids residue in all sequences. “:”= column of alignment contains conserved substitutions of amino acids. “.”= column of alignment contains semi-conserved substitutions. “-“= gaps in the amino acid sequence. The red letters indicates the amino acids contained in the catalytic domain of the Tn3 resolvases.

Figure 4-9. Phylogenetic tree of the aligned protein sequences that were related to Rvs1 and Rvs2; and the selected sequences from the Swiss protein from NCBI ftp site (2012-07-06) datasets. Klebsiella _pneumonia_135948: Tn3 resolvase Klebsiella pneumoniae (TNR4_KLEPN). Escherichia_coli_83287800: Tn3 resolvase E. coli (TNR3_ECOLX) .The scale bar corresponds to a genetic distance of 0.4 substitutions per position.

LIST OF TABLES

Table 2-1.The DNA coverage of the genomic DNA libraries of microbial community of U. australis.

Table 2-2.The antibacterial activityof clones before and after re-transformation into the same or different host cells.Codes for the bacterial target: Ss: S. sonnei; Sf: S. flexnery. Codes for the clone’s name: SL, SLe and Sln: clone library in S. lividans; Ect: clone library in E. coli; SS, SSe and SSn: cosmid isolated from Streptomyces lividans and then transformed back into S. lividans; SE, SEe and SEn: re-transformation of the cosmid isolated from S. lividans into E. coli; ES: cosmid from E. coli was transformed into S. lividans; EE: re-transformation of cosmid isolated from E. coli back into E. coli. (+): the clone inhibits the bacterial target. (-): there was no inhibition activity. Red box: clone lost the inhibition activity during re- transformation.

Table 2-3. Annotation of the open reading frames of clone SE9.Where possible, naming of the genes was performed according to the Swiss Protein database. If no information was available under the Swiss Protein database, then naming of the genes was done according to the NCBI database. (-): no s ignificant hit.The dark shaded box indicates the e-value was higher than 1 x 10 -20 under Swiss Protein database (with an e-value of 1 x 10-5 as the cut-off). The light shaded box indicates the e-value is less than 1 x 10 -20 under Swiss Protein database. White box indicates the protein is annotated as hypothetical or uncharacterized protein or no hit. For detail annotation, please refer to appendix 1.

Table 3-1: Primers used in the HERMI analysis (Yung et al, 2009).

Table 3-2.Nucleotide similarity search of the 2.8 Kb length of end sequences of B1 clone against the nucleotide collection of the NCBI. The accession number, percentage of coverage (%C), percentage of identity (%I), E- value, the name of the organism correspond to the gene as well as the Class of bacteria are presented.

Table 3-3.Nucleotide similarity search of the 16S rRNA of B1 clone against nucleotide collections of the NCBI. The accession number, percentage of coverage (%C), percentage of identity (%I), E- value, the name of the organism correspond to the gene as well as the Class of bacteria are presented.

Table 3-4. Nucleotide similarity search of the sequence flanking the kanamycin cassette of fosmid B2-B11 against nucleotide collection of the NCBI (A) and the KEGG database (B).The clone’s name, primers, gene, organism, accession number, the percentage coverage (%C) as well as the percentage of similarity (%S) are shown. FP: KanFSeq; RP: KanRSeq; (-): no sequence was obtained.

Table 3-5. Annotation of the open reading frames of clone B1.Where possible, naming of the genes was performed according to the Swiss Protein database. If no information was available under the Swiss Protein database, then naming of the genes was done according to the NCBI database. (-): no significant hit. The dark shaded box indicates the e-value was higher than 1 x 10 -20 under Swiss Protein database (with an e-value of 1 x 10-5 as the cut- off).The light shaded box indicates the e-value is less than 1 x 10 -20 under Swiss Protein database. For detail annotation, please refer to appendix 2.

Table 4-1.The growth analysis of the antibiotic-resistant clone in several concentrations of penicillin G, trimethoprim and tetracycline.B3-B10: antibiotic-resistant clones; C: the electrocompetent Escherichia coli EPI300 as the control for resistance; B2: Escherichia coli EPI300 containing pCC1FOS. (+): growth was observed; (-): no growth was observed. MIC for each antibiotic to each clone was determined from the lowest concentration of each antibiotic that showed no growth of the clone (black box).

Table 4-2: Annotation of the open reading frames of the fosmids of the antibiotic resistant clones. Where possible, the genes were named according to the Swiss protein database. If there was no information available under the Swiss protein database, the genes was named based on t he -non-redundant protein database of the National Centre for Biotechnology Information. The light shaded box indicates that the gene has an e-value lower than 1x10- 20(cutting out point 1x 10-5) under the Swiss protein database. The dark shaded box indicates the protein’s annotations are similar for two or more databases, but did not have matches to the Swiss protein or did have an e-value lower than 1x10-20 in the Swiss protein database. White box indicates the protein is annotated as hypothetical or uncharacterized protein. Please see appendix 3-7 for detail annotation.

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ABBREVIATIONS

°C: degree Celsius bp: base pair BSA: Bovine Serum Albumin DNA: Deoxyribose nucleic acid dNTPs: Deoxyribonucleotide triphosphates EDTA: Ethylenediamine-tetraacetic acid g: g- force LB: Luria Bertani MIC: minimum inhibitory concentration min: minute ml: milli liters µl: micro liters µM: micro molar ng: nano gram µg: micro gram OD: optical density PCR: Polymerase Chain Reaction PFGE: Pulsed Field Gel Electrophoresis pg: pico gram rRNA: ribosomal RNA RNA: ribonucleic acid sec: second SDS: sodium dodecyl sulfate Tn: transposon V: volts

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DEDICATION

I dedicate this work for my lovely husband: Limar Sutrisno, my sweet daughter: Rosana Valentina Sutrisno, and my beloved parents: Hadi Witarto and Menik. Thank you very much for always supporting and praying for me.

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ABSTRACT

Shigella flexnery and Shigella sonnei isolated from several countries have shown resistance to most antibiotics used for shigellosis treatment and therefore new antibiotics against the two bacteria are urgently needed. Although most antibiotics were discovered from soil microorganisms, the marine environment, such as the microbial communities of the macro- alga Ulva australis,is a potential source for finding new antibiotics.Since most of the bacteria living in the alga are not readily cultured or “not-yet” cultured, metagenomics was applied to access the uncultured properties for antibacterial activity against the two Shigella species. To optimally capture antibiotic synthesis genes, two host systems (Escherichia coli and Streptomyces lividans) were used. Moreover, to obtain a link of antibiotic functionand phylogenetic origin, Homing Endonuclease Restriction and Marker Insertion (HERMI) was applied. The functional screening of the metagenomic library expressed in the two host cells resulted in the antibacterial activity against Shigella and it is thought that the activity was due to production by a microcin-like compound by the host cell and microcin maturation through genes encoded by the cosmid. HERMI, a valuable technique for the phylogenetic analysis of metagenomic libraries, has produced a clone for phylogenetic analysis and identification of gene properties. This HERMI clone was identified as belonging to a new, uncultured bacterium with only 79% 16S rRNA geneidentity to Hyphomonas oceanitis. Five antibiotic resistant clones were obtained from the HERMI application and five genes encoding for beta- lactamases were characterized. It was revealed that both clinical and novel beta-lactamases were present in the microbial communities. The gene encoding for putative cell wall- associated hydrolase, which are distantly related to the existing sequences were also identified. Furthermore, genes for virulence (Toll/ interleukin-1 receptor protein and leucine- rich repeat protein-like protein) were identified, indicating the presence of pathogens on the alga. Overall, the microbial communities of U. australis have possibly important properties for pharmaceutical applications and further work will be needed to uncover its full potential.

ACKNOWLEDGEMENTS

Thanks to our Lord, Jesus Christ for His blessing in producing this thesis.

I would like to thank greatly: 1. Associate Professor Torsten Thomas for the support, attention and for being the best supervisor one can possibly have. Thank you so much for your excellent, brilliant suggestions and all the lessons you have taught me. Many thanks for the assembly of the shotgun sequencing data. 2. Professor Staffan Kjelleberg for the support, attention, the knowledge and being a very good Co-supervisor. 3. Professor Rick Cavicchioli and Dr. Brendan Burns for the discussions and suggestions during the annual reviews. 4. Professor D.E.A. Catcheside, Dr. Fred Bowring, Dr. Jane Yeadon and Dr. Lin Koh for the support, attention and the knowledge. 5. Dr. Cathy Burke and Dr. Maria Pui Yi Yung for the sample, discussions and suggestions. 6. Dr. Tim Carlton for the discussions and suggestions. 7. Ms. Julie Van Laarhoven (Post Award Events Coordinator, AUSAID), Ms.Elaine Kane, Ms. Jane Horgan, Mrs. Maxine Bald, Mr. George Markopoulos, Mr. Klaus Koefer, Ms. Tatjana Kroll, Mr. Matthew Byron and all AUSAID staffs for the support, suggestion and help. 8. Prof. Laura Poole-Warren (Dean of Graduate Research, the University of New South Wales/ UNSW), Mr. Gerry Reyes, Ms. Elizabeth Martens and all GRS staffs for the support and help. 9. Prof. J. William Ballard (Head of School of Biotechnology and Biomolecular Sciences/ BABS), Assosiate Prof. Ruiting Lan (BABS Postgraduate Coordinator),Ms. Kylie Jones, Ms. Penny Hamiltonand all School staffs for the support and help. 10. Ms. Ai Hong and Ms. Maria Laurentia for thesis editing. 11. Raymond Regalia for solving the Unimail problemand for helping me with the printing and binding of my thesis. Thanks for all of your kindly help and friendship. 12. Joao Pereira for the chance to work with you for your Honours degree. 13. Dr. Sofy, Melissa, Suhaila, Becca, Chris, Michael, Gee, Neil, Shaun, Sharon, Alex, Amy, Cao, Janice, Merry and everyone else (in 304, 313, 315, 616 ); thanks for the lovely friendship and help. xvi

14. Adam, Kirsty, Kim and all staffs of the teaching laboratory who have been so helpful in many different ways.

Many thanks to the Australian Government for providing me an Australian Leadership Awards (ALA) for my PhD study at the University of New South Wales, Australia.

xvii

CHAPTER 1: LITERATURE REVIEW

ANTIBIOTIC PRODUCTION AND RESISTANCE IN THE MICROBIAL COMMUNITY OF ULVA AUSTRALIS

CHAPTER 1: LITERATURE REVIEW: ANTIBIOTIC PRODUCTION AND RESISTANCE IN THE MICROBIAL COMMUNITY OF ULVA AUSTRALIS

The first available antibiotic was penicillin, which was discovered by Alexander Fleming about 80 years ago. Since then, various types of antibiotics have been found and marketed as pharmaceutical compounds for treating bacterial and fungal pathogens in humans or animals. Recently, the number of available, effective antibiotics for most pathogens has decreased dramatically due to high number of cases with antibiotic resistance (Powledge, 2004). Therefore, new compounds to fight antibiotic resistant pathogens are needed. In this chapter, an introduction to antibiotic resistance, possible natural sources for new antibiotics and the metagenomics technique will be provided. An introduction to Shigella, the target bacteria used in the antibacterial screening, will also be given.

1.1. Antibiotic resistance Antibiotic resistance is a type of drug resistance where a microorganism loses its sensitivity to the exposure of an antibiotic. A microorganism can confer resistance to an antibiotic by spontaneous mutation, induced mutation (Datta and Hughes, 1983; Martinez and Baquero, 2000) or by accepting exogenous, resistance genes via horizontal gene transfer (Davies, 1994; Andersen, 1993; Bennett, 2008; Chandrasekaran, 1985). It has been predicted that the resistance genes originated in natural antibiotic-producing bacteria (D’Costa et al, 2011; Webb and Davies, 1993; Martinez, 2008). However, this is not the case for other antibiotic resistance genes, such as beta-lactamases (Bush et al, 1995) and aminoglycoside (Lambert et al, 1999; Ainsa et al, 1997) that were identified in non-antibiotic producing bacteria. It was assumed that selective pressure given by the antibiotic therapy has resulted in the acquisition of those resistance genes (Alonso et al, 2001). Thus, antibiotic resistance genes can be of environmental or clinical origin (Davies and Davies, 2010).

Resistance genes in environmental bacteria can be transferred (via transposon or integron) to other diverse bacteria, including clinical pathogens or from clinical samples to the environment (Levy and Marshall, 2004; Sunde and Norstrom, 2006; Wright et al, 2010; Linch et al, 2011). Once resistant, a microorganism can rapidly produce progeny (Livermore, 2003). The natural environment, such as soils, rivers, and lakes, are rich sources of antibiotic resistance genes (Reid et al, 2010; Donato et al, 2010). Bacteria living in those environmental samples have been found to harbour various antibiotic resistance genes that are similar to or 1

quite distant from the ones in clinical settings (Mandomando, 2009; Perreten, 2005; Brinas et al, 2003; Tan et al, 2001; Wood, 1996; Chau et al, 1981).

Bacteria may manifest resistance to antibiotics via several mechanisms. The first resistance is caused by acquiring genes (for example, those encoding for beta-lactamases) that are able to destroy the antimicrobial agent before it can have an effect (Jacoby and Mederios, 1991; Girlich et al, 2010; Ainsa et al, 1997; Lambert et al, 1999). An example of this is the activity of erythromycin ribosomal methylase in Staphylococci (Barcs and Janoci, 1992).The second mechanism is by acquiring efflux pumps (e.g., fluoroquinolones efflux pump in Staphylococcus aureus) (Tenover, 2006; Schweiser, 2003; Poole and Srikumar, 2001). Efflux pumps remove the antibiotic from the bacterial cells before it can reach its target site within the bacterial cell (Murray et al, 1980; Perichon and Courvalin, 2004). The third type of resistance is by obtaining several genes of a m etabolic pathway, which results in altered bacterial cell wall that no longer possess the binding site of the antibiotic, for example the amino acid alterations in the penicillin-binding protein 2b in Pneumococci (Nagai et al, 2002; Wright, 2007; Bryan, 1988; Bugg et al, 1991; Keller and Perreten, 2006; Korczak et al, 2009; Felnagle et al, 2007). The fourth mechanism is by mutations that blocks access of the antibiotic to the intracellular target site through down regulation of porin genes in the bacterial cell membrane (Oliver et al, 2002, T enover, 2006). The fifth mechanism is by mutations that lead to amino acid changes in the bacterial ribosomal protein or alter its 16S rRNA primary structure. This mechanism is identified in streptomycin resistance in Mycobacterium tuberculosis (Finken et al, 1993). Gene transfer may occur between strains of the same species of bacteria, between different bacterial species in the same genera, between different genera or between different Gram of bacteria or trans Gram (Courvalin, 1994). The gene transfer may be facilitated by several mechanisms, such as transformation, transduction and conjugation. During transformation, bacteria acquire and then incorporate an extracellular DNA segment that was previously released to the environment by other bacteria (via cell lysis) (Tenover, 2006; Hannan et al, 2010). In transduction, a DNA segment or plasmid conferring resistance to antibiotic is transferred from one bacterium to another via a phage (Willi et al, 1997; Varga et al, 2012), while in conjugation, the genetic exchange is facilitated by elongation of a pilus (in conjugation among Gram-negative bacteria) or by sex pheromones production (in conjugation among Gram-positive bacteria) (Stuart et al, 1992; Tenover, 2006; Shakibaie et al, 2009).

The spread of antibiotic-resistant organisms can be driven by physical forces (wind, water) and biological forces (humans, insects, birds and other animals) (Allen et al, 2010). Thus, the resistance can be transferred easily from an environmental sample to other environments (e.g. including clinical settings) or vice versa. The easy movement of resistance antibiotic genes causes increasing public health problems as pathogens coulddevelopresistance to almost all available antibiotics. Therefore, the exploration and development of new sources of antibiotics needs to be speeded up to provide effective antibiotics at all time.

1.2. Marine macro- and micro-organisms as a reservoir for bioactive compounds Although most antibiotics are discovered from soil microorganisms (Soulides, 1965; Hansen et al, 2001; Chung et al, 2008), particularly from Actinomycetes (Nkanga and Hagedorn, 1978; Barke et al, 2010; Stevenson et al, 1956; Cundliffe, 2006; Doulll and Vining, 1990; Baltz, 2007; Matsui et al, 2012), other sources such as marine environments are a potential source for new antibiotics (Egan et al, 2008; Fiedler et al, 2005; Admi et al, 1996; Alamsjah et al, 2007; Becher et al, 2005; Berry et al, 2008; Cantillo et al, 2010; Dittmann et al, 2001; Flores and Walk, 1986; Kodani et al, 2002).

About 75% of the earth’s surface is covered by water, where ~80% of all lifeforms reside. As a consequence, the diversity of aquatic organisms is much greater than that of their terrestrial counterparts (Soliev et al, 2011; Davidson, 1995). Simple organisms such as phages, bacteria, sponges, alga, and varieties of invertebrates and other eukaryotes are the inhabitants of marine environments. Marine macro-organisms, such as algae (Alang et al, 2009; Kolanjinathan and Stella, 2011), sponges (Oliveira et al, 2006; Newbold, 1999; Muricy et al, 1993; Kim et al, 2006) and other invertebrates (Tincu and Taylor, 2004; Jian-yin et al, 2011), have been investigated to produce novel compounds, such as new drugs, enzymes, fertilizers, anti-nematodes, herbicides, anti- mosquito larvae, anti-convulsant, anti-hypertension, polysynaptic blocker, anti -oxotremorin, anti-virus, and etc. ( Vijayabaskar and Shiyamala, 2011; Agrawalet al, 2005; Blunden et al, 1981; Cocamese et al, 1981; Manilal et al, 2009; Baker, 1984, Garg et al, 1992). Remarkably, marine bioactive compounds exhibit structural features that are not found in compounds from a terrestrial origin (Carte, 1996).

1.2.1. Marine algae as macro-organisms that are a potential source of bioactive compounds and metabolites Marine alga has been extensively used for human food or animal feed (Murakami et al, 1984; Watanabe and Nisizawa, 1984). They are also good sources for the discovery of bioactive compounds or metabolites (Cocamese et al, 1981; Blunden et al, 1981).Bioactive compounds produced by marine alga include brominated phenols, terpenoids, sterol, polysaccharides, peptides, proteins and etc. Brominated phenols such as 2,3-dibromo-4,5- dihydroxybenzaldehyde, 4,5-disulphate dipotassium salt; 5-bromo-3,4- dihydroxybenzaldehyde; 3,5 -dibromo-p-hydroxybenzyl alcohol and 3-bromo-4-[2,3- dibromo-4,5-dihydroxyphenyl] methyl-5- (ethoxymethyl) 1,2-benzenediol and many other brominated phenols, h ave the capacity to inhibit the growth of several bacteria (Xu et al, 2003; Faulkner, 2002; Withus et al, 1981; Fattorusso, 1983). Besides the various brominated phenols, terpenoids from marine alga also have antibacterial activity (Fenical and Paul, 1984). Several uncharacterized compounds of marine alga have been reported to have antibacterial activity against several Gram-positive and –negative bacteria (Kandhasamy and Arunachalam, 2008). Gambieric acid A and B (polyether compounds), tolitoxin, goniodomin A, hapalindole, nostodione, nostocyclamide, tjipanazoles, and lobophorolide have been extracted from marine algae and proven to have potential antifungal activity (Abe et al, 2002; Bohm et al, 1995; Bonjouklian, 1991; Burja et al, 2001; Nagai et al, 1993; Patterson and Carmelli, 1992; Yang et al, 1993).

The macroalga U. australis has been investigated for its antimicrobial activity by Alang et al, 2009. They discovered that the extract of the alga in n-hexane, chloroform and water yielded carbohydrates, steroids and glycosides have an antimicrobial activity against several Gram- positive and -negative bacteria and also against Aspergillus niger and Candida albicans.

Another research conducted by Kim et al, 2007 found that the ethyl-ether extract of U. australis inhibits the growth of some Gram-positive and -negative bacteria, including the methicillin resistant Staphylococcus aureus (MRSA). Kolanjinathan and Stella (2011) has investigated the antibacterial activity of U. australis extracted in several organic solvents (chloroform, methanol, acetone, hexane, and ethyl acetate) against several Gram- positive and -negative bacteria in the family of Enterobacteriaceae. It can be concluded that the alga metabolites have the potential in treating infectious diseases in human and animals.

Besides having antimicrobial activity, several compounds extracted from marine algae have other pharmaceutical potentials. Several compounds, for example typoldione, fukoidan and curacin-A, are able to inhibit microtubulin polymerization during cell division (Brien et al, 1984), and thus they can block DNA replication and cell division (Yamamoto et al, 1984; Gerwick et al, 1994) and are used for anti-tumour compounds. Other compounds extracted from marine algae can be used as a cholinesterase inhibitor, hypotensive, inotropic, anti- inflammatory, sedative, neuronal death inhibitor and cholinergic compounds (Jiao et al, 2011; Sangeetha et al, 2009; Xu et al, 2003; Ma et al, 2006; Stirk et al, 2007; Serisawa et al, 2001; Fan et al, 2003; Meldahl et al, 1996; Kamei and Sagara, 2002; Vila and Przedborski, 2003; Zhao et al, 2004; Jung et al, 2009; Baker, 1984).

Marine algae are also a rich source of carrageenan, agar, alginate, antioxidative compounds and antifouling agents. Carrageenans, agar and alginates obtained from marine algae can be used for applications in the food industry and in cosmetics (Saito and Oliveira, 1990; Carte, 1996; Black et al, 1965). The antioxidatives produced by marine algae include carotenoids, fucoxanthin, phlorotannins, amino acids, glucitols, terpenoid, phospholipid, and 5-bromo-3, 4-dihydroxybenzaldehyde (Kelman et al, 2012; Liang et al, 2007; Kanedo and Ando, 1971; Fujimoto and Kaneda, 1984). Since antioxidants are used in food, cosmetics and medicines; marine algae provide important substances for applications in these areas. Several compounds from marine algae have also been found as antifouling agents, such as palisol, palisadin A, halogenated monoterpens, and phlorotannins (Bhadury and Wright, 2004; Jennings and Steinberg, 1997; Konig et al, 1999a; Steinberg et al, 1998).

Thus, marine algae offer various bioactive compounds or metabolites that can be applied in several different areas such as the food industry, cosmetics, antifouling and medical/ pharmaceuticals.

1.2.2. Antimicrobial compounds produced by bacteria associated with marine algae Marine algae are a good source for many bioactive compounds, and it has been found that the active compounds originated from the microorganisms living in association with the alga (Proksch et al, 2002; Zhang et al, 2005). Various microorganisms naturally live adherent on the surface of marine algae (Perez-Matos et al, 2007). Types of microorganisms or bacteria living in a certain marine alga are specific and differ from the types of bacteria in the sea water (Burke et al, 2011a; Jasti et al, 2005; Johnson et al, 1991). It has been observed that the 5 presence of these adherent bacteria on marine algae supports the growth of the algae (Cole, 1982). Marine algae experience major growth in the presence of these adherent bacteria rather than in the absence of them, possibly because the algae can obtain vitamins and other elements synthesized by the bacteria (Jasti et al, 2005). Additionally, the presence of certain bacteria may have a function in protecting the alga against pathogenic bacteria, as observed in Laminaria- associated bacteria (Dimitrieva et al, 2006). Certain bacteria can lyse the alga cell wall, so thus the presence of other bacteria living in association with the alga might produce antibiotic activities to the bacteria that lyse algae (Burnham et al, 1976; Cole, 1982). On the contrary, bacteria may benefit from the metabolites produced by the marine algae.

The high diversity of the microbial community in a marine alga may lead to antagonism or competition for survival (Rao et al, 2006; Flint et al, 2007; Hibbing et al, 2010). The antagonism can be regulated by quorum sensing, the system that enables bacteria to detect their density or the density of other species within the same environment. Quorum sensing may induce a grouping behaviour in bacteria such as to form a biofilm (De Kievit et al, 2001). As biofilms are produced, competition for resources (nutrients, other elements to support growth, habitat) occurs among population of bacteria living within or among the biofilm (Hojo et al, 2009). Besides biofilm induction, traits controlled by quorum sensing include surface attachment, bio-surfactant synthesis, sporulation, competence, bioluminescence, and secretion of antibiotics and virulence factors (Duerkop et al, 2009; Dunne, 2002; Peter, 2006; Grossman, 1995; Li et al, 2001; Engebrecht and Silverman, 1984; Miller and Bassler, 2001; Miller et al, 2002; Williams et al, 2000; Hentzer et al, 2003). Thus, quorum sensing in several bacteria may activate the excretion of antibiotics with the purpose to kill or inhibit the growth of other micro-organisms in the same habitat (Wiese et al, 2009; Lemos et al, 1985 and Mahasnehet al, 1995; Boyd et al, 1999; Gerard et al, 1997; Yoshikawa et al, 1997; Tran et al, 2007).

U. australis is the habitat of several different classes of bacteria (Tujula et al, 2007; Burke et al, 2011a), in which several members are known to produce antimicrobial compounds. A high diversity of microorganisms in the alga may result in antagonism (Rao et al, 2007; Kumar et al, 2011) or synergism (Burmolle et al, 2006) to increase their survival rate as epiphytes on the alga. T he antagonism activity may result in virulence development or compound excretion, such as antibiotics or toxins. Since the diversity of the microorganisms in the alga

6 is high, one can expect to harvest various types of antimicrobial compounds or antibiotics (Yung et al, 2011; Rao et al, 2007; Kumar et al, 2011).

1.3. Shigella: the organism, the disease, its antibiotic resistance and the need for new antibacterial compounds Shigella is a G ram negative bacterium, non-motile, non-spore forming, with a rod shaped cell. This genus belongs to the family Enterobacteriaceae, which also has other genera with pathogenic members such as Escherichia, Klebsiella, Enterobacter and Salmonella. There are four species of Shigella discovered so far: S. dysenteriae, S. boydii, S. sonnei and S. flexnery (Hale and Keusch, 1996).

These four species of Shigella cause the disease shigellosis, also known as bacillary dysentery in humans. The disease is transmitted through a fecal-oral route and can occur after less than 100 bacterial cells are ingested (Dupont, 1998). Both S. boydii and S. sonnei are usually associated with a short duration of mild diarrhea, while S. flexnery causes a more severe and longer-lasting diarrheal illness. S. dysenteriae causes the most severe diarrheal illness compared to the other three (Keusch and Bennish, 1989). Endemic shigellosis caused by S. boydii (Ranjbar et al, 2008), S. sonnei (Lidwin et al, 1997) and S. flexnery (Sachdev et al, 1993; Casalino et al, 1994) has been observed in developing countries.In addition, S. disenteriae may result in endemic or epidemic diseases (Keusch and Bennish, 1989; Navia et al, 1999; Rahaman et al, 1975; Sayem et al, 2011).

Shigella has been observed to have developed resistance to most antibiotics used for the treatment of the disease, such as methicillin (Hossain et al, 1989), fluoroquinolone, ampicillin, tetracycline, streptomycin, chloramphenicol (Pazhani et al, 2008; Meyer and Lerman, 1980), nalidixic acid (Seol et al, 2006), trimethoprim–sulfamethoxazole (Ashkenazi et al, 2003), ceftazidime and kanamycin (Ashtiani et al, 2009; Daniel de Paula et al, 2010). The resistance is mediated by R plasmids, transposons and integrons (Bourtchai et al, 2008; Seol et al, 2006; Frost et al, 1985; Turner et al, 2004). Since high cases of antibiotic resistance have been discovered in Shigella, and not to mention the easy transfer of the resistance elements to other bacteria, new and ffective antibiotics against these bacteria are needed

1.4. Metagenomics as a method to access unculturable bacteria in a sample Metagenomics was developed because the majority (99-99.9%) of microorganisms in a sample are not readily cultured (Handelsman et al, 1998; Torsvik et al, 1990; Kamagata and Tamaki, 2005; Rappe and Givannoni, 2003). The technique covers several steps starting with the isolation of DNA from environmental samples, followed by the size fractionation of the DNA, then DNA cloning into a suitable vector and expression into a host cell or multiple host cells (Courtois et al, 2003; Martinez et al, 2004; Brady et al, 2009; Craig et al, 2010; Kennedy et al, 2008; Schwartz, 2006; Li and Qin, 2005; Lorenz and Eck, 2005; Yun and Ryu, 2005). Then, the library is screened for a particular activity either by the sequenced- based method or by functional screening (Handelsman, 2004; Schloss and Handelsman, 2003). The diagram illustrating the steps in the metagenomics technique is provided in figure 1-1.

Figure 1-1. The diagram illustrating the steps in the metagenomics technique.The technique begins with the extraction of genomic DNA from a sample, then the ligation into a vector for cloning, then the transformation into a host cell to produce a metagenomic library for screening (functional-driven analysis or sequence-driven analysis). The picture was taken from Handelsman, 2004. As a note, the metagenomic library can be screened functionally for proteins or non-proteins. Although activities are often due to extracellular compounds secreted by the host cell, any intracellular molecule encoded by the insert DNA may also be detected by lysing of the host cell resulting in the release of active molecules.

Sequence-based screening may consist of sequencing phylogenetic marker genes (Abulencia et al, 2006; Kembell et al, 2011; Arboleya et al, 2012), DNA probing (Dumont and Murrell, 2005; Shanks et al, 2006), and PCR amplification of genes (Courtois et al, 2003).The functional screening is performed based on activities to look for key metabolic functions, such as screening for lipase activity (Henne et al, 2000), polyketide synthase (Piel, 2002), cellulose (Healy et al, 1995), antibiotics (Gillespie et al, 2002; Martinez et al, 2004), amylase (Richardson et al, 2002; Yun et al, 2004), esterases (Elend et al, 2006) and lipolytic enzyme (Lussier et al, 2011). The screening type chosen in metagenomics may influence the success in harvesting such genes. Therefore, one must be aware of the best screening type for each metagenomic library.

1.5. Host cell for metagenomics In metagenomics, the success of expressing any environmental genomic DNAs is strongly correlated with the choice of host cells. Escherichia coli has been used extensively as the host cell in metagenomics, and the bacteria has successfully expressed several enzymes (for bioremediation and industrial purposes) (Henne et al, 2000; Rees et al, 2003; Vogel et al, 2003) and pharmaceutical compounds such as anti-cancer medicines (Schirmer et al, 2005; Courtois et al, 2003) and antibiotics (Gillespie et al, 2002; Brady et al, 2004; Chung et al, 2008; August et al, 2000; MacNeil et al, 2001; Juan et al, 2006). E. coli has been used as a host cell in metagenomics because the transformation protocols of the bacteria has been well established and the bacteria have been proven as a potential host cell in several metagenomic library constructions (Rondon et al., 2000; Pfeifer and Khosla, 2001; Gillespie et al., 2002; Liles et al., 2004). Additionally, the cells grow fast and are easily used for DNA transformation without the need to develop other cell forms such as sphaeroplasts or protoplasts (Tripathi et al, 2009). Moreover, the availability of various mutants provides suitable host cells to achieve high transformation efficiencies. For example, in some strains the type 1 restriction endonuclease system has been mutated in order to increase the number of mutants by stopping the degradation of any exogenous DNA inserted (Hanahan et al, 1991).

However, it has been discovered that not all genes can be recombinantly expressed in E. coli, because of a codon bias or the lack of suitable transcription or translation initiation factors (Angelov et al, 2009; Richard et al, 2004). Genes that were not expressed in E. coli were successfully expressed in Streptomyces lividans (Lornz and Eck, 2005). Martinez et al (2004) 9 also found that genes encoding for antibiotics were expressed differently in E. coli and S. lividans. Additionally, Gabor et al (2004) stated that the use of E. coli as a host cell only covers 40% of the enzymatic activities from DNA fragments of the 32 genomes used as the DNA insert. As reported by Wang et al (2000), the cloning of microbial genomic DNA from soil and their expression in an S. lividans host cell can extend the spectrum of metabolite compounds. This is because a gene is only expressed in host systems which have all systems for the transcription and translation of the gene. It seems that E. coli does not always have all specific factors for the expression of genes, but the factors can be provided by another host cell, such as S. lividans (Steele and Streit, 2005; Li et al, 2009; Piel, 2011). Foreign expressed genes could also producea compound harmful to E. coli(Li et al, 2009), which lead to a loss of cell viability. Thus, other host cell, such as S. lividans has been employed to increase the capability of capturing potential genes from an environment (Martinez et al, 2004; Courtois et al, 2003).

S. lividans has become a popular host cell because it only produces a very low concentration of endogenous extracellular protease and is able to express numerous genes (Ayadi et al, 2007; Connell 2001; Binnie et al, 1997). Naturally, Streptomyces also produces numerous secondary metabolites such as antibiotics (Zircle et al, 2004; Atta et al, 2009; Castiglione et al, 2007; Watve et al, 2001; Ceylan et al, 2008) and other valuable metabolites (Baltz, 2007), and thus it has the translation and transcription systems for secondary metabolite biosynthesis. As a consequence, it can be a useful host cell for expressing genes which code for the synthesis of antibiotics (Niemi et al, 1993; Escribano and Bibb, 2010); Komatsu et al, 2010).

The innate secretion capacity of the Streptomyces also makes it a better host cell than E. coli for the production of many extracellular proteins originated from prokaryotes (Vrancken et al, 2010; Lorenz and Eck, 2005; Vrancken and Anne, 2009; Binnie et al, 1997; Ann and Mellaert, 1993) and eukaryotes (Ayadi et al, 2006). Additionally, the cloning procedure in S. lividansas well as shuttle vectors have been well established (Kieser et al, 2000; Petricek and Tichi, 1989; Denis and Brzezinski, 1992) and this increases the advantage of using it as a host cell for heterologous gene expression. Therefore, the use of multiple host cells may optimally express genes in a metagenomic project.

1.6. The aims of the research: The overall aims of the research are to search for antibacterial-producing, metagenomic clones able to inhibit the growth of S. flexnery and S. sonnei, to study the antibiotic resistance activity, and to gain insight into the phylogenetic and functional composition of the microbial community of U. australis.

The specific aims can be described as follows: 1) To isolate the total metagenomic DNA of the microbial community of U. australis, followed by cloning into two host cells (E. coli and S. lividans). 2) To perform a functional screening and a genetic analysis of the metagenomic library (expressed in the two host cells) for antagonism against S. flexnery and S. sonnei. 3) To obtain information that links functional genes to the phylogenetic origin of the organism using Homing Endonuclease Restriction and Marker Insertion (HERMI). 4) To identify the type of antibiotic resistance and to perform a genetic characterization of the antibiotic resistant elements. The possible transfer mechanism of the resistant elements was also studied.

CHAPTER 2

CONSTRUCTION OF A METAGENOMIC LIBRARY FROM MICROBIAL COMMUNITY OF ULVA AUSTRALIS AND SCREENING FOR ANTIBACTERIAL CLONES

CHAPTER 2: CONSTRUCTION OF A METAGENOMIC LIBRARY FROM MICROBIAL COMMUNITY OF ULVA AUSTRALIS AND SCREENING FORANTIBACTERIAL CLONES

2.1 INTRODUCTION High cases of antibiotic resistance in Shigellahave been observed mostly in developing and under-developed countries (Ashnenazo et al, 2003; Huang et al, 2005; Taneja et al, 2012; Subekti et al, 2001; Kosek et al, 2010; Talukder et al, 2006; Nguyen et al, 2005; Gaikwad and Deodhar, 1985; Ashkenazi et al, 2003; Tajbakhsh et al, 2012; Pazhani et al, 2008). A small occurrence of a Shigella outbreak has also been observed in industrialized countries (Sivapalasingam, 2006; Lewis et al, 2009; McIver et al, 2002; Nimri et al, 2009; Oglesby et al, 2005; Vila et al, 1994). Nowadays, almost all of the available antibiotics are not effectively combating the disease caused by this bacterium, and therefore new antibiotics or antibacterial compounds are urgently needed.

The large diversityof the microbial communities in U. australis can be a good source for finding antibacterial compounds because competition may occur among the micro-organisms (Rao et al, 2006). As most members of the microbial communities of the alga are not-yet cultured (Burke et al, 2011a), metagenomics will be applied to explore the properties of the uncultured micro-organisms. To increase the chance of having clones with antibacterial activity against S. sonnei and S. flexnery, two host cell systems (E. coli and S. lividans) will be used. Furthermore, to recover novel activities, functional screening will be conducted on the library.

Overall, the aims of the project written in this chapter are: 1) to generate antibacterial clone libraries; and 2) to identify the gene responsible for the antibacterial activity.

2.2. METHODS 2.2.1. Bacterial strains, media, buffer and vector used in this project The host cells were E. coli EPI300TM-T1R [F- mcrA D(mrr-hsdRMS-mcrBC) f80dlacZDM15 DlacX74 recA1 endA1 araD139 D(ara, leu)7697 galU galK l- rpsL (StrR)nupG trfA tonA and DH5α [FendA1 glnV44 thi-1 relA1 gyrA96 deoR nupG lacZdeltaM15hsdR17] and S. lividans TK24 (red and act deletion) (Martinez et al, 2004)

Lennox broth (tryptone 10 g/l, yeast extract 5g/l, NaCl 5g/l and glucose 1g/l), Lennox agar (Lennox broth plus 1.5% agar) and mannitol agar (2 g/l of soya flour, 20 g/l mannitol and 1.5% agar) were used for culturing S. lividans, while E. coli was grown on Luria Bertani (LB) broth (tryptone 10 g/l, yeast extract 5g/l and NaCl 5g/l) or Luria Bertani agar (LB with 1.5% agar). Soft nutrient agar (SNA) containing 1.5% peptones, 0.3% yeast extract, 0.6% NaCl, 0.1% D(+)glucose, 0.5% agar and kanamycin 200 µg/ml was used for overlay in the production of the metagenomic library in the S. lividans host cell TK24. Yeast Malt Extract Agar /YMEA (1.0% malt extract, 0.4% yeast extract, 0.4% dextrose and 1.5% agar) was used for the screening of the antibacterial activity of library of U. australismicrobial community expressed in S. lividans host cell. Nutrient Agar/ NA (0.5% pepton, 0.3% meat extract and 1% agar) were used as the overlay agar in the antibacterial screening. SOC

medium (yeast extract 5g/l, 0.01M NaCl, 0.0025M KCl, 0.01M MgCl2, 0.01M MgSO4, 0.02M glucose) was used in recovering the electroporated cells (E. coli EPI300 electrocompetent cells, Epicentre, Madison, WI, USA) after generating mutants via in-vitro transposon mutagenesis.

The buffer used in generating the protoplast of S. lividans was P buffer (10.3% sucrose,

0.025% K2SO4, 0.202% MgCl2.6H2O, 0.2% (v/v) trace element solution, 0.005% KH2PO4,

0.368% CaCl2.2H2O and 0.573% TES buffer/ pH 7.2). Trace element solution was made

from 0.004% ZnCl2, 0.02% FeCl3.6H2O, 0.001% CuCl2.2H2O, 0.001% M nCl2.4H2O,

0.001% Na2B4O7.10H2O) and 0.001% (NH4)6Mo7O24.4H2O). Tris-acetate-EDTA/ TAE buffer concentration 1x (4.8% Tris base, 1.1% (v/v) acetic acid, 0.001M EDTA} was used for standard gel electrophoresis while 0.5% of the buffer was used for Pulsed Field Gel Electrophoresis (PFGE).

To construct metagenomic libraries, DNA of the microbial community of the microbial community of U. australis was ligated into the pFD666 vector, a cosmid constructed by Denis and Brzezinski, 1992).

The cosmid map is given in figure 2-1.

Figure 2-1.Vector map of the pFD666 cosmid.Picture was taken from Kieser et al, 2000.

The cosmid is a shuttle vector containing the ori ColE1 and oripJV1, suitable for replication in E. coli and S. lividansrespectively. The presence of Cos provides a site for phage packaging that is essential for E. coli transfection by the l phage. Clones were selected in LB agar with 50 mg/ml kanamycin (for E. colitransformants) or Lennox Agar supplemented with kanamycin 200 mg/ml (for S. lividans).

2.2.2. Extraction of microbial genomic DNA from U. australis The total DNA o f the microorganisms living in association with U. australis was isolated using the method developed by Burke et al, 2009. The algae were collected from Coogee Beach, New South Wales, Australia on November 2007.

Algae were washed three times in sterile sea water to remove loosely-associated bacteria. Twenty grams (wet weight) of the algae were then soaked in 100 ml of calcium-magnesium- free artificial seawater (CMFSW) (0.45M NaCl, 10 mM KCl, 7 mM Na2SO4, 0.5 m M

NaHCO3containing also, 10 mM EDTA and 1 ml filter-sterilized rapid multi-enzyme cleaner (3M, North Ryde, NSW, Australia) and incubated for two hours at room temperature with shaking (80 rpm). The rapid multi-enzyme cleaner is usually used to clean surgical, medical and dental instruments from human secretions and microorganism contaminants by effectively digesting proteins, lipids, carbohydrates and mucopolysaccharides (3M Australia product catalogue.After incubation, the CMFSW containing the algae was vortexed for two minutes. The algae were then removed from the solution and the solution was centrifuged at 300 x g for 15 m inutes to further remove any algae debris. Microbial DNA was then extracted using a method described in Burke et al, 2010.

16S rRNA genes were amplified from the metagenomic DNA following the method of Lane et al, 1991. Amplification of the 18S rRNA gene was also performed to estimate the presence or absence of eukaryotic DNA. The metagenomic DNA was serially diluted to 0.09 pg; 0.9 pg; 9 pg; 90 pg; 900 pg and 9,000 pg. The PCR reaction for the 16S rRNA gene was set up as follows: 10 mM dNTPs, 2.5 µl 10x Bovine Serum Albumin (BSA), 5 pmol of each primer (27F-5’-AGAGTTTGATCMTGGCTCAG-3’ and

1429R-5’-ACGGTTACCTTGTTACGACTT-3’) (Lane, 1991), 2 µ l 10 x buffer, 1U of RedTaq DNA polymerase (Promega, Madison, WI, USA) and sterile, ultra-pure water to make a total volume of 20 µl.

PCR reaction of 18S rRNA gene was set up in a similar fashion as the 16S rRNA PCR using the primer set EK1F, 5’-CTGGTTGATCCTGCCAG-3’ (Lopez-Garcia et al, 2001) and 18sr- b, 5’-GATCCTTCYGCAGGTTCACCTA-3’ (Medlin et al, 1988). The cycling reaction for 16S and 18S rRNA was set up as follows: initial denaturation at 94°C, 3 minutes, 80°C for hot start to add the RedTaq DNA polymerase, followed by 30 cycles of 94°C, 30 seconds; 50°C, 1 m inute and 72°C, 3 m inutes with a final extension at 72°C, 6 minutes. The PCR amplification were also performed for the positive controls (E. coli genomic DNA for 16S rRNA and Saccharomyces cerevisiae genomic DNA for 23S rRNA) and a negative control (sterile ultrapure water) (Burke et al, 2010).

2.2.3. End-repairing and size selection of genomic DNA of the microbial community of Ulva australis The end-repairing reaction was set up as follows: 1 µg of the genomic DNA, 0.4 µl of end- repair enzyme mix (Epicentre, Madison, WI, USA), 1x end-repair buffer, 0.2 mM dNTPs, 1 mM ATP and ultrapure water to reach a total volume of 50 µl. The reaction was performed for 45 minutes at room temperature, followed by heat inactivation at 65°C for five minutes.

The end-repaired metagenomic DNA was run on a Clamped Homogeneous Electric Fields/ CHEF-DR II Pulsed Field Gel Electrophoresis (PFGE) system (Bio-Rad, Hercules, CA, USA). For this, 200 ml of 0.5% agarose II was prepared by dissolving 1 g of the agarose II (Amresco Inc., Solon, OH, USA) into 200 ml of 0.5 x buffer TAE. Five hundred nano-grams of the Mid-range PFG marker I (New England Biolabs, Beverley, MA, USA) and one microgram of the Lambda EcoRI+HindIII DNA ladder (Fermentas, Glen Burnie, MD, USA) were used for size determination. The PFGE condition was set as follows: switch time at initial one second to final six seconds, run time of 16 hours, voltage six V/cm, angle 120 º, buffer 0.5x TAE (40 mM Tris, 20 mM acetic acid, I mM EDTA). The size selected genomic DNA (33 KB048 Kb) was purified from the Low Melting Point/ LMP agarose using Gelase enzyme (Epicentre, Madison, WI, USA), as described in the manufacturer’s instruction.

2.2.4. Cosmid preparation before ligation

Cosmid (pFD666) was isolated from E. coli using the Illustra miniprep plasmid isolation kit (GEBio-Science Corp, NJ, USA) in accordance to the protocols developed by the manufacturer. Then, it was digested with the BamHI (Roche Applied Science, Penzberg, Germany) in a reaction containing one µg of cosmid, 1 x Buffer B (RocheApplied Science, Penzberg, Germany), 1U of restriction enzyme BamHI and sterile ultra-pure water in a total volume of 25 µl. The restriction reaction was incubated at 37°C for one hour. Then, the DNA was end-repaired.

The end-repairing reaction composed of three micrograms of DNA, 1x end-repair buffer, 0.2 mM dNTPs, 1mM ATP and 0.6 µl end-repair enzyme mix (Epicentre, Madison, WI, USA) made up t o the final volume of 50 µ l. The end-repairing reaction was kept at room temperature at 45 minutes, followed by heat inactivation at 70°C for 10 minutes.

After that, the cosmid DNA was dephosphorylated using 1 U of the Antarctic phosphatase (New England Biolabs, Beverley, MA, USA), 1x NEB Antarctic phosphatase buffer, 1.7 µg end-repaired cosmid and ultrapure water in a total volume of 25 µl. The reaction was done at 37°C for 30 minutes, followed by heat inactivation at 65°C for five minutes.

2.2.5. Ligation and transformation to generate metagenomic libraries A blunt end ligation reaction was conducted as follows: 106 ng of dephosphorylated pFD666, 848 ng of end repaired DNA, 1x ligation buffer, 5U T4 DNA ligase (RocheApplied Science, Penzberg, Germany), 30% Polyethylene glycol (PEG) 8,000 and ultrapure water to a final volume of 20 µl. The reaction was incubated at room temperature overnight. Then the ligation product was transformed into S. lividans protoplasts via the PEG-assisted transformation technique (Kieser et al, 2000) and into E. coli (using the packaging system of the Max Plax Lambda packaging extract kit; Epicentre, Madison, WI, USA).

2.2.6. Screening for antimicrobial activities from cosmid library clones The antibacterial assay for the two libraries was performed using the method described in (Brady et al, 2007). The screening was divided into two steps, the preliminary and the confirmation. The preliminary step was done by inoculating the library clones on plates of Luria Bertani agar (for E. coli clones) or yeast malt extract agar (for S. lividans clones) containing 12.5 µg/ml kanamycin, at 30°C for three days, before overlaying them with the

target bacteria. The target bacteria were grown to achieve OD600:0.5-0.6. It was then diluted (1/200) in top agar of 1% nutrient agar and was used for the overlay. The plates were incubated at 37°C for one to two days before zones of inhibition were observed. Colonies that showed zones of inhibition were assayed again on plates for the confirmation screening, using the standard paper disc method (De Beer and Sherwood, 1945). Paper discs of about 0.5 c m in diameter, which were made from filter paper, were sterilized in an autoclave. Agar plates for the assay were prepared by pouring 20 ml of Luria Bertani agar (for E. coli clones) or yeast malt extract agar (for S. lividans clones) containing a dilution

(1/200) of each target bacteria (OD600: 0.5-0.6). Paper discs were put on the agar plates and a volume of 20 µl of cell-free filtrate recovered from each clone was pipetted on the paper disc. Plates were incubated at 37°C for one to two days. The presence of bacterial growth inhibition around the paper disc indicates the antibacterial activity produced by the corresponding clone.

2.2.7. Cosmid retransformation and the antibacterial assay The cosmid of antibiotic producing clones was isolated using the protocol described in Thompson et al, 1980 (for the isolation from S. lividans). The cosmid isolation from E. coli was done using the Illustra miniprep plasmid isolation kit (GEBio-Science Corp., NJ, USA). Retransformation was then conducted into the same and into different host cell strains (E. coli EPI300 or S. lividans TK24). Retransformation into E. coli was conducted via heat shock method (Maniatis et al, 1982) using chemical-competent cells generated in accordance to the protocol described in (Maniatis et al, 1982). Transformation into S. lividans was done using the PEG-assisted transformation technique (Kieser et al, 2000).

2.2.8. Cosmid characterization A number of 27 cosmids isolated from positive clones in S. lividans and one from the E. coli positive clone was end-sequenced using primers: T7: 5’-TAATACGACTCACTATAGGG-3’, and SP6: 5’-ATTTAGGTGACACTATAGAATAC-3’. The sequencing used a reaction mixture of 100 ng cosmid DNA, two microliters Big dye terminator v.3.1 ( Applied Biosystems, Carisbad, CA, USA), one microliter 5x reaction buffer, 10 pmol of the primer, three microliters of 4M trimethylamine-N-oxide and sterile, deionized water in a total volume of 10 ml. The sequencing cycle conditions were 96°C, one minute followed by 30 cycles of 96°C, one minute; 50°C, five seconds; 60°C, four minutes and finishing at 4°C.As control, pFD666 without any DNA insert was also end-sequenced using the same reaction mixture and cycle conditions.

The sequencing products were then cleaned using EDTA and ethanol precipitation (Angela Higgins, Ramaciotti Centre of the University of New South Wales). Briefly, 10 ml of 1x TE; five microliters of 125 mM EDTA and 70 ml of 95% ethanol were added to the sequencing product and vortexed briefly for 10 seconds. The DNA was precipitated at room temperature for 15 minutes, followed by centrifugation at 3000 g, for 30 minutes at 4°C. The supernatant was aspirated, after that 60 ml of 70% ethanol was added to the DNA. The tube was centrifuged at 4°C, 1650 x g for 15 m inutes and the supernatant was aspirated again. The DNA was air-dried (covered in foil as Big- dye is light sensitive) and sequenced using the Applied Biosystems 3730 DNA analyser, Foster City, CA, USA (service was conducted by

Ramaciotti centre, UNSW). The end-sequenced products were then analysed for the sequence similarity using the blastn tool of the National Centre for Biotechnology Information/ NCBI (Altschult et al, 1997) and the Kyoto Encyclopedia of Genes and Genomes /KEGG database (Kanehisa et al, 2012; Kanehisa and Goto, 2000).

The cosmid that did not result in an end-sequencing product was then characterized by double digestion to detect the presence of the pFD666 cosmid. The double digestion was conducted with NcoI and StuI (both were from New England Biolabs, Beverley, MA, USA). A control for the double digestion was also performed using pFD666 without any DNA insert.

2.2.9. Genetic characterization of antibacterial activities via transposon mutagenesis Cosmids of positive clones in E. coli were identified for the gene responsible for the antibacterial activity. To do so, an in-vitro transposon mutagenesis was carried out using the EZ-Tn5Insertion kit (Epicentre, Madison, Wisconsin), to generate “knock-out” mutants which have lost the ability to produce antibiotics. The transposition system randomly inserts the transposon (containing a T7 promoter for gene expression and the insert primer binding sites and a trimethoprim resistance cassette) into target DNA, without the need for host cell (E. coli) factors. The transposition insertion (illustrated in figure 2-2) was performed in accordance to the protocol provided by the manufacturer (Epicentre, Madison, Wisconsin). The electrophoration into E. coli was done based on the procedure described in Sambrook and Russell, 2001.

Figure 2-2.The schematic protocol of EZ-Tn5Transposon insertion. Picture was taken from the instruction manual of Epicentre, Madison, WI, USA.

Mutant clones that lost the ability to inhibit the growth of the target bacteria were then isolated u sing the Illustra miniprep plasmid isolation kit (GE Healthcare, Piscataway, New Jersey). The sequenced analysis was conducted using DHFR-1 FP-1 forward primer (5’- GGCGGAAACATTGGATGCGG-3’) or DHFR-1 RP-1 reverse primer (5’- GACACTCTGTTATTACAAATCG-3’) provided by the manufacturer.

The flanking sequences of the transposon insertion were searched for open reading frames using Fgenes programme of Softberry (http://www.softberry.com). The ORFs were then searched against the non-redundant protein database of the National Centre for Biotechnology Information/ NCBI (Altschult et al, 1997) and against the curated (UniPotKB/ Swiss-Prot) and non-curated (UniProtKB/TrEMBL) protein database using Blastp (Donovan et al, 2002). A particular function was considered from protein sequences that had an e-value of 1x 10-20 in the Swiss Protein database; or had the same annotations in two or more databases, if the e- value from the Swiss protein database was higher than 1x 10-20 (with an e-value 1 x 10-5 as the cut -off point).

Multiple sequence alignment was conducted via the MUSCLE 3.7 p rogram (Full mode). Gblocks was used in the alignment to eliminate poorly aligned regions and divergent regions. Then, the phylogenetic tree (figure 2-8) was visualized using the PHY.FI programme accessed in http://cgi-www.daimi.au.dk/cgi-chili/phyfi/go (Fredslund, 2006). The phylogeny (figure 2-9) was analysed via PhyML 3.0 a LRT (using maximum likelihood) and the maximum likelihood tree was viewed using the TreeDyn program; all were provided in http://www.phylogeny.fr/ (Dereeper et al, 2008; Dreeper et al, 2010).

2.2.10. Analysis of whole cosmid sequences The entire fosmid of the HERMI clones was sent to the J.Craig Venter Institue for shotgun sequencing, with the procedure drafted in Rusch et al, 2007. DNA reads obtained from sequencing were trimmed for fosmid contamination and low-quality sequencing. Contigs containing sequences of fosmid forward and reverse reads were extracted and assigned to the ID for the particular fosmid of the end sequences. PCR amplification of randomly chosen DNA region of the scaffolds was performed to determine which scaffolds belong to the particular fosmid. Open reading frames were identified with the program Fgenesb from Softberry (Tyson et al, 2004). Every putative ORF was searched against the NCBI- non redundant protein database (Pruitt et al, 2007; Altschul et al, 1990), the curated/ UniProtKB/Swiss-Prot and non-curated / UniProtKB/TrEMBL (Donovan et al, 2002), the KEGG database (Kanehisa et al, 2012; Kanehisa and Goto, 2000) and Pfam database (Bateman et al, 2000) using the blastp program. A particular function was considered from protein sequences that had an e-value of 1x 10-20in the Swiss Protein database; or had the same annotations in two or more databases, if the e- value from the Swiss protein database was higher than 1x 10-20 (with an e-value 1 x 10-5 as the cut -off point).

2.3. RESULTS 2.3.1. Metagenomic DNA from Ulva australis To generate libraries of the microbial communityof U. australis, a high quantity of the metagenomic DNA was needed. DNA was successfully extracted from the algae, with a concentration of ~35 ng/ µl (as seen in figure 2-3 below) and a total yield of 7µg from 20 grams of U. australis.

Figure 2-3.Concentration calculation of Ulva australis’ microbial community.Lane 1 and 4: Lambda DNA EcoRI+ HindIII (Fermentas Inc., Glen Burnie, MD, USA) (0.2 µg); Lane 2-3: 5 ml genomic DNA of bacterial communities of U. australis. The calculation of DNA concentration in lane 2 or 3 resulted in ~35 ng/ µl. The DNA concentration was calculated based on band intensity resulted from an agarose gel electrophoresis. This method was used because the DNA can be calculated quickly by comparing the sample band intensity with that of the molecular marker. Thus, the DNA concentration can be calculated from the equation as follows: (0.2 µg/ 0.5 µg) x 87.5 ng x 5 x 1/5 µl), to give a result of ~35 ng/ µl.

To estimate the proportion of bacterial and eukaryotic DNA present in the extracted genomic DNA, PCR amplification of the 16S rRNA and the 18S rRNA using universal primers were performed. PCR amplification of the 16S rRNA resulted in a product of about 1,465 bp starting at an amount as low as 9 pg of DNA per reaction (figure 2-4A, lane 4-6). The positive control (E. coli genomic DNA, lane nine and ten) showed positive detection of the PCR product, while no DNA band was observed for the negative control (sterile ultra-pure water, lane 11). As for 18S rRNA, no positive detection was observed for 0.9- 9000 pg from the same genomic DNA (figure 2-4B, lane 2-6). This means that the metagenomic DNA extracted were mostly of bacterial origin.

To do the size fractionation of the DNA, PFGE was applied to the sample. It was observed that it has a high size of between 5 Kb and 48 Kb; with the sharp bands around 15-33 Kb (figure 2-5). Genes may need to be clustered in an operon, because this is the easiest way to control their functions under one promoter. To achieve expression for such activity in a transformant, all the genes in the operon have to be cloned. Some operon encoding for antibiotic biosynthesis can have a size of 17 Kb (Kratzschmar et al, 1989; Konz et al, 1997); therefore, a large insert DNA size may increase the coverage of the operon.

Figure 2-4. PCR amplification of 16S and 18S rRNA. Using 16S primer (figure A).Lane 1: Lambda DNA EcoRI+ HindIII (Fermentas Inc., Glen Burnie, MD, USA) (0.2 µg); Lane 2: 100 bp plus DNA marker; Lane 3-7: amplicon resulted from ~9000 pg, 900 pg, 90 pg, 9 pg, 0.9 pg of genomic DNA isolated from U. australis, respectively; Lane 8-10: positive controls (genomic DNAof E. coli); Lane 11: negative control (sterile ultra-pure water).

Using 18S primer (figure B).Lane 1: 100 bp plus DNA marker (Fermentas Inc., Glen Burnie, MD, USA); Lane 2-7: products resulted from ~9000 pg, 900 pg , 90 pg, 9 pg, 0.9 pg, 0.09 pg of the isolated genomic DNA, respectively; Lane 10: positive control (Saccharomyces cerevisiae genomic DNA); Lane 11: negative control (sterile water).

Figure 2-5. Size selection of metagenomic DNA of microbial community of U. australis. Lane 1: 10 µl Mid- range PFG marker I (New England Biolabs, Beverley, MA, USA); Lane 2: Marker (Lambda EcoRI+Hind III, Fermentas Inc., Glen Burnie, MD, USA): 1 µg; Lane 3 and 5: 10 µl (~350 ng) of the genomic DNA.

2.3.2. Library construction in S. lividans TK24 and E. coli EPI300 The two libraries were generated and resulted in a high coverage of microbial genomic DNA (see Table 2-1). Randomly picked cosmid clones have been isolated and then were subjected to restriction digest; however digestion with several different restriction enzymes did not cut some of the cosmid insert (appendix 10). Sequencing of some cosmid insert was also successfully performed (please see appendix 1). Based on the restriction digest data (figure 2- 7) and the sequencing data (appendix 1), all clones had insert with sizes ranged between 23.5 Kb -27 Kb. Different inserts had difference sizes and end-sequencing showed also different

genetic information. This shows that the library clones had distinct inserts. Given that the average genomic DNA insert was ~25 Kb (obtained from the averageof 23.5 Kb and 27 Kb) and an average bacterial genome is about 4.7 Mb (Raes et al, 2007), the two libraries covered a high number of microbial genomes (212 and 798 for the library in S. lividans and in E. coli, respectively) (table 2-1).

Table 2-1.The DNA coverage of the genomic DNA libraries of microbial community of U.australis. Host cells Approximate Transformation Estimated Estimated number of efficiency total DNA number of clones (transformants/ µg cloned (Gb) bacterial DNA) genomes covered S. lividans TK24 40,000 4 x 105 1 212 E. coli EPI300 150,000 2.9 x 106 3.75 798

2.3.3. Screening of the antibacterial activityof the library of the U. australismicrobial community Approximately 18,000 clones (E. coli) and all clones (S. lividans) were inoculated on t he screening agar. It was found that 52 c lones (expressed in S. lividans) and 12 c lones (expressed in E. coli) were able to inhibit the growth of Shigella. An early observation indicated that the host cells (S. lividans TK24 and E. coli EPI300) did not show antibacterial activities against S.sonnei and S. flexnery. In addition, it was also found that kanamycin (12.5µg/ml) did not result in growth inhibition of Shigella.

The paper disc assay (section 2.2.6.) was then employed to all clones possessing the antibacterial activity. It was observed that 30 clones (expressed in S. lividans) showed inhibition activity against one or two of the Shigella strains. In contrast, only one clone (expressed in E. coli) was able to inhibit the target bacteria (table 2-2). It was calculated that the frequency of inhibitory clones in the library expressed in E. coli and S. lividans was 0.006 % and 0.075 %, respectively.

Figure 2-6.Functional assay of the cloneagainst S. sonnei. B lack box: zone of inhibition against the target bacteria was observed.In contrast, there was no inhibition zone observed for the kanamycin 12.5 µg/ml used for the cosmid induction (as seen in the two other paper discs).

2.3.4. Retransformation of cosmid clones with inhibitory activity against Shigella and the paper disc assay Re-transformation of cosmids from 30 c lones (expressed in S. lividans); and re- transformation of a clone (expressed in E. coli) was performed. Each re-transformant was assayed for the inhibition activity and it was found that three clones (codes: SL1, SL5 and SL N15; red box) lost their activity during re-transformation. However, most of the cosmids (27 out of 30) had re-transformable, and hence stable, activities (table 2-2).

Table 2-2.The antibacterial activity of clones before and after re-transformation into the same or different host cells. Codes for the bacterial target: Ss: S. sonnei; Sf: S. flexnery. Codes for the clone’s name: SL, SLe and Sln: clone library in S. lividans; Ect: clone library in E. coli; SS, SSe and SSn: cosmid isolated from Streptomyces lividans and then transformed back into S. lividans; SE, SEe and SEn: re-transformation of the cosmid isolated from S. lividans into E. coli; ES: cosmid from E. coli was transformed into S. lividans; EE: re-transformation of cosmid isolated from E. coli back into E. coli. (+): the clone inhibits the bacterial target. (-): there was no inhibition activity. Red box: clone lost the inhibition activity during re- transformation.

Clone name Activity against Clone name Activity Clone name Activity Ss Sf against against Ss Sf Ss Sf SL1 - + SS1 - - SE1 - - SL2 + - SS2 + - SE2 + - SL3 + - SS3 + - SE3 + - SL4 + + SS4 + + SE4 + + SL5 - + SS5 - - SE5 - - SL6 + + SS6 + + SE6 + + SL7 + + SS7 + + SE7 + + SL8 - + SS8 - + SE8 - + SL9 + + SS9 + + SE9 + + SL10 + - SS10 + - SE10 + - SL11 + - SS11 + - SE11 + - SL12 + - SS12 + - SE12 + - SL19 + - SS19 + - SE19 + - SL39 + - SS39 + - SE39 + - SL41 + - SS41 + - SE41 + - SL42 - + SS42 - + SE42 - + SLe13 + - SSe13 + - SEe13 + - SLe14 + - SSe14 + - SEe14 + - SLe15 - + SSe15 - + SEe15 - + SLe16 + + SSe16 + + SEe16 + + SLe17 + + SSe17 + + SEe17 + + SLe19 + + SSe19 + + SEe19 + + SLe21 + - SSe21 + - SEe21 + - SLe22 + - SSe22 + - SEe22 + - SLe23 + + SSe23 + + SEe23 + + SLn15 + - SSn15 - - SEn15 - - SLn17 + + SSn17 + + SEn17 + + SLn19 + + SSn19 + + SEn19 + + SLn20 + + SSn20 + + SEn20 + + SLn21 + - SSn21 + - SEn21 + - Ect 7 + - ES7 + - EE7 + -

2.3.5. End-sequencing and restriction digestion of the cosmids of clones with inhibitory activity against Shigella End-sequencing was performed to the 27 clones and to one clone of the library expressed in S. lividans and E. coli respectively. Only for one clone of the library in S. lividans (SL9), clearly readable sequences (using SP6 or T7 primer) were obtained. The other 26 c lones could not be amplified with the SP6 and T7 primers and all these clones were then subjected to another sequencing reaction using primers located at the pFD666 cosmid backbone. The cosmid control amplified the backbone primers, but the 26 c lones did not amplify the backbone primers.

Characterization of the 26 cosmids from the S. lividans clones and 1 cosmid from E. coli clone was also conducted by restriction digestion using double enzymes (NcoI and StuI). The digestion on pFD666 with no insert (control; figure 2-7, lane 3) resulted in two bands of 1.3 Kb (is not clearly visible) and 3.9 K b (red box), which is consistent with the expected restriction patterns (see figure 2-1). Only one clone (SE11, figure 2-7, lane 5) showed several bands after the digestion. These bands were of a size of around 8 Kb, >10 Kb (blue box), some faint, small sized bands of about 2.5 and 2 Kb (green box) and the cosmid fragment of ~3.9 Kb (red box). The finding indicated that the genomic DNA has been inserted into the pFD666 cosmid of the clone SE11 as well as of that of the clone SL9 mentioned above.

All the other 25 a ntibacterial producing clones from S. lividans and the one antibacterial clone from E. coli (Ect7) did not amplify with all primers tested (SP6, T7 and backbone primers) and they were not cleaved by the double digestion, indicating that they did not contain pFD666. However, these clones were able to grow on plates of kanamycin and re- transformation into the two host cells (E. coli and S. lividans) was performed successfully; therefore, there is a possibility that the constructs inside these 26 antibacterial-producing clones have a kanamycin resistance gene and origin of replications for vector replication in the two host cells. Despite the effort expended on identifying the nature of these clones, it is not yet clear where they have been derived from and further research may be needed to characterize this issue.

Figure 2-7. Digestion of cosmids with NcoI-StuI.Lane1: the gene ruler 1 Kb DNA marker (Fermentas Inc., Glen Burnie, MD, USA). Lane 2: pFD666 with no insert (before the digestion); Lane 3: pFD666 with no insert (after the digestion); Lane 4: cosmid of SE11 (before the digestion); Lane 5: cosmid of SE11 (after the digestion). Blue box: upper bands >10 Kb; lower band: ~8KB; Red box: the 3.9 Kb cosmid fragment. Green box (with arrows: a band of 2 Kb and 2.5 Kb (the bands were not sharp).

2.3.6. Sequence analysis of the SE9 cosmid The cosmid SE9 contained a 1,525 bp of 16S rRNA gene.It shared 90-99% identity and 93- 100% coverage to the 16S rRNA sequences of Gammaproteobacteria of the genera Microbulbifer, Cellvibrio and Pseudomonas. The phylogenetic tree indicated that the sequence was closely related to Microbulbifer epialgicus strain F (accession number NR_041493.1) (figure 2-8).

Figure 2-8. Maximum likelihood trees of the 16S rRNA gene of SE9 and the closest representatives of culturable and uncultured from the 16S rRNA gene database (NCBI). Red box: 16S rRNA gene sequence of SE9. The scale bar indicates 0.02 divergences of the sequences. The triangle is the collapsed branch for more than 40 species of Pseudomonas.

2.3.7. Identification of the gene responsible for the inhibitory activity against Shigella The in vitro transposon mutagenesis of the SE9 clone resulted in one mutant (code P2E2) that lacked the inhibitory activity against S. flexnery. The fosmid was then sequenced withDHFR- 1 RP-1 reverse primer and DHFR-1 FP-1 forward primer. An 176 bp (from here on referred to as ORFa) was identified from nucleotide position 310-486 of the reverse sequence and another ORF (namely ORFb) encoding for 250 amino acids long (nucleotide position 3-753) was found in the reverse sequence. A conserved region of putative modulator of DNA gyrase (PmbA TldD) was identified in the amino acid position of 41-229 of the ORFb (188 amino acids; Pfam database, e-value 9.6 x 10-27).

Searches against the non-redundant protein database of NCBI indicated that the sequence of ORFb had 44-45% similarity and 81-84% coverage to the hypothetical protein of Alpha Proteobacterium (EJW21365.1), the modulator DNA gyrase of Stappia aggregata (ZP_01548115.1) and the PmbA of Loktanella vestfoldensis (ZP_01002397.1). Searches against the Swiss protein database showed that it shared 44-47% similarity and 60% coverage to the PmbA sequence of Oceanicola granulosus (TR: Q2CJH5_9RHOB) and the peptidase U62 modulator of DNA gyrase of Parvibaculum lavamentivorans (TR: A7HQS1_PARL1). The protein from ORFb was referred to as PmbA_1 (PmbA protein).

Phylogenetic analysis (figure 2-9) showed that the protein is related to the peptidase U62 modulator of DNA gyrase of Parvibaculum lavamentivorans (YP_001411911.1).

Figure 2-9. Phylogenetic tree of the aligned protein sequences that were related to PmbA of SE9; and the selected sequences from the non-redundant protein from NCBI. The scale bar corresponds to a genetic distance of 0.4 substitutions per position.

2.3.8. Sequence analysis of the entire SE9 cosmid insert DNA Shotgun sequencing and annotation were done on the cosmid of the SE9 clone to identify all the genes that were contained in the cosmid. The DNA sequence length of the cosmid was 27.1 Kb and it had 26 open reading frames (appendix 1). About 42.3% of the ORFs did not have any known functions (annotated as uncharacterized protein or had no sequence hit). The gene responsible for the antibacterial activity was identified from the shotgun sequencing data. Furthermore, some other genes for structural functions, and basic metabolism and survival functions were identified. All ORFs are listed in table 2-3.

Table 2-3. Annotation of the open reading frames of clone SE9.Where possible, naming of the genes was performed according to the Swiss Protein database. If no information was available under the Swiss Protein database, then naming of the genes was done according to the NCBI database. (-): no significant hit.The dark shaded box indicates the e-value was higher than 1 x 10 -20 under Swiss Protein database (with an e-value of 1 x 10-5 as the cut-off). The light shaded box indicates the e-value is less than 1 x 1 0 -20 under Swiss Protein database. White box indicates the protein is annotated as hypothetical or uncharacterized protein or no hit. For detail annotation, please refer to appendix 1.

ORF Location (bp) and Name of protein or gene orientation 1-1485 16 S rRNA gene ORF1 2130 – 2345 (+) Putative uncharacterized protein ORF2 2342 – 2533 (+) Putative uncharacterized protein ORF3 3049 – 3156 (-) Putative uncharacterized protein ORF4 4077-4217 (-) No hit ORF5 4509 – 5429 (-) Phosphatydilserine synthase ORF6 5496 – 6521 (-) Ketol-acid reductoisomerase ORF7 6544 – 7035 (-) Acetolactate synthase ORF8 7047-8783 (-) Acetolactate synthase ORF9 9236-9721 (+) Putative uncharacterized protein ORF10 9753-10829 (-) Putative uncharacterized protein ORF11 10858-11316 (-) Putative uncharacterized protein ORF12 11613-12785 (-) PmbA_1 ORF13 13062-13244 (+) Hypothetical protein Dshi_4011 (TRA8LU30_DINSH)

ORF14 13372-14700 (-) Putative uncharacterized protein (TR:Q5LKKI_SILPO)

ORF15 14697-15197 (-) NADH riboflavin 5’ –phosphate oxidoreductase (TR:A8TK16_9PROT)

ORF16 15194-16198 (-) Co/Zn/Cd resistance protein cZcD (TR:A8TTM6_9PROT)

ORF17 16338-16772 (+) Acetyltransferase, GNAT family protein (TR:QOFCP3_9RHOB) ORF18 16834-17589(+) Putative polyhidroxybutyratedepolymerase (TR:B9NUP3_9RHOB)

ORF19 17638-18429 (+) Amidinotransferase (TR:C8S44S_9RHOB) ORF20 18494-19651 (-) Unnamed protein product (TR:A1B8QS_PARDP)

ORF21 19797-20603 (+) 2-Dehydro-3-deoxyphosphooctonate aldolase (TR:A7HXX8_PARLI)

ORF22 20676-21167 (+) Predicted protein (TR:C3X4E1_OXAFO) ORF23 21323-22027 (+) RepressorLexA (TR:E2CCN3_9RHOB)

ORF24 22021-24011 (-) Competence protein (TR:C9D485_9RHOB)

ORF25 24140-25576 (+) Glutamyl-tRNAsynthetase (TR:B9QYE2_9RHOB)

ORF26 25654-26952 (+) Citrate synthase (TR:64R8V4_9RHIZ)

2.4. DISCUSSION 2.4.1. The quantity and quality of metagenomic DNA of the microbial communities of U. australis. The first step in a metagenomic project is to isolate the total genomic DNA from the environmental sample. The metagenomic DNA of microbial communities living in association with U. australis has been extracted from the macro-algae using the method developed by Burke et al, 2009. By comparing the result obtained in this project to that of Burke et al, 2009; it was found that the amount of metagenomic DNA extracted (per gram) from this project was much lower (1/6 less) than that of Burke et al, 2009. T he difference might be caused by the difference in sampling conditions (time, location), as well as the growth phase of the alga. Different time of sampling and location of alga might cause the difference in the amount of microbial communities or biomass per gram of alga as the temperature, pH and the abundance of nutrients may vary among different location and with different months of the year (Hellmann et al, 1996; Davis et al, 1964; Larsen et al, 2004; Besemer et al, 2007; Lyautey et al, 2005; MacNaughton et al, 1999; Sigler et al, 2002). In addition, the growth phase (the age) of the alga (Hunter et al, 2010; Larsen et al, 2004; Ercolani 1991; Hirano and Upper, 1989; Vanderhoff et al, 1981) influences the number of the microbial biomass. Since different growth phases of algae may excrete different kinds of exudates and considering that several types of algal exudates can increase the microbial rate (Murray et al, 1986), different algal phase can have different amount of microbial biomass.In addition, operator skill in handling the total microbial genomic may cause the difference in the amount of the DNA isolated from the alga.

The genomic DNA obtained in this project was of a good quality. The distribution of genomic sizes obtained (5-48 Kb) can be used to capture full genes and operon (Voget et al, 2003; Feng et al, 2007; Voget et al, 2006). The DNA insert size (with the average size of ~25 Kb) may also enable the recovery of genes for antibiotic biosynthesis since some antibiotic biosynthesis genes are between 5- <20 Kb in size (Xue et al, 1998; Martin and Gil, 1984; Schmidt and Donia, 2009). The genomic DNA also had a high ratio of bacterial DNA compared to eukaryotic DNA. This indicates that the metagenomic DNA extracted was largely of bacterial origin.

2.4.2. Metagenomic DNA coverage of the library expressed in two host cells The library of the metagenomic DNA of the microbial communities of U. lactuca in E. coli covered a total DNA of 3.75 Gb. The genome coverage in this project was smaller than that obtained in Henne et al, 1999 (5.6 Gb), but it exceeded the coverage from those of Angelov et al, 2009 (2.9 Mb); Courtois et al, 2003 (0.25 Gb) and Elend et al, 2006 (0.1 Gb). The library in S. lividans covered a total of 1GbDNA, which was achieved through a high transformation efficiency(4 x 10 5 transformant/ µg DNA), which was about ten times higher than that observed by Courtois et al (2003). The difference in the transformation efficiency might be due to the difference in vectors and in the transformation technique used in this project (PEG- assisted transformation) and that used by Courtois et al, 2003 (conjugation). This finding may suggest that pFD666 (Denis and Brzezinski, 1992) is a valuable cosmid for generating a metagenomic library in E. coli and S. lividans.

2.4.3. The U. australis microbial community as a source for antibiotics The microbial community of U. australis,a marine green macro-alga has been shown to be a potential reservoir for natural products, such as antibiotics (Yung et al, 2011; Kumar et al, 2011; Rao et al, 2007). E. coli (Gillespie et al, 2002; Schmidt and Donia, 2009; Brady et al, 2001)and S. lividans (Wang et al, 2000) have been shown to express genes encoding for antibacterial activities.The percentage of recovery in E. coli library (0.006%) and in S. lividans library (0.075%) was higher than those reported in Lim et al, 2005 (1.76 x 10-5) and Gillespie et al, 2002 (0.0001%): both were metagenomic libraries of soil microbial communities. T his indicates that the microbial communities of U. lactuca can be a good source for finding antibacterial clones. The higher percentage of expression of the antibacterial genes in S. lividans (0.075%) than that in E. coli (0.006%) may indicate that S. lividans as a host cell was able to provide the requirements for the expression of genes (such as codon preference, transcriptional factor and translational factor) better than E. coli. Several reports (McMahon et al, 2012; Grosjean and Fiers, 1982; Angelov et al, 2009; Martinez et al, 2004) have informed that the use of multiple host cells can increase potential gene recoveries, and this project further supported the theory of the superior ability of S. lividans in expressing genes for antibacterial activity.

Two clones containing pFD666- insert DNA with antibacterial activity against Shigella have been observed. One clone (SE11) was not characterized yet for its antibacterial gene because the transposon mutagenesis did not work for this clone. It is possible that the 37

antibacterialactivity is not only encoded by the DNA inserted into the cosmid, but also by the chromosomal DNA of the host cells. Thus, the phenotype may be resulted from the synergism activity of the DNA segment in the cosmid insert and the DNA segment in the host cells’ chromosome. The possibility that the phenotype is only encoded by the host cell’s chromosomes is not possible as both host cells did not inhibit the growth of Shigella in the absence of the cosmid construct. It may however be possible that a DNA segment inserted into the cosmid activates the host cells’ chromosome to express the phenotype. Furthermore, the failure to do the transposon mutagenesis in SE11 clone might be caused by the size of the gene which is very small so that there are not enough transposon mutants generated to hit it.There is no possibility that the activity was encoded by the host cell’s chromosome since re-transformation into different host cells showed antibacterial activity while the host cells alone (without the DNA insert) showed no antibacterial activity.

Another clone (SE9) has been characterized for the antibacterial gene via an In-vitro transposon mutagenesis and it was revealed that the transposon has been inserted into a sequence that shares a similarity to PmbA (thus in this project it is named as pmbA_1). The pmbA gene (Allali et al, 2002) has a function in cleaving the 26- amino- terminal leader peptide of pro-microcin during microcin maturation (Allali et al, 2002; Rodriquez Sainz et al, 1990). Microcin is an antibiotic with a mode of action by inhibiting DNA replication of target bacteria (Vizan et al, 1991), with several types of microcin have been shown to have antibacterial activity against Shigella and other Gram- negative bacteria (Cursino et al, 2006; Zschuttig et al, 2012; Eberhart et al, 2012; Milne et al, 1999).

GC percentage of an exogenous gene can affect its expression in a h ost cell (Welch et al, 2009). The nucleotide sequence of pmbA_1 has 62.8 % GC content and it was expressed in both E. coli and S. lividans. This is interesting since both host cells have different GC contents in their genomes. E. coli is a low GC content organism (40-51%) (Oliver and Marin, 1996; Gabor et al, 2004), while S. lividans has >70% GC content (Ohama et al, 1990). The ability of S. lividans in expressing the pmbA_1 gene is not surprising, because this host cell can express genes from either high GC or low GC organisms (for example from Chryseobacterium indoltheticum, a bacterium with 33.8% GC content) (McMahon et al, 2012; Gallego et al, 2006). The gene was also expressed in E. coli, possibly because microcin is produced naturally by some E. coli strains (Lavina et al, 1990; Poey et al, 2006), and thus the factors for the transcription and translation might be provided by the host cell. 38

Deletion of pmbA results in the absence of mature microcin in the supernatant of mutant growth media (Allali et al, 2002), and thus the compound cannot alter target bacteria. Since the pmbA -like sequence of SE9 (pmbA_1) was disrupted by the transposon, the loss of antibacterial activity can be caused from the lack of mature microcin inside the mutant clone. pmbA is located in the chromosomal DNA of microcin producing bacteria (Rodriquez-Sainz et al, 1990; Allali et al, 2002; Murayama et al, 1996), although the genes for its biosynthesis can be encoded in a plasmid (Baquero and Moreno, 1984) or in the genome (Lavina et al, 1990; Poey et al, 2006; Lagos et al, 2001; Grosdanov et al, 2004). The gene encoding for the microcin-processing enzyme has not been identified yet in the whole fosmid sequence of SE9 although the antibacterial activity was observed. As only the gene encoding for the maturation of microcin was identified in the shotgun sequencing data of SE9, there is a possibility that the E. coli EPI300 used in this project has got the gene encoding for the microcin biosynthesis; therefore, the presence of the pmbA gene may result in the maturation of the microcin and thus the antibacterial activity was observed in the assay.

2.4.4. The presence of a plasmid containing the antibacterial genes in the microbial community of U. australis Natural vectors are abundant in the environment and they may contain genes for a diverse range of functions, such as resistance to heavy metals, resistance to heat, resistance to antibiotics, antibiotic biosynthesis, virulence and others (Dahlberg, 1997; Sobecky et al, 1997; Thomas, 2005; Kobayashi and Balley, 1994; Zolgharnein, 2007). Most of the antibacterial clones obtained in this project (except for SE11 and SE9) were possibly encoded by natural plasmids. This is because an attempt to do end-sequencing and restriction digest to the vector were not successful. In contrast, the positive control (from pFD666 without DNA insert) and the SE9 cosmid were successfully end-sequenced. Additionally, restriction digestion was performed successfully in the SE11 cosmid, resulting in a cosmid backbone and several bands for the DNA inserts.

There is a possibility that a natural plasmid was isolated together with the microbial genomic DNA. It seems that the natural plasmid also contained a kanamycin resistance gene, because they were able to grow in plates supplemented with kanamycin 200 µg/ ml. The presence of a plasmid containing the antibacterial gene is not surprising as it is a kind of “weapon” used by the microorganism to suppress the growth of other microorganisms, possibly in response to the limitation of the attachment site to the U. australis and the limitation of the available 39

nutrients and also to prevent invasion from other microbes (Hibbing et al, 2010; Wiener, 1999). It seems that the natural plasmids also have the origin of replication in both host cells because they could replicate in both host cells. Further analysisof the plasmid sequences will provide information on the possible route of transfer of the plasmid.

2.4.5. Phylogenetic analysis of SE9 clone Phylogenetic analysis of the 16S rRNA gene revealed the clone SE9 was derived from a bacterium related to Microbulbifer epialgicus strain F (99% identity, 100% coverage). The sequence shared an identity of 96% and 95% coverage to the new Microbulbifer isolated from marine sediment (Wang et al, 2009). Compared to other Microbulbifer species with known 16S rRNA sequences, it showed between 94-97% identity and 93-98% coverage. Bosshard et al, 2003 i ndicated that micro-organisms are grouped into a genus if the 16S rRNA sequences share an identity of > 95% to >99%. Based on t his assumption, the bacterium identified in this project is a new bacterium belonging to the genus Microbulbifer.

2.4.6. Other genes identified in SE9 clone. The cosmid sequence of the clone SE9 covered 27.1 Kb of genomic DNA consisting of 26 open reading frames (ORFs). Several genes were identified, for example, the genes encoding for phosphatidylserine synthase. The protein catalyzes the synthesis of phospholipids and it is bound either in the bacterial ribosomes (Raetz and Kennedy, 1972) or the cellular membrane (Okada et al, 1994; Cousminer et al, 1982; Dutt and Dowhan, 1981, Hawrot and Kennedy, 1975). Genes for basic metabolism such as the gene encoding for the ketol-acid- reductoisomerase, the gene for the acetolactate synthase and the regulator for acetolactate synthase gene were also found.

Ketol-acid reductoisomerase, also known as acetohydroxy acid isomeroreductase, functions in valine and isoleucine biosynthesis (Tyagi et al, 2005). Acetolactate synthase (ALS), also known as acetohydroxy acid synthase (AHAS), plays a role in the biosynthesis of branched- chain amino acids valine and leucine(Tyagi et al, 2005; Duggleby and Pang, 2000; Pang et al, 2004). Thus, the bacterium was able to synthesize amino acids for its growth.

Genes encoding for proteins need for bacterial survival such as the LexA protein and the protein for resistance to some heavy metals such as Co, Zn and Cd were identified. The protein LexA works together with RecA in response to DNA damage. In the event of DNA 40

damage, RecA interacts with the LexA repressor to cause autocleavage of the LexA repressor and thus a r elease from the operator (Little and Mount, 1982). The autocleavage process induces the activation of SOS genes to produce enzymes for DNA repair, switching on the expression of the gene encoding for virulence factor (Bisogno et al, 2004; Mellies et al, 2007; Little, 1993) or the gene encoding for antibiotics (Kamensek et al, 2010; Justice et al, 2004; Lewis and Vulic, 2009). When DNA repair is complete, the production of RecA is switched off and the SOS system is repressed by LexA (LexA binds to its promoter).

The gene encoding for resistance to heavy metals (Co/ Cd/ Zn) was found in the genomic DNA fragment of SE9. These heavy metals are needed by bacteria in a sufficient amount and they play a role as coenzymes, cofactors, catalysts or as structural stabilizers of enzymes and DNA –binding proteins (Hugher et al, 1991; Xiong and Jayaswal, 1998; Huckle et al, 1993). However, an excessive intracellular amount can cause a poisonous effect to the bacteria (Beard et al, 1995; Kleiner, 1978).

To conclude, the functional screening of the metagenomic library of microbial communities of U. lactuca has successfully identified the presence of a gene encoding for microcin maturation. This gene seems to process the immature microcin coded by the host cell’s chromosome to produce mature microcin.

CHAPTER 3: PHYLOGENETIC STUDY OF METAGENOMIC LIBRARIES OF MICROBIAL COMMUNITY OF ULVA AUSTRALIS USING HOMING ENDONUCLEASE RESTRICTION AND MARKER INSERTION

3.1 INTRODUCTION The amplification of rRNA genes followed by the comparison of the gene sequence with the sequence references available in various databases is a powerful tool for the phylogenetic analysis of bacteria (Weisburg et al, 1991).

Fox et al (1980) have successfully applied the 16S rRNA gene sequence to establish the phylogeny of prokaryotes. 16S rRNA is an important gene for phylogenetic analysis because it is distributed universally in prokaryotes and it may have some highly conserved sequence or sequence variability among prokaryotes (Lane et al, 1985; Huysman and Wachter, 1986; Gray et al, 1984; Dams et al, 1988).

The 23S rRNA gene has also been used for studying bacterial phylogeny, because it has conserved and variable sequence areas, which make it useful for phylogenetic and taxonomic analysis (De Rijk et al, 1995; Pei et al, 2009). The 23S rRNA has a longer nucleotide length than the 16S rRNA and it contains unique insertions and or deletions (Ludwig et al, 1994). The 23S rRNA also contains conserved regions that permit the design of universal primers that cover a broad range of bacteria (Hunt et al, 2006). To gain more accurate bacterial phylogeny, both 23S rRNA and 16S rRNA can be used (Bavykin et al, 2004; Badger et al, 2005).

Though phylogenetic marker genes (16S and 23S rRNA) can be useful molecules for bacterial phylogeny, they do not provide any information on t he functional genes of the organisms. In contrast, metagenomics combined together with shotgun sequencing is an important technique to explore properties of genes from both cultured and “uncultured” microorganisms (Riesenfeld et al, 2004; Courtois et al, 2003; Richardson et al, 2002; Henne et al, 2000; Rondon et al, 2000; Knietsch et al, 2003; Gabor et al, 2004). However, limited success in read assembly of rich samples (Tringe et al, 2005) often results in functional genes being detached from the phylogenetic marker genes. As a result, the information of the phylogeny for functional genes (especially for the “unculturable”) cannot be provided.

A new screen for phylogenetic markers in metagenomic DNA, called as Homing Endonuclease and Restriction Marker Insertion (HERMI), has been developed by Yung et al, 2009. The technique employs restriction of metagenomic libraries with the homing endonuclease I-CeuI. The enzyme recognizes specifically the 26 bp of the conserved part of the intron-free 23S rRNA gene within the clone libraries, and then cut the region to produce a four nucleotide (CTAA) 3’overhang (Marshall and Lemieux, 1992). After digestion with the I-CeuI, a kanamycin cassette as the antibiotic marker for clone selection is inserted. The vectors construct containing the kanamycin cassette were then transformed into the E. coli host cell, followed by a process of clone selection on plates containing kanamycin. The use of HERMI in the library of the microbial community of the sponge Cymbastella concentrica (Yung et al, 2009) has resulted in the analysis of six HERMI clones revealing several potential genes with a defined phylogenetic origin.

In this project, HERMI was applied to reveal the phylotype-function relationships of the library of the U. australis microbial community. Overall, the aims are 1) to construct HERMI transformants of the library of the microbial community of U. australis; and 2) to study the phylogenetic and gene properties of the HERMI clone.

3.2. METHODS

3.2.1. Bacterial strains and media and culture conditions E. coli EPI300 [F- mcrA D(mrr-hsdRMS-mcrBC) f80dlacZDM15 DlacX74 recA1 endA1 araD139 D(ara, leu) 7697 g alU galK l- rpsL (StrR) nupG trfA tonA and DH5α [FendA1 glnV44 thi-1 relA1 gyrA96 deoR nupG lacZdeltaM15 hsdR17] strains were used in the experiment. Clones of the Ulva australis microbial community generated in pCC1FOS (Yung et al, 2010) were grown on the media supplemented with 12.5mg/ml chloramphenicol. The media used in the project were LB broth (1% tryptone, 1% NaCl, 0.5% yeast extract); LB containing 1.5% agar and SOC media (2% Bacto tryptone; 0.5% yeast extract; 0.01 M NaCl;

0,0025 M KCl; 0.01 M MgCl2; 0,01 M MgSO4; 0.02 M glucose). Both chloramphenicol (12.5 mg/ml) and kanamycin (50 mg/ml) were added for growth media for HERMI clones. The fosmid map of pCC1FOS is given in the figure3-1.

Figure 3-1.The map of pCC1FOS (Epicentre, Madison, WI, USA). The fosmid contains Ori2 and OriV for single copy and multicopy replication in E. coli host cell, respectively. The presence of Cos enables the construct of DNA insert and the fosmid to be packaged in lambda bacteriophage system. Antibiotic selection marker (Choramphenicol resistance gene) is present in the fosmid for clone selection. The picture was taken from the manufacturers manual (Epicentre, Madison, WI, USA).

3.2.2. Extraction of fosmid DNAs from the pooled library of an U. australis microbial community, and illustration of steps in performing HERMI The metagenomic library was pooled from all clones grown on t he transformation plates.Cells were then inoculated in a 50 ml Falcon tube of 10 ml of LB broth containing 12.5 mg/ml chloramphenicol, 0.01% arabinose or 20 µl of the 500 x copy control induction fosmid auto induction solution (Epicentre, Madison, WI, USA). The tube was then incubated at 37°C with shaking at 200 rpm, overnight, to induce the high copy number fosmid within the cells. After incubation, bacterial cells were pelleted by centrifugation at 4,500 rpm for 15 minutes.

The pooled fosmid DNAs were isolated using a QIAprep spin miniprep kit (Qiagen, Hilden, Germany) in a bench microcentrifuge. The isolation was conducted in accordance to the protocol provided in the kit.

The fosmid was then treated to generate HERMI clones. The diagram showing how HERMI works is provided in figure 3-2 below.

3.2.3. Homing Endonuclease Restriction and Marker Insertion (HERMI) The HERMI process in this experiment was performed as described by Yung et al. 2009, but with the following modification: One microgram of pooled fosmid DNAs of the U.australismicrobial community was digested with 2 units of I-CeuI enzyme (NEB).I-CeuI digestion products were then dephosphorylated using Antarctic phosphatase (New England Biolabs, Beverly, MA, USA). The kanamycin cassette (~1 Kb) was isolated from the pBHS- Kan by the alkaline lysis method (Sambrook and Russell, 2001) and it was then extracted from the gel using Gelase enzyme in accordance to the protocol provided by the manufacturer (Epicentre, Madison, Wisconsin).

Ligation for HERMI was carried out in a reaction of 1:30 vector/insert molar ratio using T4 DNA ligase (New England Biolabs, Beverly, MA, USA) at room temperature overnight.As a control, I-CeuI digested pBHS (has been dephosphorylated) was ligated to the kanamycin cassette as described in Yung et al, 2009. Ligation products (For HERMI and the control) were transformed into 50 µl of electrocompetent E. coli EPI300 (Epicentre, Madison, Wisconsin) using the Ec2 programme (2.5 kV, 2.0 m m cuvette) (Sambrook and Russell, 2001), in a Biorad micropulser (Biorad, Hercules, CA, USA).

HERMI clones were selected onto LB agar containing of 12.5 mg/ml chloramphenicol and 50 mg/ml kanamycin while the control was plated on L B agar supplemented with 50 mg/ml kanamycin and 100 mg/ml ampicillin. HERMI transformants that grew on the selective media were purified and isolated for the fosmid using the DNA QIAprep spin miniprep kit, QIAGEN, Hilden, Germany (performed in accordance to the manufacturer’s protocol). End sequencing of the fosmid clones was conducted as directed in section 2.2.8.

3.2.4. Phylogenetic analysis of the HERMI clones

Phylogenetic analysis of the HERMI clones was done by PCR amplification of the 23S rRNA gene followed by sequencing using universal primers for 23S rRNA (table 3-1) with a protocol described inHunt et al, 2006.The PCR amplification reaction contained 12.5 ml of EconoTaq plus green 2x master mix (Lucigen, Middleton, WI, USA), 20 pm ol of each primers (23S-129Fand 23S-2241R), 10 ng of fosmid DNA and sterile, ultrapure water in a total volume of 25 ml. Thermal cycling conditions for the PCR were 94°C, three minutes 47

(initial denaturation) followed by 30 cycles of 94°C, one minute ; 57°C, one minute ; 72°C, three minutes then final extension at 72°C, 10 minutes and finishing at 4°C. Amplicon of 23S rRNA was then purified using the QIAquick PCR purification microcentrifuge kit (QIAGEN, Hilden, Germany) in accordance to the manufacturer’s instructions.

Sequencing of the 23S rRNA gene was conducted using the big dye terminator v3.1 c ycle sequencing protocol. The reaction mixture was composed of 30 ng of the DNA, 20 pmol of either 23S-129F primer or 23S-2241R primer (table 3-1), 1 x big dye buffer, 1 ml big dye terminator v3.1 (Applied Biosystems, Carisbad, CA, USA) and sterile, ultrapure water in a total volume of 15 ml. The sequencing cycle conditions were: 96°C, one minute followed by 25 cycles of 96°C, 10 seconds; 50°C, five seconds; 60°C, four minutes and finishing at 4°C. The products were then cleaned and sent for sequencing as described in section 2.2.8.

The sequencing result was analysed using the Blastn (Altschult et al, 1997) program provided by the NCBI: it w as compared to NCBI’s nucleotide database. The phylogenetic tree was drawn using the Phyfi programme (Fredslund, 2006) online tool, from the Newick tree resulted via the distance tree of Blastp analysis from the National Centre for Biotechnology Information. The genetic distances for the phylogenetic marker gene (16S and 23S rRNA) and the annotated ORF were calculated using Jukes-Cantor method (Jukes and Cantor, 1969) and Kimura’s method (Kimura, 1983) respectively. The Newick tree was generated from the distance matrices using the fast minimum evolution (FastME) (Desper and Gascuel, 2002).

3.2.5. Analysis of the sequence of the flanking region of the kanamycin cassette The sequence flanking the kanamycin cassette was obtained by end sequencing using a mixture of 50 ng DNA template, 20 pmol of each primer (KanFSeq and Kan RSeq primer),

250 mM dNTPs, 1x Red Taq buffer (10 mM Tris-Cl, pH 8.3, 50 mM KCl, 1.1 mM MgCl2), one unit of Red Taq DNA Polymerase (Promega, Madison, WI, USA) and sterile, ultrapure water in a final volume of 20 ml. The PCR cycling conditions were 94 for three minutes, followed by 30 cycles of 94°C for one minute, 57°C for one minute, 72°C for three minutes with a final extension at 72°C, ten minutes. The resulting sequence was analysed for the similarity using the Blastn programme from the NCBI (Altschul et al, 1990) and the KEGG database (Kanehisa et al, 2012; Kanehisa and Goto, 2000).

3.2.6. Amplification of the kanamycin cassette and analysis of the sequence PCR amplification of the kanamycin cassette used a reaction mixture of 10 ng of DNA template, 20 pmol of each KanFCeu and KanRCeu primer (table 3-1), 12.5 ml Econotaq plus green 2x master mix (Lucigen, Middleton, WI, USA), and sterile, ultrapure water in a total volume of 25 ml. Thermal cycling conditions were 94°C, two minutes followed by 30 cycles of 94°C, 30 seconds; 53°C, 30 seconds; 72°C, one minute then a final extension of 72°C, 10 minutes and finishing at 4°C. Bioinformatic analysis: Blastp and Blastn was performed through the facility provided by National Centre for Biotechnology Information/ NCBI (Altschul et al, 1990). The primers used in the projects are listed in table 3-1.

Table 3-1: Primers used in HERMI analysis (Yung et al, 2009).

Primers’ name Sequence (5’-3’) epiFos F GGATGTGCTGCAAGGCGATTAAGTTGG

23S-129F CYGAATGGGRVAACC

23S-2241R ACCGCCCCAGTHAAACT

KanFSeq GACCGTTCCGTGGCAAAG

KanRSeq GCTCGATGAGTTTTTCTAATC

KanFCeu GTCCTAAGGTAGCGAGGTTGATGAGAGCTTTGTTG

KanRCeu AGGACCGTTATAGTTAAAGTCAGCGTAATGCTCTGC

3.2.7. Whole fosmid sequencing and analysis of genomic DNA of the HERMI clone To study gene properties of the HERMI clones, the entire fosmid of the HERMI clone was sent to the J.Craig Venter Institute for shotgun sequencing, with the procedure presentedin Rusch et al, 2007. Please refer to section 2.2.10 for more details on the analysis.

3.3. RESULTS 3.3.1. Construction of transformants via HERMI technique and clone characterization Eleven transformants were able to grow on LB agar plates supplemented with 12.5 mg/ml chloramphenicol and 50 mg/ml kanamycin. All transformants were then subjected to PCR amplification using the universal 23S primers. It was found that only one clone: B1 (figure 3.3, lane 2) amplified the 23S universal primers to produce the expected final amplicon of about 3.1 Kb (including the size of the kanamycin cassette). Other clones (B2-B11) showed a PCR product, which came from the E. coli background.

Figure 3-3: The PCR amplification of 23S rRNA gene using universal primers. Lane 1 and 11: 1 Kb DNA ladder (Fermentas Inc., Glen Burnie, MD, USA); lane 2: B1 clone, it produced around 3.1 Kb of a mplicon; lane 3 t o 10: B2 to B9 clone; lane 12 a nd 13: B10 and B11, respectively. B2-B11 produced ~2.1 Kb PCR product.

3.3.2.1. Clone B1 The data on figure 3-3 lane 2 indicated that B1 fosmid was able to amplify the 23S universal primers. Sequencing of B1 fosmid using 23S rRNA primers (23S-129F or 23S-2241R) and primers for the flanking region of the kanamycin cassette (KanFseq or KanRSeq primer) was performed and the whole sequences were assembled to form a nucleotide of 2843 bp i n length.

The nucleotide sequence was then analyzed for the nucleotide identity against the nucleotide collection from NCBI (table 3-2.).The analysis indicated that the nucleotide sequence of the B1 fosmid has approximately 89% identity and 100 % coverage to the 23S rRNA gene of Hyphomonas neptunium (table 3-2). The sequence similarity of the 16S rRNA obtained from

the shotgun sequencing (table 3-3) indicated that it shared >91% identity and >76% coverage to several 16S rRNA sequences of Hyphomonadaceae. Furthermore, the I-CeuI site in the fosmid was also identified from the sequencing of the region flanking the kanamycin cassette.

Accession Gene and organism Class %C %I E- number value CP000158.1 23S rRNA Hyphomonas Alphaproteobacteria 100 89 0.0 neptunium

CP001678.1 23S rRNA Hirschia baltica Alphaproteobacteria 96 86 0.0

EU795193.1 23S rRNA uncultured bacterium Not classified 96 84 0.0 HF0500_31I04

AP009384.1 23S rRNA Azorhizobium Alphaproteobacteria 96 84 0.0 caulinodans

CP000449.1 23S rRNA Maricaulis maris. Alphaproteobacteria 89 86 0.0

Table 3-3.Nucleotide similarity search of the 16S rRNA of B1 clone against the nucleotide collection of the NCBI. The accession number, percentage of coverage (%C), percentage of identity (%I), E- value, the name of the organism correspond to the gene as well as the Class of bacteria are presented.

Accession Gene and organism Class %C %I E- number value NR_024941.1 16S rRNA Hyphomonas Alphaproteobacteria 79 93 0.0 oceanitis

NR_044573.1 16S rRNA Maribaculum Alphaproteobacteria 79 93 0.0 marimum

NR_024939.1 16S rRNA Hyphomonas Alphaproteobacteria 79 92 0.0 hirschiana

NR_025325.1 16S rRNA Hyphomonas Alphaproteobacteria 77 93 0.0 polymorpha

NR_044345.1 16S rRNA Henriciella Alphaproteobacteria 77 93 0.0 marina

The phylogenetic tree (figure 3-4) indicated that the 23S rRNA sequence of B1 is closely clustered with the sequence of H. neptunium while the 16S rRNA sequence was related to the 16S rRNA sequences of Ponticaulis koreensis, Maribaculum marinum, and Henriciella marina(figure 3-5).

Figure 3-4. Maximum likelihood tree of the 23S rRNA gene of B1and the closest representatives of microorganisms from the nucleotide database (NCBI). Red box: 23S rRNA sequence of B1. The scale bar indicates 0.07 divergences of the sequences.

Figure 3-5. Maximum likelihood tree of the 16S rRNA gene of B1and the closest representatives of microorganisms from the 16S rRNA database (NCBI). Red box: 16S rRNA sequence of B1. The scale bar indicates 0.04divergence of the sequences.

3.3.2.2. Clone B2-B11 While B1 is the HERMI clone and has the 23S rRNA gene in its fosmid, the other clones (B2- B11) did not show the expected 3.1 Kb product (Figure 3.3., lane 3-10; 12 and 13). All clones were able to grow on kanamycin plates as their fosmids had a kanamycin resistance sequence. The flanking region of their kanamycin resistance gene sharesimilarity to those of beta lactamases, chloramphenicol acetyltransferase, transposase/resolvase, and beta-D- galactosidase (table 3.4.).

Clone Pri Gene, organism and % C % S Pri Gene, organism and %C %S mer accession number mer accession number B2 FP Beta-D-galactosidase, 98 98 RP Chloramphenicol 90 98 vector cloning pCC1FOS acetyltransferase cloning (EU140751.1) vector pCC1FOS (EU140751.1) B3 FP Transposon Tn3 resolvase, 76 87 RP Tn-1 resolvase plasmid RP4 100 96 shuttle vector pAY201 (AAA26419.1) (AB526842.1) B4 FP Beta –D-galactosidase vector 77 85 RP Chloramphenicol 88 94 cloning pCC1FOS acetyltransferase cloning (AB526842.1) vector pCC1FOS (EU140751.1) B5 FP Transposon Tn3 resolvase and 88 95 RP Transposon Tn3 resolvase 90 100 beta lactamase of shuttle and beta lactamase of shuttle vector pAY201 vector pAY201 (AB526842.1) (EU140751.1) B6 FP - - - RP - - - B7 FP Transposon Tn3 resolvase and 81 94 RP Transposon Tn3 resolvase and 96 92 beta lactamase of shuttle beta lactamase of shuttle vector pAY201 vector pAY201 (AB526842.1) (EU140751.1) B8 FP - - - RP Chloramphenicol 94 94 acetyltransferase of cloning vector pCC1FOS (EU140751.1) B9 FP Beta-D-galactosidase cloning 84 93 RP Chloramphenicol 87 96 vector pCC1FOS acetyltransferase of cloning (EU140751.1) vector pCC1FOS (EU140751.1) B10 FP Transposon Tn3 resolvase and 94 90 RP Aminoglycoside 98 100 beta lactamase of shuttle phosphotransferase and vector pAY201 transposon Tn3 resolvase of (AB526842.1) shuttle vector pAY201 (EU140751.1) B11 FP Transposon Tn3 resolvase of 68 93 RP Region of cloning vector 96 97 shuttle vectorpAY201 pHUGE-LjMtNFS (AB526842.1) (JN874483.1)

Pri Pri Gene, organism and accession Clone Gene, organism and accession number % S %S mer mer number Chloramphenicol acetyltransferase Beta-D-galactosidase Shigella sonnei B2 FP 98 RP Acinetobacter baumannii 96 (SSON53_01755) (abn:AB57_0280) Transposase, IS4 family protein Transposon Tn3 resolvase B3 FP Shewanella sp. 94 RP Acinetobacter baumannii 95 (shw:Sputw3181_0433) (abn:AB57_0282) Beta-D-galactosidase Chloramphenicol acetyltransferase B4 FP Escherichia coli O157:H7 EDL933 91 RP Acinetobacter baumannii 94 (ece:Z0440) (abn:AB57_0280) IS102 transposase Transposon Tn3 resolvase B5 FP Citrobacter rodentium 94 RP Acinetobacter baumannii 99 (cro:ROD_p2471) (abn:AB57_0282) B6 FP - - RP - - Transposon Tn3 resolvase IS102 transposase Citrobacter rodentium B7 FP 93 RP Acinetobacter baumannii 100 (cro:ROD_p2471) (abn:AB57_0282) Chloramphenicol acetyltransferase B8 FP - - RP Acinetobacter baumannii 97 (abn:AB57_0280) Chloramphenicol acetyltransferase Beta-D-galactosidase Shigella sonnei B9 FP 96 RP Acinetobacter baumannii 94 (ssj:SSON53_01755) (abn:AB57_0280) IS102 transposase Transposon Tn3 resolvase B10 FP Citrobacter rodentium 94 RP Acinetobacter baumannii 97 (cro:ROD_p2471) (abn:AB57_0282) Transposase, IS4 family protein Transposon Tn3 resolvase B11 FP Shewanella sp. 94 RP Acinetobacter baumannii 95 (shw:Sputw3181_0433) (abn:AB57_0282)

The data of the analysis of the fosmid sequences of clone B2-B11 (table 3-4) revealed that the three clones (B2, B4, and B9) were actually sister clones. B2-B11 clones did not possess the 23S rRNA which was disrupted with the kanamycin cassette/ kanamycin resistance gene, but they can grow on t he transformation plates of LB agar containing 12.5 mg/ml chloramphenicol (the antibiotic used for transformant selection in pCC1FOS system, Epicentre, Madison, WI, USA) and 50 mg/ml kanamycin. Their ability to grow in plates supplemented with kanamycin was possibly caused by the presence of a natural kanamycin resistance gene in the fosmid insert DNAs.

The presence of the natural kanamycin resistance gene on the three fosmids was supported by three lines of evidence. First was by the success of the sequencing of the flanking region of the kanamycin resistance gene using a KanRSeq or KanFSeq primer (as seen in table 3-4). Second was by the ability to amplify the kanamycin resistance gene using KanFCeu and KanRCeu (primer for the amplification of the kanamycin cassette). The PCR amplification resulted in a fragment of about 1 Kb (the size of the inserted kanamycin cassette) (figure 3-6, lanes 3 to 12).

Figure 3-6.The PCR amplification of kanamycin cassette.Lane 1: 1 Kb DNA ladder (Fermentas Inc., Glen Burnie, MD, USA). Lane 3-12: Kanamycin amplicon of fosmid B2-B11. All fosmid amplified KanFCeu and KanRCeu to result in a 1 Kb amplicon.

Third was the difference in sequence of the kanamycin resistance gene of the three fosmids compared to the sequence of the kanamycin cassette (pBHS-Kan). The sequence similarity analysis via blastn programme of the NCBI showed that the kanamycin resistance sequence of the fosmids of B2, B4 and B9 shared 20-96% similarity and 16-84% coverage to the

sequence of the kanamycin cassette from pBHS-Kan. Clones B3, B5, B6, B7, B8, B10 and B11 have kanamycin resistance genes with sequence similarities between 94% and 99% and coverage of 65% to 83% to the inserted kanamycin (pBHS-Kan) sequence. Sequence difference below 1% might be due to sequencing error (Hoff, 2009). However the higher sequence difference observed here make it likely that natural kanamycin resistance genes have been recovered in this project.

To sum up, one HERMI clone (B1) was obtained. The clone’s fosmid has the kanamycin resistance cassette originated from the pBHS-Kan as well as the phylogenetic marker genes. In contrast, B2-B11 clones obtain the kanamycin resistance genes from the natural environment and did not possess the phylogenetic marker gene. Therefore, B2-B11clones were not HERMI transformants.

3.3.3. Whole genome sequencing of fosmid B1 To study the gene properties of the HERMI clone, the B1 clone’s fosmid was completely sequenced and a total of 18 op en reading frames in a 20 K b insert (appendix 2) was recovered. About 22.2% of the ORFs were annotated as a hypothetical protein. The annotated proteins had similarities to sequences belonging to several class of bacteria such as the class of Alphaproteobacteria (71.4%), Betaproteobacteria (7.1%), Gammaproteobacteria (14.2%) and another 7.3% of unclassified bacteria. In addition, two ORFs had no m atches in the NCBI, EBI, KEGG and Pfam databases (table 3-5).

The whole sequencing of fosmid B1 revealed the presence of genes for 5S rRNA, 23S rRNA as well as for 16S rRNA that showed a similarity to the phylogenetic marker genes of bacteria within the Class Alphaproteobacteria. The recognition site for the I-CeuI cutting point was discovered in position 17,280-17,306 bp a nd in position 17,939-17,963 bp. T he schematic diagram of the position of each phylogenetic marker gene is provided in figure 3.7.

Table 3-5. Annotation of the open reading frames of clone B1.Where possible, naming of the genes was performed according to the Swiss Protein database. If no information was available under the Swiss Protein database, then naming of the genes was done according to the NCBI database. (-): no significant hit. The dark shaded box indicates the e-value was higher than 1 x 10 -20 under Swiss Protein database (with an e-value of 1 x 10-5 as the cut-off). The light shaded box indicates the e-value is less than 1 x 1 0 -20 under Swiss Protein database. For detail annotation, please refer to appendix 2.

Location (bp) Orien- Name of protein or gene Taxonomic assignment (class- tation level) based on blast hit 7-465 Minus Glycolate oxidase, iron sulphur sub unit Alphaproteobacteria 470-649 Minus Glycolate oxidase FAD binding sub unit Alphaproteobacteria 806-1333 Plus Endopeptidase ClpP Alphaproteobacteria 1391-1981 Plus Histidine kinase Alphaproteobacteria 2059-3690 Plus Hypothetical protein Alphaproteobacteria 3691-4254 Minus Hypothetical protein Alphaproteobacteria 4402-5316 Plus Ton B-dependent receptor Alphaproteobacteria 5721-8462 Plus Hypothetical protein Uncharacterized bacteria 8524-8904 Minus Holdfast attachment protein hfaD Alphaproteobacteria 9037-10296 Minus Holdfast attachment protein hfaB Alphaproteobacteria 10299-11324 Minus Cystathionine beta-synthase Alphaproteobacteria 11317-11745 Minus Putative uncharacterized protein Beta Proteobacteria 11987-12418 Plus - - 12756-14813 Plus 16S rRNA gene - 15728-15913 Minus Putative cell wall-associated hydrolase Gammaproteobacteria 15383-18928 Plus 23S rRNA gene - 18321-18779 Minus - - 19318-19398 Plus 5S rRNA gene - 19658-19987 Plus Mg 2+ transporter protein Gammaproteobacteria

Analysis of the annotation of the genes of B1 revealed the presence of a structural gene encoding for proteins for the production of extracellular polysaccharides that function in adhering to surfaces, such as host tissues and non-biological surfaces. T he genes are clustered in a complete operon of hfaABD (Toh et al, 2008; Janakiraman and Brun1999; Kurtz and Smith, 1992). T he protein sequences of HfaA had 50% similarity and 100% coverage to the HfaA protein sequence of Hirschia baltica (YP_003059050.1). Additionally, the HfaB protein sequence showed 71% similarity and 93% coverage to the HfaB protein sequence of H.baltica (YP_003059049.1). The HfaD protein was of 26% similarity and 92% coverage to the HfaD protein of Oceanicaulis alexandrii (ZP_00958003.1).

A complete operon of glcDEF encoding for proteins for the oxidation of glycolate into glyoxylate (Hansen and Hayasi, 1961) was detectedtoo. The glycolate oxidase F shared 78% similarity with 97% coverage to the protein sequence of Maritimibacter alkaliphilus (ZP_01012214.1), while the glycolate oxidase sub unit D and E has more than 65% similarity to those ofAurantimonas manganoxidans (ZP_01227860.1), Rhodospirilum centenum (TR:B62U02_RHOCS) and manganese oxidizing bacterium strain S185-9A1 (TR:Q1YH39_MOBAS).

A gene encoding for adenine phosphoribosyl transferase that is involved in the formation of adenosine-5-monophosphate/AMP from adenine and 5-phosphoribosyl-1-pyrophosphate (Alfonso et al, 1995; Park and Taylor, 1988) was identified. The conserved region was positioned in amino acid 1-175. The sequence has more than 60% similarity to the adenine phosphporibosyl transferase of four species of bacteria (Maricaulis maris, Bradyrhizobium sp, Nitrobacter winogradskyi and Nitrobacter sp) - all are grouped in the Alphaproteobacteria class.

A gene encoding for protein whose use is in responding to environmental changes (histidine kinase) was present. The conserved domain for histidine kinase was found in the position of amino acid 150 to 200. The histidine kinase-like ATPase domain was observed in amino acid 270-375. Clustal 2.1 a nalysis of B1 histidine kinase protein sequence compared to several selected sequences shows the presence of conserved regions of kinase domain (N, G1, G2, G3 and F) and conserved region of H box (Kim and Forst, 2001; Kofoid and Parkinson, 1988; Stock et al, 1988; Garzon and Parkinson, 1996; Robinson and Stock, 1999).The H-box domain contained the histidine site of phosphorylation. In contrast, N, G1, G2, G3 and F were the ATP-binding kinase domain. Based on the conserved amino acid contained in the H-box domain, the histidine kinase of B1 belongs to the histidine kinase subtype I.

Figure 3-8. Multiple protein sequence alignments of the histidine kinase identified from B1 with its closest representatives according to the NCBI database. M. maris: Maricaulis maris, O. alexandrii: Oceranicaulus alexandrii, P. bermudensis: Parvularcula bermudensis and H. neptunium; Hyphomonas neptunium. S7.5: the histidine kinase of B1. Accession numbers of the protein sequences are shown next to the organism to which the protein sequence belongs. Red letter was the conserved amino acid for each domain.Code for the column: (*): column of the alignment contains identical amino acid residues in all sequences. (: ): column of the alignment contains conserved substitution of amino acids. (.): column of the alignment contains semi conserved amino acids.

Another gene encoding for protein for uptake of vitamin B12, iron and maltodextrins, named as Ton-B-dependent receptors (Blanvillain et al, 2007) was also observed. The Ton-B- dependent/ ligand-gated channels domains were present in amino acid position of 75-400 and 650-913. The best match of the protein sequence Ton-B-dependent receptors of B1 was to the protein of Sphingopyxis alaskensis (38% similarity and 97% coverage).

CorA gene encoded for protein transport (Mg2+ transporter protein) was also identified. The protein encoded by CorA has a function in the transport of Mg2+ across the membrane cell. In bacteria, there are two types of Mg2+protein transports, i.e. CorA or MgtE in which CorA is the main supplier for Mg2+ (Essenwine et al, 1995; Smith et al, 1995). The CorA protein sequence showed homology of 51% similarity and 98% coverage to the Mg2+ transporter protein (CorA family protein) of Psychromonas ingrahamii (YP_944135.1).

The gene encoding for transcriptional regulators CBS (named after cystathione beta synthase) domain protein was present in B1. The protein have very divergent functions, such as for metabolic enzymes, transcriptional regulators (in cystathionine beta synthase), binding site for adenosyl groups (AMP, ATP or s-adenosylmethionine), and ion channels and transporters (Ignoul and Eggermont, 2005; Ono et al, 1988). The CBS conserved domain was found in the amino acid position 12-124. The best match for the CBS protein sequence was to the correlated sequence of Hyphomonas neptunium (TR: Q0BYV1_HYPNA) with 59% similarities and 74% positives.

A gene encoding for putative cell wall-associated hydrolase was identified in the fosmid. The protein sequence of cell wall hydrolase obtained in B1 shared a high similarity of over 75% and more than 80% coverage to the same sequences of Brucella abortus (ZP_05822976.1), Brucella suis (ZP_05839398.1), Roseobacter sp. (ZP_01900972.1), and Haemophylus haemoliticus (TR:F9GFT8_HAEHA).

3.4. DISCUSSION The application of Homing Endonuclease Restriction and Marker Insertion (HERMI) on the metagenomic library of the microbial community of U. australis has resulted in one HERMI clone (namely B1). The HERMI application has also resulted in ten other clones (B2-B11) containing natural kanamycin resistance genes. The HERMI clone (B1) has the kanamycin cassette (pBHS-Kan), and the fosmid data revealed the presence of a s et of phylogenetic marker genes (5S rRNA, 16S rRNA and 23S rRNA), as well as the recognition site of I-CeuI.

3.4.1. The specificity of the recognition site of I-CeuI I-CeuI is a homing endonuclease, which specifically recognizes and cuts the 26 bp conserved region of the intron free 23S rRNA to produce a four-nucleotide 3’overhang (CTAA) (Marshall and Lemieux, 1992). The enzyme has been used to determine the number of rrn operons in bacteria using Pulsed Field Gel Electrophoresis/ PFGE (Wulff et al, 2008; Toda and Itaya, 1995; Rakin and Heesemann, 1995; Liu et all, 1993).

I-CeuI is used in generating HERMI clones in Yung et al (2009) and in this project. Yung et al (2009) has reported the specificity of the enzyme in detecting and cutting the 23S rRNA within three HERMI transformants obtained in their experiment. This project also identified the specificity in the enzyme recognition and cut in fosmid B1. On the other hand, the clones obtained in this project (clone B2-B11) did not possess the 23S rRNA genes, so therefore the fosmids were not digested by the I-CeuI and as a result no kanamycin cassette (pBHS-kan) was inserted into the fosmids.

3.4.2. The coverage of HERMI clone in this project The application of I-CeuI in generating HERMI transformants has resulted in four unique phylogenetic clones recovered from a total of 6,500 clones (Yung et al, 2009). In contrast, one HERMI clone out of 59,000 clones was obtained in this project. Technical errors in generating HERMI clones, such as sub optimal vector/ insert molar ratio as well as other technical issues such as the incomplete digestion (caused by old enzymes, etc.) of the fosmids by the I-CeuI might cause the low coverage.

The high number of false positive clones obtained in this project may indicate that the HERMI system (using the antibiotic resistance as the selectable marker) is not specific enough to capture clones containing the phylogenetic gene. This system is impaired to 64 recover phylogenetic clones from environments rich of natural antibiotic resistance genes. To increase HERMI sensitivity, the use of a non-antibiotic resistance gene (for example the gene encoding for fluorescence) as the selection marker is suggested.

3.4.3. HERMI as a tool to study the phylogenetic and potential functions of uncultured microorganisms HERMI application in the metagenomic DNA library of the microbial community of U.australishas provided an insight of the phylogenetic origin and functions of an uncultured bacterium. Based on the sequences of the 23S rRNA, the genomic insert was coming from an uncultured bacterium with a high identity of 89% and 100% coverage to the Hyphomonas neptunium, a Gram negative marine bacterium that often causes biofouling in a marine environment (Baier et al, 1983; Quintero and Weiner, 1995). The closest identity of the 16S rRNA of B1 was to the 16S rRNA sequence of Hyphomonas oceanitis (79% identity), which means that it is distantly related to known 16S rRNA sequences. Therefore, a new, unculturable microorganism was identified in this project.

Several interesting genes and functions were identified as potential properties of the new, uncultured micro-organism (B1). Firstly, it was predicted that the bacteria is able to attach to surfaces, including the U. australis’ surface. This is because it had an operon hfaABD which encodes for proteins to synthesize extracellular adhesive polysaccharide (Quintero et al, 1998; Smith et al, 2003; Toh et al, 2008). The extracellular adhesive polysaccharide plays a role in the attachment of the bacterial cell to different substrates or surfaces (Kurtz and Smith, 1992). T his attachment may be important to the bacterium because it f acilitates the absorption of any needed nutrients from the algae as well as protection to the bacterium from being washed away by ocean waves. In addition to the attachment function, the extracellular polysaccharide may provide protection against environmental stress (osmotic pressure, alkalinity, antibiotics, and host immune responses) (Costerton et al, 1999; Singh et al, 2006; Ahimou et al, 2007).

The presence of genes (glc DEF) encoding for glycolate oxidasein B1 clone may indicate that the bacterium absorbs glycolate from the alga. The glycolate could then be converted into glyoxylate by the enzyme glycolate oxidase (Lord, 1972; Sallal and Nimer, 1989). The energy resulted from this compound conversion could then be used by the bacterium for its 65

metabolism and growth (Hansen and Hayashi, 1961; Pellicer et al, 1996; Lord, 1972). This finding indicated that the bacterium prefers to live in the alga because the alga normally secretes glycolate from its photorespiration (Paver and Kent, 2010).

The gene encoding for TonB-dependent receptor was also present in the B1 fosmid insert DNA. The protein functions in the active transport of iron siderophore complexes in bacteria (Blanvillain et al, 2007). Bacteria transport the iron siderophore complexes as iron is needed for several processes such as respiration, nitrate reduction, nitrogen fixation, and detoxification of oxygen radicals or for chlorophyll synthesis in photosynthetic bacteria (Tortell et al, 1999; Guan et al, 2001). Additionally, the binding of iron into siderophore by bacteria converts the insoluble stable Fe3+- oxides into a more soluble form (Guerinot and Yi, 1994; Tortell et al, 1999). The soluble iron is then captured by the alga because it is used as a co-factor in the algal photosynthesis systems (photosystem I and II, the cytochrome b6/f

complex and cytochrome c6)(Raven, 1988; Singh et al, 2003; Vassiliev et al, 1995). The presence of two genes (glc DEF) and the gene encoding for TonB-dependent receptor may indicate that the bacterium lives in the alga as a mutualistic symbiont, with both parties obtaining benefits from the interaction.

The bacterium possessesthe gene encoding for histidine kinase subtype I.Histidine kinase plays a role in the adaptative response of bacteria to environmental stimuli. Under certain stimuli, the conserved histidine residue of the dimerick histidine kinase proteins can undergo transautophosphorylation (Dutta et al, 1999). The phosphoryl groups are then transferred to an Asp residue in the receiver domain of the response regulator protein, leading to the expression of target genes or cellular behavior such as swimming motility, chemotaxis, production of exocellular scavenging enzymes and antibotics, proteolysis regulation, and sporulation (Hoch and Silhavy, 1995; Msadek et al, 1998; Mascher et al, 2006; Hoch and Silhavy, 1995; Msadek et al, 1998; Dutta et al, 1999; Suzuki et al, 2005) to keep bacterial survival.

The gene encoding for cell wall hydrolases was present in the fosmid. Cell wall hydrolases are divided into four different groups based on the substrate specificity, i.e. the lysozymes/ lytic transglycosylases, which hydrolyse the β-(1,4)-glycosidic linkage between N- acetylmuramic acid and N-acetylglucosamine; the endopeptidases, which degrade the peptide bonds in the amino acid side chains connecting the parallel glycan strands; the 66

carboxypeptidases, which hydrolyse the C-terminal amino acids of peptide chains; and the amidases, which cleave between N-acetylmuramic acid and the first residue (L-Ala) of the peptide side chain (Haiser et al, 2009; Vollmer et al, 2008). These proteins work together with cell wall synthases for regulating bacterial growth and daughter cell separation (Vollmer et al, 2008). They also function in autolysis to remove damaged cells from the population (Rice and Bayles, 2003). The ability of cell wall hydrolases to attack components of the peptidoglycan of the bacterial cell wall (Haiser et al, 2009; Vollmer et al, 2008) may have a function in attacking the cell wall of gram-positive bacteria (Parisien et al, 2008; Dhalluin et al, 2005). Therefore, its production by the bacterium may also increase its competition against gram-positive bacteria in the communities.

In conclusion, HERMI application has resulted in the success in recovering and studying the gene properties of a new, uncultured bacterium.

CHAPTER 4

NATURAL ANTIBIOTIC RESISTANCE GENES IN METAGENOMIC LIBRARY OF ULVA AUSTRALIS

CHAPTER 4: NATURAL ANTIBIOTIC RESISTANCE GENES IN METAGENOMIC LIBRARY OF ULVA AUSTRALIS

4.1 INTRODUCTION Antibiotics have been used worldwide for treating infectious diseases, promoting growth or improving feed in farming and agriculture (Vidaver, 2002). However there is a growing concern that their excessive use in clinical settings and in farming/ agriculture is supporting the emergence and spread of antibiotic-resistant bacteria (Perreten et al, 2005; Levy, 1992; Weigel et al, 2003; Tenover et al, 2004; Walsh et al, 2003; Blanc et al, 1999; Levy et al, 1988; Wang et al, 2001; Sundeand Norstrom, 2006; Cortes et al, 2011).

Besides the overuse of antibiotics in both clinical and agricultural settings, the emergence of antibiotic resistance is also a result ofthe fact that antibiotic-producing bacteria in nature often possess genes for antibiotic resistance (Hopwood, 2007; Tahlan et al, 2007). These resistance genes can be transferred to other bacteria in the environment by transposons, integrons (Levesque et al, 1995; Stokes and Hall, 1989), plasmids or by natural competence (Manson et al, 2004; Bischoff et al, 2005; Nezbo et al, 2005). The transfer systems allow mobilization of the antibiotic resistance gene to bacteria of the same or different species (Andersen, 1993; Kruse and Sorum, 1994; Silva et al, 2006; Chaturvedi et al, 2008; Salyers et al, 2004; Zhang et al, 2011, Allen et al, 2010). Additionally, the resistance elements are easily dispersed into different environments by physical (wind, water) and biological forces (humans, animals, insects) (Allen et al, 2010).

The study of novel antibiotic resistance genes sourced from natural environments may increase our understanding of the mechanisms and of the origin of antibiotic resistance genes. Several techniques have been used to study antibiotic resistance genes from the natural environment, including techniques such as PCR (Knapp et al, 2010) and metagenomics, which were applied to activated sludge, soil, and river (Mc.Garvey et al, 2012; Hernandez et al, 2012; Mori et al, 2008; Donato et al, 2010; Cortes, 2011; Canton, 2009; Hellweger et al, 2011; Girlich et al, 2010). Novel antibiotic resistance genes have been recovered from these approaches, including genes coding for beta-lactamases (Hernandez et al, 2012), aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases (Mc.Garvey et al, 2012), and tetracycline resistance proteins (Sunde and Norstrom, 2006; Mc.Garvey et al, 2012; Knapp et al, 2010). 69

In this chapter, the identification of antibiotic resistance genes from a metagenomic DNA library of the microbial community of U. australis will be presented. As described in chapter 3, the sequence analysis of several clones generated from the Homing Endonuclease and Restriction Marker Insertion (HERMI) (table 3-3) indicated the presence of antibiotic resistance genes and transposons originated from the natural environment. Overall, the aims of this chapter are: 1) to functionally determine the growth activity of clones B3, B5, B6, B7, and B10 in growth media supplemented with several different concentrations and types of antibiotics; 2) to obtain the gene sequence responsible for the antibiotic resistance via the generation of transposon mutants and 3) to study the gene properties of the clones through shotgun-sequencing of the fosmids.

4.2. METHODS 4.2.1. Bacterial strains and media and culture conditions Growth media used for culturing all antibiotic-resistant clones was Luria Bertani (LB) containing 1% tryptone, 1% NaCl, 0.5% yeast extract, supplemented with 5% agar (LBA), 12.5 µg/ ml chloramphenicol and 100 µ g/ml kanamycin. Penicillin G, tetracycline and trimethoprim were used as indicated below (please see section 4.2.2.).

Electrocompetent Escherichia coli EPI300 [F- mcrAD(mrr-hsdRMS-mcrBC) f80dlacZDM15 DlacX74 recA1 endA1 araD139 D(ara, leu) 7697galUgalKl- rpsL (StrR) nupGtrfAtonAand DH5α [FendA1 glnV44 thi-1 relA1 gyrA96 deoRnupGlacZdeltaM15 hsdR17] was used for generating mutants which lost the ability to grow in media containing the suitable antibiotics. The transposon kit used was EZ-Tn5Insertion kit (Epicentre, Madison, Wisconsin). The kit uses tetracycline as the antibiotic for transformants selection. Therefore, mutant transformants were cultured on LB agar containing tetracycline (10µg/ ml), chloramphenicol (12.5 µg/ ml) and kanamycin (100 µg/ ml) as the selectable markers.

4.2.2. Resistance analysis and the minimum inhibitory concentration (MIC) determination of the clones in media containing antibiotics One loop of each antibiotic-resistant clone was streaked on a 35mm x 10 mm plate of LB agar supplemented with antibiotics (penicillin G or trimethoprim or tetracycline) to a final concentration of 6.25, 12.5, 25, 50, 100, 200, 400, and 800 µg/ ml. The plates were incubated at 37°C for 24-48 hours and observed for the presence/ absence of the bacterial growth. The clone was considered as an antibiotic-resistant clone if the growth was observed in all triplicates. As the first control, the electrocompetent E. coli was also streaked in plates containing the tested antibiotics and the assay showed its sensitivity to all the antibiotics. The second control was performed using B2 clones containing the pCC1FOS (with chloramphenicol resistance gene) and natural kanamycin resistance gene.

The minimum inhibitory concentration was performed in accordance to the standard method used in Donato et al, 2010, with modification in the antibiotics concentration. Briefly, serial dilutions at two fold concentration increase (50, 100, 200, 400, 800, 1,600 µg/ ml) of penicillin G or trimetophrim were made in Luria Bertani. For tetracycline, the concentrations

used were 6.25, 12.5, 25, 50, 100, a nd 200µg/ ml. The assay was conducted in 96-well plates with a number of ~1 x 105 CFU of the antibiotic-resistant clone. As a control (for the sterility of media and antibiotics), broth media containing each concentration of the antibiotics (without the inoculated bacteria) were added to the 96-well plate. Another control (to assess the bacterial ability to grow in the media) was done by inoculating the bacteria into the media (without antibiotics). All 96-well plates were incubated at 37°C, 125 rpm, for 24 hours. The minimum inhibitory concentration was determined from the lowest concentration of the corresponding antibiotic that showed no cell growth in all triplicates.

4.2.3. Genetic characterization and whole sequence analysis of the antibiotic resistance gene via transposon mutagenesis Clones able to grow in media supplemented with antibiotics (on section 4.2.2.) were considered as antibiotic-resistant clones. The antibiotic resistance genes in the fosmid inserts were then identified by generating knockout mutants of the fosmids using the EZ-Tn5Insertion kit (Epicentre, Madison, Wisconsin). The transposase reaction was then transformed into electrocompetent E. coli (sensitive to 10 µg/ml tetracycline) via electrophoration (Sambrook and Russell, 2001). The transformants were selected on plates of LB agar supplemented with chloramphenicol (12.5 µg/ ml), kanamycin (100 µg/ ml) and tetracycline (10µg/ ml).

Mutant fosmids sensitive to 200 µg/ml penicillin G or 400 µg/ml trimethoprim or 50 µg/ml tetracycline were then isolated using the Illustra miniprep plasmid isolation kit (GE Healthcare, Piscataway, New Jersey). The end sequenced analysis was conducted using TET- 1 FP-1 forward primer (5’-GGGTGCGCATGATCCTTAGAGT-3’) and TET-1 RP-1 reverse primer (5’-TAAATTGCACTGAAATCTAGAAATA-3’) in accordance to the protocols provided by the company (Epicentre, Madison, Wisconsin).

Phylogenetic analysis was performed using PhyML programme as well as Blast Explorer provided inhttp://www.phylogeny.fr/ (Dereeper et al, 2008; Dreeper et al, 2010). Multiple sequence alignment was conducted via MUSCLE 3.7 pr ogram. Gblocks was used in the alignment to eliminate poorly aligned regions and divergent regions. Then, the phylogeny was analysed via PhyML 3.0 a LRT (using maximum likelihood) and the maximum likelihood tree was viewed using the TreeDyn programme.

Sequencing results were searched for the open reading frames (ORFs) via Fgenesb programme provided by Softberry (http://www.softberry.com). Every putative ORF was searched against the NCBI- non redundant protein database (Pruitt et al, 2007; Altschul et al, 1990), the curated UniProtKB/Swiss-Prot, the non-curated UniProtKB/TrEMBL (Donovan et al, 2002), and the KEGG database (Kanehisa et al, 2012; Kanehisa and Goto, 2000) using the blastp program. A particular function was assigned to a protein sequence that had an e- value of 1x 10-20in the Swiss Protein database; or had the same annotations in two or more databases, if the e- value from the Swiss protein database was higher than 1x 10-20 (with an e- value 1 x 10-5 as the cut -off point).

The whole sequences of fosmid of the antibiotic-resistant clones were sequenced by J.Craig Venter Institute (Rusch et al, 2007) as described in section 2.2.10. The ORFs analysis was conducted in the same manner as described in 3.2.12.

4.3. RESULTS 4.3.1. Analysis of growth and minimum inhibitory concentration (MIC) of the antibiotic- resistant clones Antibiotic resistance against penicillin, tetracycline and trimethoprim was determined for clones B3, B5, B6, B7 and B10 to understand if they encode for one or multiple resistances. All clones were resistance to penicillin G (200 µg/ ml) or trimethoprim (400 µg/ ml), but were sensitive to all tetracycline concentration tested (table 4-1). The MIC of penicillin G and trimethoprim in all clones was at 400 µg/ ml and 800 µg/ ml respectively.

There was no resistance cassettes for penicillin G and trimethoprim in the control containing the empty fosmid pCC1FOS (figure 3-1.). This may indicate that the resistance to both antibiotics might be encoded by the insert DNA fosmids.

4.3.2. Identification of the gene responsible for the antibiotic resistance activity To identify the genes responsible for the antibiotic resistance, the fosmid of each clone was isolated and used in in-vitro transposon mutagenesis using the EZ-Tn5Insertion kit (Epicentre, Madison, WI, USA).

No mutant lost the ability to grow in trimethoprim-supplemented media. However, mutants for five clones (B3, B5, B6, B7, and B10) that lacked the ability to grow in penicillin G supplemented media were obtained.

4.3.2.1. Clone B3 Transposon mutagenesis of clone B3 yielded one mutant (B3C12) that lost the ability to grow in 200 µg/ ml penicillin G. The fosmid was then sequenced with TET-1 FP forward primer and TET-1-RP-1 reverse primer. An 800 bp (from here on referred to as ORF1) was identified from nucleotide position 10-810 of the forward sequence and another ORF (namely ORF2) encoding for 50 amino acids long (nucleotide position 1-153) was found in the reverse sequence (see figure 4-1).

Figure 4.1.Schematic diagrams of the ORFs of B3 mutant (namely B3C12). The direction of the primers is shown by arrows. ORF2: unknown function (Unk), 50 amino acids. ORF1: beta-lactamase (Bel1), 266 amino acids. T: transposon insertion site. Grey line: intergenic region.

The protein sequence encoded by ORF1 contained a conserved beta-lactamase domain (with an e-value 1.3 x 10 -38) at amino acids position 49-247, according to the Pfam database. Searches against the non-redundant protein of the NCBI revealed that the protein sequence had 98% for similarity and coverage to a TEM beta-lactamase 197 from Shigella sonnei (accession no. AFE48833.1). The sequence similar to the S. sonnei TEM beta-lactamase did not result from contamination since no work with them was performed at the time of the transposition reaction and the sequencing. A search against the non-curated part of the Swiss protein database showed that the protein sequence had 94 % similarity and 94% coverage to a beta-lactamase TEM from Escherichia coli (SP:BLAT_ECOLX). Searches against the curated part of Swiss protein database indicated that the protein sequence shared 99% similarity and 99% coverage to a beta-lactamase of Escherichia coli (TR: D5L073_ECOLX). The protein identified from ORF1 will be referred to as Bel1 (Beta lactamase). On the contrary, there was no hit found for the protein sequence encoded by ORF2.

A multiple sequence alignment of the protein sequence of Bel1 and selected sequences (appendix 8) indicated that it contained the conserved S*XXK motif in amino acid positions 68-71 (box 1), with serine as the active site residue (Paradkar et al, 1996). The Bel1 protein sequence also contained the S(Y) XN motif and the K (H) T(S) G motif in amino acid positions 128-130 (box 2) and 232-234 (box 3) respectively. All three motifs are present in penicillin-binding proteins and beta lactamases (Paradkar et al, 1996).

4.3.2.2. Clone B5 Transposon mutagenesis yielded four mutants (namely B5P2A12; B5P1H9; B5P2E3; and B5P1E10) that were sensitive to penicillin G 200 µg/ ml. The four mutants were sister clones since the transposon was inserted into the exact same site. The reverse primer resulted in an ORF (from here on r eferred to as ORF3) containing 228 a mino acids long. Another ORF (namely ORF4) encoding for 185 amino acids long (nucleotide position 196-753) was found in the forward sequence (see figure 4-2).

The protein sequence encoded by ORF3 contained a conserved region of beta-lactamase 2 (with e-value 5.7 x 10-33) between amino acid positions 49-201.Searches against the non- curated part as well as against the curated part of the Swiss protein database indicated that the protein sequence shared 100% for both similarity and 100% coverage to beta-lactamases TEM from Enterobacteriaceae. The protein from ORF3 will be referred to as Bel2 (Beta lactamase).

The protein sequence encoded by ORF4 contained a conserved region for resolvase (N terminal domain) on amino acid positions 28-137 and a conserved region of helix-turn-helix domain of resolvase (with e -value 7 x 10-11) on amino acid positions 141-181, according to the Pfam database. The protein of ORF4 was named as Rvs1 (Resolvase). A search against the non-redundant protein indicated that the protein had 93% similarity, with 94% coverage,

76 to a resolvase protein from Acinetobacter baumannii (accession no.ZP_08442906.1). Protein sequences with 98% similarity and 98% coverage to Rvs1 (based on the Swiss protein database) were found in transposon resolvases of bacteria in family Enterobacteriaceae, Moraxellaceae and Pasteurellaceae.

4.3.2.3. Clone B6 Six mutants (namely B6P3B5, B6P3H12, B6P3A5, B6P4B4, B6P4C5, and B6P4C6) that have lost the ability to grow in plates containing penicillin G 200 µg/ml were obtained from the transposon mutagenesis of fosmid B6. Two open reading frames were identified from the reverse sequence. The ORFs identified from the 1272 bp of the reverse sequence were the 362 bp (from here on referred to as ORF5a) from nucleotide position 41-403 bp (120 amino acids long) and another ORF (namely ORF6) positioned at 586-987 bp (133 amino acids long). The forward sequence contained two open reading frames. They were located on nucleotide position 110-592 (160 amino acids long) and 953-1132 (59 amino acids long) and were named as ORF5b and ORF7, respectively (see figure 4-3).

The protein sequence encoded by ORF5a contained a co nserved beta-lactamase 2 domain (with e-value 1.3 x 10-07) between amino acid positions 49-109 (Pfam database). A conserved domain of beta-lactamase (with e-value 9.6 x 10-22, between amino acid positions 2-131) was also contained in the protein sequence encoded by ORF5b (Pfam database). As ORF5a and ORF5b code for the same protein (beta-lactamase), the protein sequences of the two were merged as a sequence named as Bel3.Searches against the non-redundant protein of the NCBI showed that the protein sequence had 94% similarity and 100% coverage to beta lactamases from E. coli and Neisseria gonorrhoeae. Searches against the curated part and non-curated part of the Swiss protein database indicated that the protein sequence had 94% similarity and 94% coverage to beta lactamases from Enterobacteriaceae (Salmonella typhi, Salmonella typhimurium and E. coli), Haemophilus influenzae and Acinetobacter baumannii.

The protein sequence encoded by ORF6 had a conserved resolvase N terminal domain (with e-value 1.8 x 10-25) between amino acid positions 2-85 (Pfam database). Searches against the non-redundant protein of theNCBI, both curated and non-curated part of the Swiss protein database showed that the protein sequence had 100% similarity and 100% coverage to

78 resolvases from Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae), Moraxellaceae (Acinetobacter baumannii), and Neisseriaceae (Neisseria meningitidis).The protein from ORF6 will be named as Rvs2 (Resolvase).

The protein sequence encoded by ORF7 shared 95% similarity and 98% coverage (with e-value 6 x 10-22) to a predicted protein of Populus trichocarpa (an Angiosperms plant, Accession no. TR: 89NE13_POPTR). The protein from ORF7 will be referred to as Ppr1 (predicted protein).

4.3.2.4. Clone B7 Transposon mutagenesis of clone B7 has resulted in three mutants (namely B7P3H2, B7P1A8 and B7P1B2), which lost their ability to grow in media supplemented with 200 µg/ml penicillin G. Two open reading frames were identified from the longest reverse sequence. The first ORF identified from the 1145 bp of the reverse sequence was 494 bp (from here on referred to as ORF8a) from nucleotide positions 55-549 bp (164 amino acids long) and the second (namely ORF9) was positioned at 954-1118 bp (54 amino acids long). Two open reading frames were obtained from the forward sequence, and they were located on nucleotide positions 235-873 (212 amino acids long) and 891-1064 (57 amino acids long), and are named as ORF8b and ORF10 respectively (see figure 4-4).

Figure 4-4.Schematic diagrams of the ORFs of B7 mutant. The direction of the primers is shown by arrows. ORF8a: beta-lactamase (Bel4), 164 amino acids. ORF8b: beta-lactamase (Bel4), 212 amino acids. ORF9: unknown protein (Unk2), 54 a mino acids. ORF10: unknown protein (Unk3), 57 a mino acids T: transposon insertion site. Grey line: intergenic region.

The protein sequence encoded by ORF8a contained a conserved region of beta-lactamase (with e-value 3.4 x 10-22) between amino acid positions 5-135 (Pfam database). In addition, the protein sequence encoded by ORF8b had a beta-lactamase conserved domain (with e- value 1.9 x 10-32) between amino acid positions 3-183. As ORF8a and ORF8b code for the same protein (beta-lactamase), the protein sequences of the two were merged as a sequence named as Bel4.Searches against the curated and non-curated part of the Swiss protein database showed that the protein sequence had 98% similarity and 98% coverage to TEM beta lactamases from Enterobacteriaceae (E. coli, Salmonella typhimurium, Salmonella typhi and Klebsiella oxytoca). There was no hi t found for the protein sequence encoded by ORF9 and ORF10 and are therefore classified as hypothetical proteins.

4.3.2.5. Clone B10 One mutant (namely B10B4) which lost the capacity to grow in media with penicillin G 200 µg/ml was obtained and end-sequenced. A 417 bp (from here on r eferred to as ORF11a) encoding for 139 a mino acids long was identified from nucleotide positions 1-418 of the forward sequence.Another ORF (namely ORF11b) encoding for 160 amino acids long (nucleotide position 100-582) was found in the 1193 bp of the reverse sequence (see figure 4-5).

Figure 4-5.Schematic diagrams of the two ORFs of B10 mutant. The direction of primers is shown by arrows. ORF11a: beta-lactamase (Bel5), 160 amino acids. ORF11b: beta-lactamase (Bel5), 139 amino acids. T: transposon insertion site.

The protein sequence encoded by ORF11 contained a conserved beta-lactamase domain (with an e-value 3.7 x 10-10) at amino acid positions 49-128, according to the Pfam database. Additionally, the protein sequence encoded by ORF14 had a beta-lactamase conserved domain with an e-value 9.6 x 10-22 (Pfam database). As ORF11 and ORF11 code for the same protein (beta-lactamase), the protein sequences of the two were assembled as a sequence named as Bel5. Searches against the curated and non-curated part of the Swiss protein database showed that the protein sequence had 100% similarity and 100% coverage to TEM beta lactamases from Enterobacteriaceae (E. coli and Salmonella typhi).

4.3.3. Comparison of beta-lactamase sequences Five beta-lactamase sequences (Bel1-Bel5) were found in this project. Multiple sequence alignment (figure 4-6) revealed the presence of the conserved S*XXK motif (code 1 in figure 4-6) in all sequences (Bel1-Bel5). Additionally, the S(Y) XN motif (code 2) was identified in Bel1-Bel4. In contrast, Bel5 did not contain the S(Y) XN motif, but it had a motif deviation of STR instead. Moreover, the K (H) T(S) G motif (code 3) was present in Bel1, Bel3 and Bel4.

Bel2 ----MSIQHFRVALIPFFAAF-CLPVFAH------PETLVKVKDAEDQ----- Bel1 ----MSIQHFRVALIPFFAAF-CLPVFAH------PETLVKVKDAEDQ----- Bel3 ----MSIQHFRVALIPFFAAF-CLPVFAH------PETLVKVKDAEDQ----- Bel4 VYKRQSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPA Bel5 ----MSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPA * : *:. :.. *..* * ** : : * :

Bel2 ------LGAR---VGYIELDLNSGKILESFRPEERF------Bel1 ------LGAR---VGYIELDLNSGKILESFRPEERF------Bel3 ------LGAR---VGYIELDLNSGKILESFRPEERF------Bel4 AMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRG Bel5 AMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRG *.:* :.::* * :* :* * * .*

Bel2 ------Bel1 ------Bel3 ------Bel4 IIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA------Bel5 IIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHWMSIQHFRVALIPFFAA

(1)

Bel2 ------PMMSTFKVLLCG Bel1 ------PMMSTFKVLLCG Bel3 ------PMMSTFKVLLCG Bel4 ------EIGASLIKHWVA Bel5 FCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCG : ::: .

(2)

Bel2 AVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLL Bel1 AVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLL Bel3 AVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEK------PVSYTHLNHHRMSDNTAANLL Bel4 PVLSRVDAGQEQSVARIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLL Bel5 AVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCLSLIHISTRSC---- .*********** ****************** .* :* .:.

Bel2 LTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGEL Bel1 LTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGEL Bel3 LTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGEL Bel4 LTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGEL Bel5 ------NEEHG------*:*..

(3)

Bel2 LTLASRATINRLGWRRI-KLQDHFCARPFRLGW------Bel1 LTLASRQQL--IDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKLS Bel3 LTLASRQQL--IDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPS Bel4 LTLASRQQL--IDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPS Bel5 ------GW------**

Bel2 ------Bel1 LIHISIISLKKT------Bel3 RIVVIYTTGSQATMDERNRQIAEIGASLIKHW Bel4 RIVVIYTTGSQATMDERNRQIAEIGASLIKHW Bel5 ------

Figure 4-6. Protein sequence alignment of the Bel1 with sequences of Bel2, Bel3, Bel4 and Bel5. Symbols:”*”= column of the alignment contains identical amino acids residue in all sequences. “:”= column of alignment contains conserved substitutions of amino acids. “.”= column of alignment contains semi-conserved substitutions. “-“= gaps in the amino acid sequence.Code (1) indicates the active site (S*XXK motif), red letters. Code (2) indicates the S(Y) XN motif, red letters. Code (3) indicates the K (H) T(S) G motif, red letters. Blue letters in (2): deviation in the S (Y) XN motif.

Phylogenetic analysis of Bel1 to Bel5 was performed using closely-related sequences (figure 4-7). The tree indicated that Bel1 to Bel5 arised from the same ancestor as do the beta- lactamase TEM sequence from E. coli and beta-lactamase TEM-12 from Klebsiella oxytoca.

4.3.4. Comparison of resolvase sequences Sequence similarity analysis of protein Rvs1 to Rvs2 indicated that both sequence shared 100% similarity in 71% coverage. A multiple sequence alignment of Rvs1-Rvs2 was generated by comparing both sequences with the resolvase sequences of Escherichia coli and Klebsiella pneumoniae (figure 4-8). It was observed that all four sequences were highly conserved.

Figure 4-8. Protein sequence alignment of the Rvs1, Rvs2 with Tn3 resolvases sequences of E. coli (TNR3_ECOLX) and Klebsiella pneumoniae (Accession no. TNR4_KLEPN). K.: Klebsiella pneumoniae. E.: Escherichia coli. Symbols:”*”= column of the alignment contains identical amino acids residue in all sequences. “:”= column of alignment contains conserved substitutions of amino acids. “.”= column of alignment contains semi-conserved substitutions. “-“= gaps in the amino acid sequence. The red letters indicates the amino acids contained in the catalytic domain of the Tn3 resolvases.

A phylogenetic tree of Rvs1 and Rvs2 was constructed (figure 4-9) using sequences that were closely related. This revealed that Rvs1 and Rvs2 are very closely related to the Tn3 resolvase sequences of E. coli and Klebsiella pneumoniae.

4.3.5. Genomic context of resistance clones Shotgun sequencing and auto-annotation were conducted on the clones B3, B5, B6, B7, and B10 to identify the genomic DNA context of the resistance genes as well as to determine the possible phylogenetic origin of the fosmid insert DNA.

The DNA sequence lengths of the antibiotic resistant clones were 9.3 Kb for B3; 39.8 Kb for B5; 15.2 Kb for B6; 6.6 Kb for B7 and 16.4 Kb for B10.Fosmid clone B3 had 20 ORFs and its sequence overlapped with the sequence of the fosmid B5, from ORF 25-ORF39 (relative to B5’s ORF position). Fosmid B5 contained 39 ORFs and it also overlapped with the sequence of the fosmid B6, from ORF1-ORF19 (relative to all ORF positions of B6) (table 4- 86

2.A). Fosmid clone B10 had 15 open reading frames and its sequence overlapped with the sequence of the fosmid clone B7, from ORF1-ORF9 (relative to B7’s ORF position) (table 4.2.B). The full annotations of the B3-B10 fosmids are given in appendix 3-7. The bel genes (bel1 to bel5) were not recovered in the shotgun sequence data of the fosmids. A possible explanation for this is that the sequences of bel1 to bel5 were similar to the sequences in Enterobacteriaceae, including E. coli, and therefore were accidently removed in the quality- control of the sequencing data. This quality-control involved removing sequences from common lab contaminants, such as E. coli.

The B3 and B5 clones contained several genes that encode for proteins responsible for cell survival, such as LexA, protein resistance to heavy metals, competence protein, and the peptidase U62 modulator of DNA gyrase. All the genes were orientated in the same direction in both fosmids. The peptidase U62 modulator of DNA gyrase shared a 43-49% similarity (e- value: 3.10 x 10 -96 to 2.2 x 10 -102) to some microcin-processing peptidase 1 of several bacteria.

In addition, genes properties for aerobic cellular respiration (such as genes encode ATP synthase subunit 6, c ytochrome C oxidase sub unit I, cytochrome oxidase sub unit 2 a nd NADH ubiquinone oxidoreductase chain) and degradative enzymes (polyhydroxybutyrate depolymerase and 2-hydroxypentadienoic acid hydratase) were identified in fosmid B5. All the aerobic cellular respiration genes shared 96-99% similarity and 100% coverage to sequences from fungi species (Ustilago maydis and Sporisorium reilianum). Moreover, genes encoding for protein involved in transport (two ORFs for molybdate ABC transporter), protein against oxidative stress (superoxide dismutase), and proteins for attacking innate immune systems in host cells (Toll/ Interleukin-1 like receptor/ TIR protein and Leucine rich repeat (LRR) protein-like protein) were detected in both B5 and B6 clones.

B10 and B7 had genes that were identified to be encoding for transposase and proteins responsible for the synthesis of the isoprenoids precursor (table 4-2). The protein transposase shared 46% similarity (83% coverage) to the transposase sequence of Candidatus Nitrospira defluvii (YP_003799838.1). Additionally, the conserved regions of winged helix-turn helix (with e-value 3.8 x 10-10) and integrase core domain (e-value 3.2 x 10-17) were identified on the annotation of the shotgun sequencing data (based on Pfam database).

Table 4-2: Annotation of the open reading frames of the fosmids of the antibiotic resistant clones. Where possible, the genes were named according to the Swiss protein database. If there was no information available under the Swiss protein database, the genes was named based on the -non-redundant protein database of the National Centre for Biotechnology Information. The light shaded box indicates that the gene has an e-value lower than 1x10-20(cutting out point 1x 10-5) under the Swiss protein database. The dark shaded box indicates the protein’s annotations are similar for two or more databases, but did not have matches to the Swiss protein or did have an e-value lower than 1x10-20 in the Swiss protein database. White box indicates the protein is annotated as hypothetical or uncharacterized protein. Please see appendix 3-7 for detail annotation.

Location and ORF B3 Location and Location and B5 ORF Name of protein and accession number B6 ORF orientation orientation orientation ORF1 203-967 (-) ATP synthase subunit 6 ( TR:E6ZZ97_SPORF) ORF2 2595-4073 (-) Cytochrome C oxidase subunit 1 (SP:COX1_USTMA) ORF3 5605-6222 (+) Putative uncharacterized protein (TR:Q4P923_USTMA) NADH ubiquinone oxidoreductase chain 1 ORF4 6223-6967 (-) (SP:NU1M_USTMA) Cytochrome C oxidase subunit 2 ORF5 8294-8920 (-) (SP:COX2_USTMA) 2-keto-4-pentenoate hydratase/ 210 -380 (-) ORF6 9130 -9300 (-) 2-oxohepta-3-ene-17-dioic acid hydratase ORF1 (TR:Q3J118_RHOS4) ORF7 10666 - 11618 (+) Putative uncharacterized protein (TR:F7ZEA9_ROSLO) ORF2 1746 - 2318 (+) 2387 -2980 (-) ORF8 11687 -12280 (-) Dihydroxyaciddehydratase (TR:A9DBJ3_9RHIZ) ORF3 Dihydroxyaciddehydratase 3953 - 4171 (-) ORF9 13253 - 13471 (-) ORF4 (TR:A71GU9_XANP2) ORF10 13769 -14956 (+) TIR protein (TR:B4S3R6_PROA2)E value: 4e-5 ORF5 4469 -5656 (+) Leucine-rich repeat (LRR) protein-like protein 5425 -5949 (+) ORF11 14725 -15249 (+) ORF6 (TR:Q0AX68_SYNWW) Inositol 2-dehydrogenase 5969 -6922 (-) ORF12 15269 -16222 (-) ORF7 (SP:M12D_RHIME) SMP-30/ gluconolaconase/LRE-like region family protein 7003 -7416 (-) ORF13 16303 -16716 (-) ORF8 (TR: E3H171_ACHXA) Molybdate ABC transporter, ATP binding protein 8789 -9109 (-) ORF15 18089 -18409 (-) ORF10 (TR:Q2CBP2_9RHOB) Binding-protein-dependent transport system 9061 -9759 (-) ORF16 18361 -19059 (-) inner membrane (TR:C9XZ07_CROTZ) ORF11

30S ribosomal protein S2 10013 -10348 (+) ORF17 19313 -19648 (+) ORF12 (TR:D0D246_9RHOB) 30S ribosomal protein S2 10702 - 10980 (+) ORF18 20002 - 20280 (+) ORF13 (TR:E8L2S7_9RHIZ) Elongation factor Ts 11095 -12030 (+) ORF19 20395 -21330 (+) ORF14 (TR;F21Y16_POLGS) Superoxide dismutase, Fe 12234 -12836 (+) ORF20 21534 -22136 (+) ORF15 (TR:A3SQA9_9RHOB) Chloramphenicol-sensitive protein RarD 12919 -13065 (+) ORF21 22219 -22365 (+) (sit:TM1040_0071) ORF16

14006 – 14470 (+) ORF22 23306 – 23770 (+) RarD (TR:Q5LQZ4_SILPO) ORF17 Decarboxylase, putative 14410 -14964 (-) ORF23 23710 -24264 (-) ORF18 (TR:A9E8B4_9RHOB) DEAD/DEAH box helicase domain protein 15022 - 15234 (-) ORF24 24322 - 24534 (-) ORF19 (TR:A7HY74_PARL1) Location and ORF B3

orientation 98 -1432 (-) ORF1 Peptidase U62 modulator of DNA gyrase ORF25 24632 -25966 (-) (TRA7HQS1_PARLI) 1746 -1928 (+) ORF2 ORF26 26280 -26462 (+) Hypothetical protein Dshi_4011 (TRA8LU30_DINSH) 2056 -3384 (-) ORF3 Putative uncharacterized protein ORF27 26590 -27918 (-) (TR:Q5LKKI_SILPO) 3381 -3881 (-) ORF4 NADH riboflavin 5’ –phosphate oxidoreductase ORF28 27915 -28415 (-) (TR:A8TK16_9PROT) 3878 -4882 (-) ORF5 ORF29 28412 -29416 (-) Co/Zn/Cd resistance protein cZcD (TR:A8TTM6_9PROT) 5022 -5456 (+) ORF6 Acetyltransferase, GNAT family protein ORF30 29556-29990 (+) (TR:QOFCP3_9RHOB) 5518 -6273(+) ORF7 Putative polyhidroxybutyrate depolymerase ORF31 30052-300807(+) (TR:B9NUP3_9RHOB) 6322 -7113 (+) ORF8 ORF32 30856-31647 (+) Amidinotransferase (TR:C8S44S_9RHOB) 7178 -8335 (-) ORF9 Unnamed protein product ORF33 31712 -32869 (-) (TR:A1B8QS_PARDP) 8481 -9287 (+) ORF10 2-Dehydro-3-deoxyphosphooctonate aldolase ORF34 33015 -33821 (+) (TR:A7HXX8_PARLI) 9360 -9851 (+) ORF11 ORF35 33894 -34385 (+) Predicted protein (TR:C3X4E1_OXAFO) 10007 – 10711 (+) ORF12 RepressorLexA ORF36 34541– 35245(+) (TR:E2CCN3_9RHOB) 10705-12695 (-) ORF13 Competence protein ORF37 35239-37229 (-) (TR:C9D485_9RHOB)

12824 -14260 (+) ORF14 Glutamyl-tRNAsynthetase ORF38 37358-38794 (+) (TR:B9QYE2_9RHOB) 14338 - 15636 (+) ORF15 ORF39 38872 - 40170 (+) Citrate synthase (TR:64R8V4_9RHIZ) 15695-16603 (+) ORF16 Beta-ketoacyl-acyl-carrier protein synthase III

(TR:C6XS10_HIRBI) 16643-17197 (+) ORF17 Hypothetical protein PHZ_c1374

(YP_002130217.1) 17221-17532 (+) ORF18 Flippase (TR:Q4KOH6_STRPN)

17620-18780 (+) ORF19 Quinolinatesynthetase complex A sub unit (TR: C6XRJ4_HIRBI)

18900-19301 (+) ORF20 Putative uncharacterized protein (TR:A4A7JI_9GAMM)

Location and Location and ORF B10 Name of protein and accession number ORF B7 orientation orientation 14-535 (+) ORF1 1-deoxy-D-xylulose-5-phosphate synthase ORF1 14-535 (+) (TR:A4ANK7_MARSH) Putative uncharacterized protein 495 -1577 (+) ORF2 ORF2 495 -1577 (+) (TR:E6XCSU_CELAD) Deoxyguanosine triphosphate triphosphohydrolase 1587-2366 (-) ORF3 ORF3 1587-2366 (-) (TR:G2PQ12_9FLAO) Diguanylatecyclase/ phosphodiesterase with PAS/PAC sensor (s) 2396 - 2635 (-) ORF4 ORF4 2396 - 2635 (-) (TR: A6TSA7_ALKMQ) 2657 - 3394 (-) ORF5 Ribonuclease 3 (TR:D9PK52_9ZZZZ) ORF5 2657 - 3394 (-) N utilization substance protein B homolog 3513 -3977 (-) ORF6 ORF6 3513 -3977 (-) (TR:E4M0V9_9FIRM) 50S ribosomal protein L32 4075 -4269 (-) ORF7 ORF7 4075 -4269 (-) (TR:B8JEX7_ANAD2) Putative transposase 4435 -5310 (-) ORF8 ORF8 4435 -5310 (-) (TR:D8P8T1_9BACT) 5416 - 6606 (-) ORF9 Predicted glycosyltransferase ORF9 5416 - 6606 (-) AlanyltRNAsynthetase(TR:F4KWP4_HALHI) 6685-6774 (+) ORF10

Putative uncharacterized protein 8805- 9032 (+) ORF11 (TR:A0Y568_LYNSP) Aminoacyl-histidine dipeptidase 9498- 10954 (-) ORF12 (TR:A8UG77_9FLAO) 11146 - 14259 (-) ORF13 Putative uncharacterized protein (TR:A6EP04_9BACT) Ubiquitin carboxyl-terminal hydrolase 14267 - 14542 (-) ORF14 (TR:F0ZN32_DIRPU) Secreted protein containing N-terminal Zinc-dependent carboxypeptidase 14775 - 16427 (-) ORF15 related domain (TR:A1ZPV4_9BACT)

To predict the phylogenetic/ taxonomical origin of the fosmid of the five antibiotic resistant clones, sequences were analysed using the Phylopythia algorithm (McHardy et al, 2007).

The Phylopythia algorithm predicted that the fosmid insert DNA of clone B3, B5, and B6 (appendix 9) belonged to the order Rhodobacterales. The analysis also indicated that B7 belonged to the phylum Proteobacteria, while B10 was grouped into the domain Bacteria.

4.4. DISCUSSION A functional metagenomic screening of the microbial community from U. australis indicated the presence of resistance activities to penicillin G (200 µg/ ml) and trimethoprim (400 µg/ ml), in addition to kanamycin (as described in chapter 3). To further understand the genes responsible for the antibiotic resistance, mutants were generated in which the resistance genes have been inactivated. Mutants sensitive to trimethoprim were not knocked out during the in-vitro mutagenesis and this could be due to chance or due to the genes being very small. The genes responsible for the resistance to beta-lactam antibiotics have been identified for five antibiotic resistant clones. The genes are discussed in the next section.

4.4.1. The presence of beta-lactamase genes in the microbial community of U. australis Sequences of Bel1-Bel5 have the S-X-X-K motif, which is present not only in the beta- lactamase family, but also in the VII carboxylesterases family (Puneet et al, 2011). The family VII carboxylesterases are also characterised by the presence of GXSXG, HGGG and HGF motifs, they do not belong to the carboxylesterases. Since the Bel1-Bel5 protein sequences did not contain these three additional motifs described in Puneet et al, 2001, they were not grouped into carboxylesterases. The presence of beta-lactamase conserved domains (Pfam database analysis) and three motifs{S*XXK, S(Y) XN and K ( H) T(S) G}(Paradkar et al, 1996; Tanaka et al, 2001; Matagne et al, 1998; Singh et al, 2009) in all Bel proteins indicated that they were beta-lactamases. Bel1-Bel4 proteins were grouped into Class A beta-lactamases because they had the S*XXK and S(Y) XN motifs (Tanaka et al, 2001). Bel5 also had the S*XXK motif but it had the STR motif instead of the S(Y) XN. The presence of the STR motif has not been observed before, and thus a novel motif in beta-lactamases was identified in this project.

The presence of beta-lactamases in the microbial community of U. australis indicates the distribution of the resistance genes in marine environments, as was also reported by Tanaka et al, 2001. Beta-lactamase resistance genes have evoled during the past 60 years (Lachmayr et al, 2009; Hall and Barlow, 2004; Gniadkowski, 2008), and numerous novel sequences have been described (Bou et al, 2000; De Champs et al, 2001). Three beta- lactamases, (Bel1, Bel3 and Bel4, found in this project) were sequenced and high quality

93 sequences were obtained and used for the blast analysis. The three beta-lactamases only share 94-98% similarity and 94-100% coverage to known beta-lactamases, and therefore they were novel sequences. Two other sequences (Bel2 and Bel5) were identical (100% similarity and 100% coverage) to the TEM beta-lactamases of E. coli and Salmonella typhi. Thus, both clinical and novel beta-lactamases were present in the microbial communities of U. lactuca isolated from Coogee Beach, Sydney, Australia.

Beta-lactamase genes can spread from one bacterium to another by transposons (Mendonca et al, 2007; Ouellette et al, 1987; Weber and Goering, 1988; Poirel et al, 2005). In this project, transposons are believed to be responsible for the transfer of bel2 and bel3 genes because sequences encoding for resolvases (namely as Rvs1 and Rvs2) were detected adjacent to them. Both resolvase sequences shared a similarity to several existing Tn3 resolvases, with the closest to the sequences belonging to clinical samples (E. coli and Klebsiella pneumonia). The clones containing the Bel2 and Bel3 sequences were identified as belonging to the order Rhodobacterales. This finding supports the notion that horizontal gene transfer has occurred in the microbial communities of U. lactuca, causing the micro-organisms to obtain the beta-lactamase genes.

The resolvases identified in this project contained the catalytic domain of Tn3 transposase such as β2, αβ, β3, αD, β4, β5, and αE (Ollorunniji and Stark, 2009; Gaj et al, 2011). The presence of the resolvase identical to clinical samples (Rvs2) and the sequence (Rvs1) that shared a high similarity (>92% similarity with >93% coverage) may indicate the spread of transposons from a clinical setting into the marine environment.

4.4.2. Predicted proteins identified from the uncultured Alphaproteobacteria of the microbial community of U. australis Bel1 was contained in a genomic DNA fragment of an Alphaproteobacteria and this organism also had other genes for survival, such as the gene encoding for competence protein. The presence of the gene encoding for competence protein is interesting as this protein has been observed to be involved in DNA uptake and transformation in bacteria (Zhu and Lau, 2011), for example in taking up a ntibiotic resistant genes from the environment (Dowson et al, 1989; Morrison and Lee, 2000; Claveris et al, 2009; Dowson

94 et al, 1977). Alternatively, extracellular DNAs taken up can be used as a carbon or energy source (Finkel and Kolter, 2001). Although the gene encoding for a competence protein was identified in this project, expression analysis and further characterisation are required to fully understand its function. .

The genomic fragment of Bel2 was identified as belonging to an Alphaproteobacteria. This bacterium had genes encoding for 2-dehydro-3-deoxyphosphooctonate aldolase (DAHP synthase) and for dihydroxyacid dehydratase. The DAHP synthase protein functions as the first enzyme in the shikimate pathway for the biosynthesis of phenylalanine, tyrosine, and tryptophan (Light et al, 2012; Ikeda, 2006; Panina et al, 2003; Hermann, 1999; Salcher and Lingens, 1980). Though the gene encoding for the DAHP synthase has been found in this bacterium, it can not be absolutely concluded that the bacterium can synthesis the amino acids described above, as the presence of other enzymes in the pathway, like the shikimate dehydrogenase and the chorismate synthase, are also required. Dihydroxyacid dehydratase is the key enzyme in the biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine) (Singh et al, 2011; Myers, 1961; Kanamori and Wixom, 1963; Huser et al, 2005). However, since the genes encoding for branched chain amino acids transferase (Oliver et al, 2012), the protein which works together with the dihydroxyacid dehydratase, has not been identified in the cosmid, it cannot be concluded that this bacterium is able to provide the branched amino acids by itself.

Additionally, the gene encoding for Toll/interleukin-1 receptor (TIR) protein and Leucine-rich repeat (LRR) –like protein were present in the genome fragment Bel2. Both proteins can suppress the innate immune response of plant (Weinberger, 2007) and animal host cells (Newman et al, 2006; Salcedo et al, 2008; Radhakrishnan et al, 2009; Cirl et al, 2008; O’Neil, 2008; Chan et al, 2009; Novatchkova et al, 2003), and thus provide bacteria with the ability to escape phagocytosis (Yutin et al, 2009; Salcedo et al, 2008).

Another beta-lactamase (Bel3) and several other genes were identified in the genome fragment of an Alphaproteobacteria. This bacterium has got a gene encoding for dihydroxyacid dehydratase, LLR and TIR protein; those proteins have been described in the previous paragraph. In addition a gene encoding for superoxide dismutase which plays

95 an important role in cell protection against reactive oxygen species (Chaudiere and Ferrari-Iliou, 1999; Scott et al, 1987; Han et al, 2006) was identified in the cosmid This gene was quite divergent from those in existing databases with the closest hit being to the gene of Roseovarius nubinhibens (70% identical).

Bel4 was identified in a genomic DNA fragment of a Proteobacteria. The bacterium had the gene encoding for a glycosyltransferase, an enzyme synthesizing disaccharides, oligosaccharides and polysaccharides. Saccharides are needed for several functions in bacteria, such as for peptidoglycan synthesis (Ha et al, 2001) and for glycocalyx production (Narimatsu et al, 2004).

The last beta-lactamase obtained in this project (Bel5) was identified in a genomic fragment that also contained the gene encoding for aminoacyl-histidine dipeptidase. This protein catalyzes the hydrolysis of various peptide substrates (Chang et al, 2009; Wang et al, 2008) and is able to maintain bacterial cell wall homeostasis against various antibiotics (White et al, 2011; White et al, 2010). Additionally, a gene encoding for Zinc-dependent carboxypeptidase, an enzyme that cleaves proteins and peptides (Makarova and Grishin, 1999), was found. The presence of both genes might increase the adaptability of the bacterium to antibiotics, such as peptide antibiotics. The genes encoding for proteins for aerobic respiration (citrate synthase, NADH ubiquinone oxidoreductase, cytochrome C oxidase sub unit 1 and 2 and ATP synthase sub unit 6) were also present, which might indicate that the organism is aerobic (Castresana et al, 1994). Interestingly, their sequences did not show a similarity to the genes from bacteria, but instead were related to fungal protein. Gene transfer between organisms of different domains can occur (Schmitt and Lumbsch, 2009; Fitzpatrick et al, 2008; Richards et al, 2005 and Moran and Jarvik, 2010; Wenz et al, 2005), and the presence of those fungal genes in the bacterial genomic context might be one example of this.

In conclusion, genes for various beta-lactamases and transposases have been identified in the bacterial community of U. australis. The beta-lactamases were of clinical origin and or divergent from other known sequences (i.e. novel), and thus marine environment possess various types of beta-lactamases. Moreover, for two beta-lactamase genes there is

96 evidence that they were incorporated into the bacterial genomes via horizontal gene transfer.

The metagenomic library obtained in this project contained a large number of clones (40,000 clones in S. lividans and 150,000 clones in E. coli) and, in the future, could be screened for resistance against other antibiotics not tested in this PhD study. Functional screening rather than the PCR-based method should be applied to isolate the antibiotic resistance genes from the metagenomic library, as the functional screening may offer the discovery of novel genes. In contrast, a PCR-based approach is limited in that only gives to access to genes that are similar to known sequences (Aminov et al, 2001). Since here resistance to kanamycin has been observed in several clones recovered from the library, the functional screening should be conducted in LB media containing inhibitory concentration of several aminoglycosides. Furthermore, clones should be screened for their resistance to several groups of antibiotics, for example fluoroquinolones, glycopeptides, rifamycin and polypeptides. The antibiotic resistance genes can then be identified by in vitro mutagenesis using the EZ-Tn5 Insertion kit (Epicentre, Madison, Wisconsin). Such studies would then further address the hypothesis that surface- associated, marine communities are rich in antibiotics and respective resistance mechanisms.

CHAPTER 5

GENERAL DISCUSSION, CONCLUDING REMARKS AND FUTURE DIRECTION

CHAPTER 5: GENERAL DISCUSSION, CONCLUDING REMARKS AND FUTURE DIRECTION

Overall, this thesis has presented findings on t he bacterial community of U. australis, with discoveries on antibacterial activities, antibacterial resistance elements and other biological functions.

5.1. The presence of antibiotic biosynthesis and resistance elements in bacterial communities of U. australis U.australis is a habitat with a diverse range of bacteria, which live as epiphytes on the algae. The high diversity of bacteria on the alga causes a competitive environment for the bacteria. This can theoretically lead to the production of numerous antibiotics to inhibit the growth or to kill other bacteria that live in the same habitat (the alga) (Rao et al, 2006). Furthermore, antibacterial activity of the bacterial community can inhibit the growth of clinical samples, such as Shigella sonnei, Shigella flexnery (both observed in this project), Staphylococcus aureus, Klebsiella pneumoniae (Yung et al, 2011), Neisseria canis and, Candida albicans (Penesyan et al, 2009). This indicated that the bacterial community can be a good source for finding antibacterial compounds against human or animal pathogens.

The functional analysis conducted in this project has identified the likely presence of an antibacterial compound related to microcin. Microcin is a b actericidal antibiotic that inhibits DNA replication of the target bacteria (Herrero and Moreno, 1986; Vizan et al, 1991). This antibiotic has been observed to kill the food borne pathogens E. coli O157:H7 (Eberhart et al, 2012), uropathogenic E. coli (Azpiroz et al, 2009), Salmonella enteritidis (Portrait et al, 1999), S. flexnery (Salomon and Farias, 1992), Citrobacter, and Klebsiella (Papagianni, 2003). Certain microcin (microcin E492) do not function only as an antimicrobial compound, but it also has anti- tumour activity against malignant human cell-lines (Lagos et al, 2009).

Fosmid sequencing of the antibiotic-resistant clone and the HERMI clone also identified sequences encoding for an antibacterial compound related to cell wall hydrolase, which were not identical to existing protein sequences. Cell wall hydrolases together with

99 synthases are needed for bacterial growth and division (Wyckoff et al, 2012). However, they can also act as toxins that degrade the component of the peptidoglycan of cell walls of Gram-positive bacteria (Haiser et al, 2009; Vollmer et al, 2008), for example Enterococci (Dhalluin et al, 2005; Rodriquez et al, 2007). Therefore, the bacterial community of U. lactuca is a potential source for obtaining antibacterial compounds against Gram-positive and -negative bacteria.

Transposases have been observed in various microbial communities (Sundin et al, 1995; Hooper et al, 2009), and some are responsible for the transfer of antibiotic resistance genes among the microbial communities (Kristiansson et al, 2011; Zhang et al, 2011). This project indicates that transposons may also play a role in the transfer of the beta- lactamase genes in the microbial communities of U. lactuca. The presence of transposons carrying antibiotic resistance genes, such as the ones encoding for beta-lactamases can increase the survival rate of the bacteria in the microbial communities (as described previously in this thesis and in Yung et al, 20011; Penesyan et al, 2009). Moreover, the presence of natural competence genes observed in the microbial communities in this project may facilitate the transposition event in the communities. The presence of transposases in the bacterial community is alarming as the transposon containing the resistance elements can be transferred into other bacteria in the same habitat (the alga) or even to bacteria of other environments via biological or physical forces (Allen et al, 2010).

Antibiotic resistance genes can be transferred from an environment to clinical microorganisms or vice versa (Wright et al, 2010; Linch et al, 2011). It has been reviewed in several papers (D’Costa et al, 2011; Webb and Davies, 1993; Martinez, 2008) that natural antibiotic-producing bacteria have the gene encoding for the biosynthesis of an antibiotic as well as the gene encoding for the resistance to the corresponding antibiotic. As beta- lactamases are discovered in non-antibiotic producing bacteria (Bush et al, 1995), it is likely that the genes encoding for resistance to beta-lactams are originated from a non- natural environment. Additionally, it is observed that resistance to beta-lactamase antibiotics occurs in clinical settings because of the exposure of the microorganisms to beta-lactam antibiotics (Alonso et al, 2001). Therefore the antibiotic resistance genes recovered in this project came from clinically relevant organisms and have been

100 transferred into environmental micro-organisms via horizontal gene transfer (as described previously).

5.2. The presence of virulence genes in the antibiotic resistant clones Two antibiotic resistant clones (B5 and B6) had the gene encoding for toll/interleukin-1 receptor like/TIR and the Leucine-rich repeat/ LLR proteins. Both proteins are natural mediators of the innate immune response in animals and plants and are produced in recognition to a bacterial or fungal infection (Weinberger, 2007; Newman et al, 2006; Medzhitov, 2001). The finding suggests that genes virulent to animals and plants have been recovered in this project. It also indicates that some algae might contain potential bacteria with these virulence genes.

The presence of pathogenic micro-organisms has been reported in the natural microbial communities of several plants and animals (Flores et al, 2011; Hirano and Upper, 2000; Smith et al, 2012; Lu et al, 2006). Algae, as also other plants, have also the mechanism to detect invading micro-organisms via their innate immune response (Weinberger, 2007). Two proteins (TIR and LLR) play a role in the innate immune system to cause the host cell to activate its gene for the production of a toxin against the invading micro-organisms (Nihorimbere et al, 2011). Since the bacterial genomic DNA observed in this project had genes encoding for TIR and LLR proteins, it seems that they are able to synthesize both TIR and LLR proteins to inactivate the algal signalling molecules. Therefore, the bacterium may escape the host cell’s innate immune response as the presence of this bacterium may not be detected. The gene encoding for TIR has been reported in pathogens of terrestrial plants (Smith et al, 2007). This project is the first to report the presence of the virulence genes encoding for TIR and LLR occurring in bacterial communities of alga.

5.3. Management systems to avoid the spread of antibiotic resistance genes Several ways to control the spread of antibiotic resistance in clinical settings are performed by effective infection control practices (isolating patient), and effective hygiene barriers (destruction of contaminated devices, hospital-waste management) (Amyes, 2005). Education for physicians on t he proper use of antibiotics for disease treatment (Paterson, 2006) and strict regulations on selling un-prescribed antibiotics can

101 also be conducted to reduce antibiotic resistance in clinical micro-organisms. As various natural environments such as soils and marine environments in different parts of the world (McGarvey et al, 2012; Heritier et al, 2004; Livermore, 1995; Allen et al, 2009; Tanaka et al, 2001; Teo et al, 2000; Bahry et al, 2012; Simm et al, 2001; Henriques et al, 2006) have been identified to have antibiotic resistance genes, a good management system in clinical settings alone might not be sufficient to control and reduce the spread of the antibiotic-resistance element. A good management system in minimizing the transfer of specimens collected from areas with the resistance elements might be beneficial in lowering the possible transfer of the resistance elements to the broader environment. A study of the antibiotic resistance genes sourced from environmental samples may provide information about the affected area, and thus it w ill assist the authorities to perform the proper action to eliminate their spread. Furthermore, information on novel antibacterial resistance proteins may support data for the design of an inhibitor against the corresponding resistance elements, so that the available antibiotics might be used in combination with the antibiotic resistance inhibitor.

5.4. Concluding remarks and future direction To sum up, t his bacterial community of Ulva australis contains resistance to several clinical antibiotics, which has possibly been acquired through transferable elements. Potential genes (encoding for the microcin-like antibiotic; cell-wall hydrolase) have been found in the metagenome of the microbial communities of U. lactuca and further research such as the cloning and the expression of the potential genes in multiple host cells, functional analysis, bioactive compounds isolation and compound determination will be needed in the future.

102

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APPENDIX

Appendix 1. Annotation of clone SE9 (clone with antimicrobial activity against Shigella) according to several databases. The encoded protein was obtained from NCBI NR, Swiss Prot, KEGG and Pfam. Confidence values are shown for each database. No hit: unsignificant match (with an e-value of 1 x 10-5 as the cut-off).

ORF Position Encoded protein and accession number E value

ORF1 2130 - 2345 Hypothetical protein Oceanospirillales bacterium HFO130 (ADI17717.1) 3e-11 Putative uncharacterized protein Uncultured Oceanospirilalesbacterium HF0130_25G24 8e-7 (TR:EOXTH6_9GAMM) No hit - No hit - ORF2 2342 - 2533 Hypothetical protein Pseudomonas syringae (ZP_03400294.1) 2e-37 Putative uncharacterized protein Pseudomonas syringae pv. Tomato T1 5e-28 (TR:E2MMZ5_PSEUB) Hypothetical protein Coxiella burnetii RSA 331(COXBURSA331_A0274) 7e-21 Pfam B 1237 2.2e-18

ORF3 3049 - 3156 Hypothetical protein VIS19158_09392 Vibrio scophthalmi (ZP_08747662.1) 5e-13 Putative uncharacterized protein Vibrio scophthalmi TR:F9RN43_9VIBR) 2e-8 Ribosomal protein S10 Medicago truncatula (MTR_1g006120) 2e-5 No hit -

ORF4 4077-4217 No hit - No hit - No hit - No hit -

ORF5 4509 – 5429 Phosphatidylserine synthase Cellvibrio japonicas Ueda107 (YP_001981189.1) 3e-110 Phosphatydilserine synthase Marine gamma proteobacterium HTCC2143 (TR:AOYG54_9GAMM) 1e-87 Phosphatidylserine Cellvibrio japonicas 1.5e-55

134

CDP-OH P transf ( CDP alcohol phosphatidyltransferase) 1e-16

ORF6 5496 – 6521 Ketol-acid reductoisomerase Thioalkalivibrio sulfidophilus (YP_002514223.1) 0.0 Ketol-acid redsuctoisomerase Methylococcus capsulatus ATCC33009(SP:ILVC_METCA) 0.0 Ketol-acid reductoisomerase Thioalkalivibrio sp. HL-EbGR7 (Tgr7_2156) 0.0 IlvN and IlvC (Acetohydroxy acid isomeroreductase catalytic domain) 2e-106

ORF7 6544 – 7035 Acetolactate synthase 3 regulator subunit Alteromonas sp S89 (ZP_09503949.1) 2e-106 Acetolactate synthase, small subunit Saccharophagusdegradans strain 2-40/ ATCC 4396 1e-70 (TR:Q21HM6_SACD2) Acetolactate synthase 3 regulatory subunit Saccharophagus degradans (Sde_2543) 6e-75 ACT 5 (ACT domain); 1e-18 ALS C ( Small sub unit of acetolactate synthase) 8.4e-29

ORF8 7047-8783 Unnamed protein product Saccharophagus degradans (YP_528016.1) 0.0 Acetolactate synthase Saccharophagus degradans (TR:Q21HMS_SACD2) 0.0 Acetolactate synthase 3 catalytic subunit Saccharophagus degradans (Sde_2544) 0.0 TPP enzyme N (Thiamine pyrophosphate enzyme, N- terminal TPP binding domain) 3.1e-60 TPP enzyme M (Thiamine pyrophosphate enzyme, central domain); 1.2e-41 TPP enzyme C ( Thiamine pyrophosphate enzyme, C-terminal TPP binding domain) 2.2e-51

ORF9 9236-9721 Hypothetical protein Pseudomonas aeruginosa (EGM20324.1) 1e-17 Putative uncharacterized protein Pseudomonas aeruginosa strain PA7 (TR:AGVCF1_PSEA7) 3e-17 Hypothetical protein Pseudomonas aeruginosa strain PA7 (PSPA7_5412 ) 2.5e-9 No hit -

ORF10 9753-10829 Hypothetical protein Oceanobacter sp. (ZP_01307192.1) 8e-50 Putative uncharacterized protein Bermanella marisrubri (TR:Q1N1T4_9GAMM) 1e-41 Hypothetical protein Leptospira biflexa serovar Patoc Patoc 1 (Ames) (lbf:LBF_0832) 3e-5 No hit -

135

ORF11 10858-11316 Hypothetical protein Oceanobacter sp. (RED65_04210) 4e-61 Putative uncharacterized protein Bermanella marisrubri (TR:Q1N1T3_9GAMM) 2e-50 Hypothetical protein Marinobacter aquaeolei (maq:Maqu_1681) 9.9e-4 No hit -

Scaffold 5 ORF12 98 -1432 Putative Zn-dependent protease, pmb A-like protein Azospirillum brasilense (CCC98942.1) 2e-145 Peptidase U62 modulator of DNA gyrase Parvibaculum lavamentivorans strain DS-1/DSM 13023 1e-120 (TRA7HQS1_PARLI) 1e-106 Putative peptidase, PmbA-like; K03592 PmbA protein ( Azospirillum lipoferum ali:AZOLI_2345) 1.4e-63 Putative modulator of DNA gyrase

ORF13 1746 -1928 Hypothetical protein Dshi_4011 Dinoroseobacter shibae DFL12 (YP_001542219.1) 1e-16 Hypothetical protein Dshi_4011 Dinoroseobacter shibae DFL12 (TRA8LU30_DINSH) 1e-12 Hypothetical protein Dinoroseobacter shibae (dsh:Dshi_4011) 1e-12 Toxin with endonuclease activity yhaV 5.3e-16

ORF14 2056 -3384 Putative oxidoreductase Marine bacterium 01-004080 (ACA21512.1) 3e-95 Putative uncharacterized protein Silicibacter pomeroyi strain ATCC 700808 (TR:Q5LKKI_SILPO) 1e-78 Hypothetical protein (sil:SPOA0380) 9e-74 FAD-dependent oxidoreductase 5.7e-57

ORF15 3381 -3881 NADH riboflavin 5’ –phosphate oxidoreductase α Proteobacterium BAL199 (ZP_02187032.1) 3e-40 NADH riboflavin 5’ –phosphate oxidoreductase α Proteobacterium BAL199 (TR:A8TK16_9PROT) 9e-33 Flavin reductase domain-containing protein Parvibaculum lavamentivorans (pla:Plav_2888) 2e-27 Flavin reductase like domain 7.3e-31

ORF16 3878 -4882 Co/Zn/Cd cation transporter Hahella chejuensis KCTC2396 (YP-435164.1) 2e-110 Co/Zn/Cd resistance protein cZcD α Proteobacterium BAL199 (TR:A8TTM6_9PROT) 4e-90 Co/Zn/Cd cation transporter Hahella chejuensis (hch:HCH_04023) 1e-78

136

Cation efflux family 1.8e-50

ORF17 5022 -5456 Acetyltransferase, GNAT family protein Rhodobacterales bacterium HTCC2255 (ZP_01447824.1) 8e-42 Acetyltransferase, GNAT family protein Rhodobacterales bacterium HTCC2255 (TR:QOFCP3_9RHOB) 5e-31 GCN5-related N-acetyltransferase Ruegeria sp.(sit:TM1040_2995) 2e-28 Acetyltransferase (GNAT) family 3.3e-10

ORF18 5518 -6273 Putative polyhidroxybutyrate depolymerase Rhodobacteraceae bacteriu (ZP-05123306.1) 6e-6 Putative polyhidroxybutyrate depolymerase Rhodobacteraceae bacterium (TR:B9NUP3_9RHOB) 6e-55 Polyhydroxybutyrate depolymerase Pseudovibrio sp. (psf:PSE_0849) 3e-23 Alpha/beta hydrolase family 3e-10

ORF19 6322 -7113 NG,NG-dimethyl arginine dymethylaminohydrolase Rhodobacterales bacterium HTCC2150 (ZP_01741609.1) 1e-118 Amidinotransferase Rhodobacterales bacterium HTCC2150 (TR:C8S44S_9RHOB) 3e-93 NG,NG-dimethylarginine dimethylaminohydrolase Silicibacter pomeroyi (sil:SPOA0064) 2e-90 Amidinotransferase 2.4e-19

ORF20 7178 -8335 Unnamed protein product Paracoccus denitrificans PD 1222 (YP_917595.1) 0.0 Unnamed protein product Paracoccus denitrificans PD 1222 (TR:A1B8QS_PARDP) 1e-148 Hypothetical protein Paracoccus denitrificans (pde:Pden_3833) 1e-135 AAA domain 4.6e-21 Domain of unknown function (DUF 4143) 3.6e-11

ORF21 8481 -9287 2-Dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (YP_001414418.1) 7e-136 2-Dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (TR:A7HXX8_PARLI) 1e-103 2-dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (pla:Plav_3155) 1e-102 DAHP synthetase 1 family 5.4e-74

ORF22 9360 -9851 Predicted protein Oxalobacter formigenes (ZP_04577115.1) 5e-22 Predicted protein Oxalobacter formigenes (TR:C3X4E1_OXAFO) 7e-20 Peptidoglycan-associated lipoprotein Candidatus Protochlamydia amoebophila (cu:pc0606) 2e-5 Glycine sipper 6e-5

ORF23 10007 - 10711 RepressorLexA Roseibium sp. Trich SKD4 (ZP_07657520.1) 2e-90 RepressorLexA Roseibium sp. Trich SKD4 (TR:E2CCN3_9RHOB) 1e-68 SOS-response transcriptional repressors Polymorphum gilvum (pgv:SL003B_2110) 1e-58

137

LexA DNA binding domain 2.7e-15 Peptidase S24 –like 5.8e-14

ORF24 10715-12695 Competence protein Silicibacter sp. Trich CH4B (ZP_05742713.1) 8e-115 Competence protein Silicibacter sp. Trich CH4B (TR:C9D485_9RHOB) 1e-110 Glutamyl-tRNA synthetase Polymorphum gilvum (pgv:SL003B_2108) 1e-175 Competence protein 2.4e-64 Domain of unknown function (DUF 4131) 4.7e-10

ORF25 12824 -14260 Glutamyl-tRNA synthetase Labrenzia alexandrii DFL-11 (ZP_05112465.1) 0.0 Glutamyl-tRNA synthetase Labrenzia alexandrii DFL-11 (TR:B9QYE2_9RHOB) 1e-175 Glutamyl-tRNA synthetase Polymorphum gilvum (pgv:SL003B_2108) 1e-175 tRNA synthetases class I (E and Q) catalytic domain 5e-96 Pfam B1171 4e-7

ORF26 14338 - 15636 Unnamed protein product Pelagibacterium halotolerans B2 (YP+004899121.1) 0.0 Citrate synthase Pelagibacterium halotolerans B2 (TR:64R8V4_9RHIZ) 0.0 Citrate synthase Pelagibacterium halotolerans (phl:KKY_1349) 0.0 Citrate synthase 4.1e-131

138

Appendix 2. Annotation of the HERMIcloneB1 according to several databases. The encoded protein was obtained from NCBI NR, Swiss Prot, KEGG and Pfam. Confidence values are shown for each database. No hit: no significant match (with an e-value of 1 x 10-5 as the cut-off).

ORF Position Encoded protein and accession number E value

Scaffold 7 ORF 1 7-465 Glycolate oxidase, iron sulphur subunit Maritimibacter alkaliphilus (ZP_01012214.1) 2e-80 Glycolate oxidase, iron- sulphur subunit Maritimibacter alkaliphilus (TR:A3VC50_9RHOB) 9e-65 Glycolate oxidase, iron- sulphur subunit Jannaschia sp. CCS1 (Jann_0693) 3e-64 Fer4 8 (4Fe 4S dicluster domain) 1.4e-11

ORF 2 470 - 649 FAD linked oxidase-like protein Alphaproteobacteria BAL199 (ZP_02187379.1) 2e-16 Glycolate oxidase subunit glc E Rhodospirillum centenum ATCC 51521 (TR:B61U02_RHOCS) 3e-14 Glycolate oxidase subunit glc E Azospirillum sp. B510 (azl:AZL_004980) 8e-12 FAD linked oxidases, C terminal domain 3.3e-6

ORF 3 806 -1333 Unnamed protein product Maricaulis maris MCS10 (YP_755974.1) 1e-74 Adenine phosphoribosyltransferase Maricaulis maris MCS10 (SP:APT_MARMM) 1e-58 Adenine phosphoribosyltransferase Bradyrhizobium sp. BTAi1 (bbt:BBta_2310) 4.1e-38 Phosphoribosyl transferase domain 3.3e-16

ORF 4 1391 -1981 Endopeptidase Clp Hirschia baltica ATCC49814 (YP_003059143.1) 9e-119 ATP dependent Clp protease proteolytic subunit 1 Hirschia baltica ATCC 49814(TR:C6XPGO_HIRBI) 4e-88 Endopeptidase Clp Hirschia baltica ATCC 49814 (Hbal_0752) 2e-92 Clp protease 3.7e-59

ORF 5 2059 -3690 Unnamed protein product Hyphomonas neptuniumATCC 15444 (YP_759273.1) 2e-134 Sensor histidine kinase/ response Hyphomonas neptunium ATCC 15444 (TR:QOC4SO_HYPNA) 2e-134 Sensor histidine kinase/ response Hyphomonas neptunium (HNE_0543) 5.6e-65 His Kinase A (Phosphor-acceptor domain); Histidine kinase, DNA gyrase; and HSP90-like ATPase; 6.6e-16;2.7e-25; Response regulator receiver domain 8.6e-14

139

ORF 6 3691 - 4254 Hypothetical protein Hirshia baltica ATCC 49814 (YP_003059046.1) 2e-34 Hypothetical protein HbaI_0647 Hirschia baltica ATCC 49814 (TR:C6XNU6_HIRBI) 3e-26 Hypothetical protein HbaI Hirschia baltica (Hbal_0647) 1.1e-19 PAS domain 2.9e-17

ORF 7 4402 - 5316 Hypothetical protein Hba I_0649 Hirschia baltica ATCC 49814 (YP-003059047.1) 9e-64 Hypothetical protein HbaI_0649 Hirschia baltica ATCC 49814 (TR:C6XNU7_HIRBI) 8e-53 Hypothetical protein HbaI Hirschia baltica ATCC 49814 (Hbal_0649) 7.9e-37 No hit - ORF 8 5721 - 8462 Ton-B dependent receptor Sphingopyxis alaskensis RB2256 (YP_616958.1) 0.0 Ton-B dependent receptor Sphingopyxis alaskanensis strain DSM 13593 (YP_616958.1) 1e-159 Ton B-dependent receptor Sphingopyxis alaskanensis (Sala_1913) 2e-77 TonB -dependent receptor plug domain; TonB dependent receptor 3.3e-14; 2.4e-19

ORF 9 8524 - 8904 Hypothetical protein Uncultured marine bacterium MedDCM-OCT-SO5-C259 (ADD93792.1) 3e-09 Hypothetical protein Uncultured marine bacterium MedDCM-OCT-SOS-C259 (TR:D6PDJ1_9BACT) 2e-8 No hit - Domain of unknown function 9.5e-9

ORF 10 9037 -10296 Cell wall surface anchor family protein Oceanicaulis alexandrii (ZP_009580031) 6e-08 Holdfast attachment protein HfaD Caulobacter crescentus strain NA1000/CB15N 9e-25 (TR:B8HOVO_CAUCN) - No hit - No hit -

ORF 11 10299 -11324 Holdfast attachment protein precursor HfaB Hirschia baltica ATCC 49814 (YP_003059049.1) 1e-159 Holdfast attachment protein, precursor Hirschia baltica ATCC 49814 (TR:C6XNU9_HIRBI) 1e-159 Holdfast attachment protein HfaB (Hirschia baltica (Hbal_0651) 1e-118 Curly production assembly/ transport component CsgG 7.3e-13

ORF 12 11317 - 11745 Holdfast attachment protein Hfa A Hirschia baltica ATCC 49814 (YP_003059050.1) 3e-37 Holdfast attachment protein HfaA Hirschia baltica ATCC 49814 (TR:CGXNO_HIRBI) 2e-30 Holdfast attachment protein HfaA Hirschia baltica (Hbal_0652) 6e-26 No hit -

140

ORF 13 11987 - 12418 Unnamed protein product Hyphomonas neptunium ATCC 15444 (YP-761342.1) 2e-52 CBS domain protein Hyphomonas neptunium ATCC 15444 (TR:QOBYV1_HYPNA) 1e-39 Hypothetical protein Hyphomonas neptunium (HNE_2660) 6e-41 CBS domain; CBS domain 1.1e-07; 4.6e-14

ORF 14 15728 -15913 Hypothetical protein Csp_D29560 Curvibacter putative symbiont of Hydra magnipapillata 2e-08 (CBA31926.1) 6e-7 Putative uncharacterized protein Curvibacter putative simbiont of Hydra magnipapillata - (TR:C9YEFO_9BURK) - No hit No hit

ORF 15 18321 - 18779 Conserved hypothetical protein Aurantimonas manganoxydans (ZP_01227369.1) 2e-80 Putative cell wall associated Hydrolase Haemophilus haemolyticusM 19501 (TR:F9GLT8_HAEHA) 1e-54 Hypothetical protein Haemophilus parainfluenzae (PARA_08330) 4e-47 Pfam B83 2.9e-35

ORF 16 19658 - 19987 Mg2+ transporter protein, CorA family protein Psychromonas ingrahamii (YP_944135.1) 6e-31 Mg2 transporter protein Shewanella frigidimarina strain NCIMB 400 (TR:QO87T7_SHEFN) 1e-24 Hypothetical protein; K03284 metal ion transporter, MIT family Azorhizobium caulinodans (AZC_3056) 6e-17 CorA-like Mg 2+ transporter protein 3e-22

141

Appendix 3. Annotation of shotgun sequencing of fosmid B3 via Blastp analysis compared to several databases.The data on column two (encoded protein and accession number) consists of annotated resulted from four different database; the first was from nucleotide collection NCBI; the second was from UniProtKB/TrEMBL and UniProtKB/Swiss-Prot (EBI); the third was from KEGG genes (KEGG database) and the fourth was from Pfam database.The name of the protein was given as its name in UniProtKB, if necessary. A particular function was determined from protein sequences that had an e-value of less or similar to 1x 10-20, or had the same annotations under two or more databases (if the e-value resulted from UniProtKB was larger than 1x 10-20).

ORF Position Encoded protein and accession number E value (bp) Scaffold 5 ORF1 98 -1432 Putative Zn-dependent protease, pmb A-like protein Azospirillum brasilense (CCC98942.1) 2e-145 Peptidase U62 modulator of DNA gyrase Parvibaculum lavamentivorans strain DS-1/DSM 13023 1e-120 (TRA7HQS1_PARLI) 1e-106 Putative peptidase, PmbA-like; K03592 PmbA protein ( Azospirillum lipoferum ali:AZOLI_2345) 1.4e-63 Putative modulator of DNA gyrase

ORF2 1746 -1928 Hypothetical protein Dshi_4011 Dinoroseobacter shibae DFL12 (YP_001542219.1) 1e-16 Hypothetical protein Dshi_4011 Dinoroseobacter shibae DFL12 (TRA8LU30_DINSH) 1e-12 Hypothetical protein Dinoroseobacter shibae (dsh:Dshi_4011) 1e-12 Toxin with endonuclease activity yhaV 5.3e-16

ORF3 2056 -3384 Putative oxidoreductase Marine bacterium 01-004080 (ACA21512.1) 3e-95 Putative uncharacterized protein Silicibacter pomeroyi strain ATCC 700808 (TR:Q5LKKI_SILPO) 1e-78 Hypothetical protein (sil:SPOA0380) 9e-74 FAD-dependent oxidoreductase 5.7e-57

ORF4 3381 -3881 NADH riboflavin 5’ –phosphate oxidoreductase α Proteobacterium BAL199 (ZP_02187032.1) 3e-40 NADH riboflavin 5’ –phosphate oxidoreductase α Proteobacterium BAL199 (TR:A8TK16_9PROT) 9e-33 Flavin reductase domain-containing protein Parvibaculum lavamentivorans (pla:Plav_2888) 2e-27 Flavin reductase like domain 7.3e-31

ORF5 3878 -4882 Co/Zn/Cd cation transporter Hahella chejuensis KCTC2396 (YP-435164.1) 2e-110 Co/Zn/Cd resistance protein cZcD α Proteobacterium BAL199 (TR:A8TTM6_9PROT) 4e-90 Co/Zn/Cd cation transporter Hahella chejuensis (hch:HCH_04023) 1e-78 Cation efflux family 1.8e-50

142

ORF6 5022 -5456 Acetyltransferase, GNAT family protein Rhodobacterales bacterium HTCC2255 (ZP_01447824.1) 8e-42 Acetyltransferase, GNAT family protein Rhodobacterales bacterium HTCC2255 (TR:QOFCP3_9RHOB) 5e-31 GCN5-related N-acetyltransferase Ruegeria sp.(sit:TM1040_2995) 2e-28 Acetyltransferase (GNAT) family 3.3e-10

ORF7 5518 -6273 Putative polyhidroxybutyrate depolymerase Rhodobacteraceae bacteriu (ZP-05123306.1) 6e-6 Putative polyhidroxybutyrate depolymerase Rhodobacteraceae bacterium (TR:B9NUP3_9RHOB) 6e-55 Polyhydroxybutyrate depolymerase Pseudovibrio sp. (psf:PSE_0849) 3e-23 Alpha/beta hydrolase family 3e-10

ORF8 6322 -7113 NG,NG-dimethyl arginine dymethylaminohydrolase Rhodobacterales bacterium HTCC2150 (ZP_01741609.1) 1e-118 Amidinotransferase Rhodobacterales bacterium HTCC2150 (TR:C8S44S_9RHOB) 3e-93 NG,NG-dimethylarginine dimethylaminohydrolase Silicibacter pomeroyi (sil:SPOA0064) 2e-90 Amidinotransferase 2.4e-19

ORF9 7178 -8335 Unnamed protein product Paracoccus denitrificans PD 1222 (YP_917595.1) 0.0 Unnamed protein product Paracoccus denitrificans PD 1222 (TR:A1B8QS_PARDP) 1e-148 Hypothetical protein Paracoccus denitrificans (pde:Pden_3833) 1e-135 AAA domain 4.6e-21 Domain of unknown function (DUF 4143) 3.6e-11

ORF10 8481 -9287 2-Dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (YP_001414418.1) 7e-136 2-Dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (TR:A7HXX8_PARLI) 1e-103 2-dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (pla:Plav_3155) 1e-102 DAHP synthetase 1 family 5.4e-74

ORF11 9360 -9851 Predicted protein Oxalobacter formigenes (ZP_04577115.1) 5e-22 Predicted protein Oxalobacter formigenes (TR:C3X4E1_OXAFO) 7e-20 Peptidoglycan-associated lipoprotein Candidatus Protochlamydia amoebophila (cu:pc0606) 2e-5 Glycine sipper 6e-5

ORF12 10007 - RepressorLexA Roseibium sp. Trich SKD4 (ZP_07657520.1) 2e-90 10711 RepressorLexA Roseibium sp. Trich SKD4 (TR:E2CCN3_9RHOB) 1e-68 SOS-response transcriptional repressors Polymorphum gilvum (pgv:SL003B_2110) 1e-58 LexA DNA binding domain 2.7e-15 Peptidase S24 –like 5.8e-14

143

ORF13 10715- Competence protein Silicibacter sp. Trich CH4B (ZP_05742713.1) 8e-115 12695 Competence protein Silicibacter sp. Trich CH4B (TR:C9D485_9RHOB) 1e-110 Glutamyl-tRNA synthetase Polymorphum gilvum (pgv:SL003B_2108) 1e-175 Competence protein 2.4e-64 Domain of unknown function (DUF 4131) 4.7e-10

ORF14 12824 - Glutamyl-tRNA synthetase Labrenzia alexandrii DFL-11 (ZP_05112465.1) 0.0 14260 Glutamyl-tRNA synthetase Labrenzia alexandrii DFL-11 (TR:B9QYE2_9RHOB) 1e-175 Glutamyl-tRNA synthetase Polymorphum gilvum (pgv:SL003B_2108) 1e-175 tRNA synthetases class I (E and Q) catalytic domain 5e-96 Pfam B1171 4e-7

ORF15 14338 - Unnamed protein product Pelagibacterium halotolerans B2 (YP+004899121.1) 0.0 15636 Citrate synthase Pelagibacterium halotolerans B2 (TR:64R8V4_9RHIZ) 0.0 Citrate synthase Pelagibacterium halotolerans (phl:KKY_1349) 0.0 Citrate synthase 4.1e-131 Scaffold 10 ORF16 59-967 Beta-ketoacyl-acyl-carrier protein synthase III Hirschia baltica (YP_003061548.1) 1e-162 Beta-ketoacyl-acyl-carrier protein synthase III Hirschia baltica (TR:C6XS10_HIRBI) 1e-123 3-oxoacyl (acyl-carrier-protein/ ACP) synthase III 2.6e-21 3-oxoacyl (acyl-carrier-protein/ ACP) synthase III C terminal 4e-19

ORF17 1007-1561 Hypothetical protein PHZ_c1374 Phenylobacterium zucineum HLKI (YP_002130217.1) 2e-84 Putative uncharacterized protein Phenylobacterium zucineum (TR:B4R9M4_PHEZH) 4e-19 HD domain 1.7e-08 PfamB 697 1.8e-40

ORF18 1585-1896 No hit - No hit - No hit - No hit -

ORF19 1984-3144 Quinolinate synthetase complex subunit alpha Hirschia baltica ATCC 49814 (YP_003059523.1) 6e-174 Quinolinate synthetase complex A sub unit Hirschia baltica (TR: C6XRJ4_HIRBI) 1e-136 Quinolinate synthetase complex subunit alpha Hirschia baltica (hba:Hbal_1134) 1e-138 NfeD-like C-terminal partner binding 4.7e-05

144

ORF20 3264-3665 Bacterial protein of unknown function DUF937Congregibacter litoralis KT71 (ZP_01102546.1) 2e-21 Putative uncharacterized protein Congregibacter litoralis (TR:A4A7JI_9GAMM) 4e-24 Hypothetical protein Rhodoferax ferrireducens (rfr:Rfer_2429) 5e-4 Bacterial protein of unknown function (DUF937) 2.5e-14

145

Appendix 4. Annotation of shotgun sequencing of fosmid B5 via Blastp analysis compared to several databases.The data on column two (encoded protein and accession number) were resulted from annotation from four different databases; the first was from nucleotide collection NCBI; the second was from UniProtKB/TrEMBL and UniProtKB/Swiss-Prot (EBI); the third was from KEGG genes (KEGG database) and the fourth was from Pfam database. The name of the protein was given as it was written in UniProtKB, if necessary. A particular function was considered from protein sequences that had an e-value of less or similar to 1x 10-20, or had the same annotations under two or more databases (if the e-value resulted from UniProtKB was larger than 1x 10-20).

ORF Position Encoded protein and accession number E value Scaffold 4 ORF1 203-967 ATP synthase subunit 6Sporisorium reilianum SRZ2 (CBQ72554.1) 3e-176 ATP synthase subunit 6Sporisorium reilianum (TR:E6ZZ97_SPORF) 1e-135 ATP synthase subunit 6 Ustilago maydis (uma:UsmafMp06) 1e-117 ATP synthase A chain 3.8e-54

ORF2 2595-4073 Cytochrome C oxidase subunit 1 Ustilago maydis(YP_762688.1) 0.0 Cytochrome C oxidase subunit 1 Ustilago maydis (SP:COX1_USTMA) 0.0 Cytochrome c oxidase subunit1Ustilago maydis (uma:UsmafMp08) 0.0 Cytochrome C and quinol oxidase polypeptide 1 7.7e-166

ORF3 5605-6222 Hypothetical protein Ustilago maydis (XP _759537.1) 1e-7 Putative uncharacterized protein Ustilago maydis(TR:Q4P923_USTMA) 3e-9 Hypothetical protein Ustilago maydis (uma:UM03390.1) 7e-7 NUMOD 1 (DNA-binding helix-turn-helix 1.8e-5

Scaffold 5 ORF4 98-1432 Putative Zn-dependent protease, pmb A-like protein Azospirillum brasilense (CCC98942.1) 2e-145 Peptidase U62 modulator of DNA gyrase Parvibaculum lavamentivorans strain DS-1/DSM 13023 1e-120 (TRA7HQS1_PARLI) Putative peptidase, PmbA-like; K03592 PmbA protein Azospirillum lipoferum (ali:AZOLI_2345) 1e-106 Putative modulator of DNA gyrase 1.4e-63

ORF5 1746 -1928 Hypothetical protein Dshi_4011 Dinoroseobacter shibae DFL12 (YP_001542219.1) 1e-16 Hypothetical protein Dshi_4011 Dinoroseobacter shibae DFL12(TRA8LU30_DINSH) 1e-12 Hypothetical protein Dinoroseobacter shibae (dsh:Dshi_4011) 1e-12 Toxin with endonuclease activity yhaV 5.3e-16

146

ORF6 2056 -3384 Putative oxidoreductase Marine bacterium 01-004080 (ACA21512.1) 3e-95 Putative uncharacterized proteinSilicibacter pomeroyi strain ATCC 700808 (TR:Q5LKKI_SILPO) 1e-78 Hypothetical protein Silicibacter pomeroyi strain ATCC 700808 (sil:SPOA0380) 9e-74 FAD-dependent oxidoreductase 5.7e-57

ORF7 3381 -3881 NADH riboflavin 5’ –phosphate oxidoreductase α Proteobacterium BAL199 (ZP_02187032.1) 3e-40 NADH riboflavin 5’ –phosphate oxidoreductase α Proteobacterium BAL199 (TR:A8TK16_9PROT) 9e-33 Flavin reductase domain-containing protein Parvibaculum lavamentivorans (pla:Plav_2888) 2e-27 Flavin reductase like domain 7.3e-31

ORF8 3878 -4882 Co/Zn/Cd cation transporter Hahella chejuensis KCTC2396(YP-435164.1) 2e-110 Co/Zn/Cd resistance protein cZcD α Proteobacterium BAL199 (TR:A8TTM6_9PROT) 4e-90 Co/Zn/Cd cation transporter Hahella chejuensis (hch:HCH_04023) 1e-78 Cation efflux family 1.8e-50

ORF9 5022 -5456 Acetyltransferase, GNAT family protein Rhodobacterales bacterium HTCC2255(ZP_01447824.1) 8e-42 Acetyltransferase, GNAT family protein Rhodobacterales bacterium HTCC2255 (TR:QOFCP3_9RHOB) 5e-31 GCN5-related N-acetyltransferase Ruegeria sp.(sit:TM1040_2995) 2e-28 Acetyltransferase (GNAT) family 3.3e-10

ORF10 5518 -6273 Putative polyhidroxybutyrate depolymerase Rhodobacteraceae bacterium (ZP-05123306.1) 6e-66 Putative polyhidroxybutyrate depolymerase Rhodobacteraceae bacterium (TR:B9NUP3_9RHOB) e-55 Polyhydroxybutyrate depolymerase Pseudovibrio sp (psf:PSE_0849) 3e-23 Alpha/beta hydrolase family 3e-10

ORF11 6322 -7113 NG,NG-dimethyl arginine dymethylaminohydrolase Rhodobacterales bacterium HTCC2150 1e-118 (ZP_01741609.1) Amidinotransferase Rhodobacterales bacterium HTCC2150 (TR:C8S44S_9RHOB) 3e-93 NG,NG-dimethylarginine dimethylaminohydrolaseSilicibacter pomeroyi (sil:SPOA0064) 2e-90 Amidinotransferase 2.4e-19

ORF12 7178 -8335 Unnamed protein product Paracoccus denitrificans PD 1222 (YP_917595.1) 0.0 Unnamed protein product Paracoccus denitrificans PD 1222 (TR:A1B8QS_PARDP) 1e-148 Hypothetical protein Paracoccus denitrificans (pde:Pden_3833) 1e-135 Domain of unknown function (DUF 4143) 4.6e-21

147

ORF13 8481 -9287 2-Dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans (YP_001414418.1) 7e-136 2-Dehydro-3-deoxyphosphooctonate aldolase Parvibaculum lavamentivorans(TR:A7HXX8_PARLI) 1e-103 2-dehydro-3-deoxyphosphooctonate aldolaseParvibaculum lavamentivorans (pla:Plav_3155) 1e-102 DAHP synthetase 1 family 5.4e-74

ORF14 9360 -9851 Predicted protein Oxalobacter formigenes (ZP_04577115.1) 5e-22 Predicted protein Oxalobacter formigenes (TR:C3X4E1_OXAFO) 7e-20 Peptidoglycan-associated lipoprotein Candidatus Protochlamydia amoebophila (cu:pc0606) 2e-5 Glycine sipper 6e-5

ORF15 10007 - 10711 Repressor Lex A Roseibium sp. Trich SKD4 (ZP_07657520.1) 2e-90 RepressorLexA Roseibium sp. Trich SKD4 (TR:E2CCN3_9RHOB) 1e-68 SOS-response transcriptional repressors Polymorphum gilvum (pgv:SL003B_2110) 1e-58 LexA DNA binding domain 2.7e-15 Peptidase S24 –like 5.8e-14

ORF16 10715-12695 Competence protein Silicibacter sp. Trich CH4B (ZP_05742713.1) 8e-115 Competence protein Silicibacter sp. Trich CH4B (TR:C9D485_9RHOB) 1e-110 Glutamyl-tRNA synthetase(Polymorphum gilvumpgv:SL003B_2108) 1e-175 Competence protein; Domain of unknown function (DUF 4131) 2.4e-64; 4.7e-10

ORF17 12824 -14260 Glutamyl-tRNA synthetase Labrenzia alexandrii DFL-11 (ZP_05112465.1) 0.0 Glutamyl-tRNA synthetase Labrenzia alexandrii DFL-11 (TR:B9QYE2_9RHOB) 1e-175 Glutamyl-tRNA synthetase Polymorphum gilvum (pgv:SL003B_2108) 1e-175 tRNA synthetases class I (E and Q) catalytic domain; Pfam B1171 5e-96; 4e-7

ORF18 14338 - 15636 Unnamed protein product Pelagibacterium halotolerans B2 (YP+004899121.1) 0.0 Citrate synthase (Pelagibacterium halotolerans B2 TR:64R8V4_9RHIZ) 0.0 Citrate synthase Pelagibacterium halotolerans (phl:KKY_1349) 0.0 Citrate synthase 4.1e-131

Scaffold 6 ORF19 1-745 NADH ubiquinone oxidoreductase subunit 1 Ustilago maydis(YP_762697.1) 1e-169 NADH ubiquinone oxidoreductase chain 1Ustilago maydis(SP:NU1M_USTMA) 1e-130 NADH:ubiquinone oxidoreductase subunit 1Ustilago maydis (uma:UsmafMp10) 1e-124 NADH dehydrogenase 2e-66

148

ORF20 2072-2698 Cytochrome C oxidase subunit 2 Ustilago maydis(YP_762685.1) 7e-145 Cytochrome C oxidase subunit 2 Ustilago maydis (SP:COX2_USTMA) 1e-112 Cytochrome c oxidase subunit 2 Ustilago maydis (uma:UsmafMp05) 1e-105 Cytochrome C oxidase subunit II, transmembrane domain 7.6e-30 Cytochrome C oxidase subunit II, periplasmic domain 4.9e-36

Scaffold 12 ORF21 210 -380 Ureidoglycolate lyase Leadbetterella byssophilia DSM 17132 (YP_003997993.1) 4e-16 2-keto-4-pentenoate hydratase/ 2-oxohepta-3-ene-17-dioic acid hydratase Rhodobacter sphaeroides 6e-13 (TR:Q3J118_RHOS4) Ureidoglycolate lyaseLeadbetterella byssophilia (lby:Lbys_1938) 8e-13 Fumarylacetoacetate (FAA) hydrolase family 2.6e-11

ORF22 1746 - 2318 Hypothetical protein RL0149_c026590 Roseobacter litoralis Och 149 (YP_004691585.1) 1e-34 Putative uncharacterized protein Roseobacter litoralis(TR:F7ZEA9_ROSLO) 2e-28 Hypothetical proteinRoseobacter litoralis (rli:RLO149_c026590) 2e-22 No hit -

ORF23 2387 -2980 Dihydroxyacid dehydratase Hoeflea phototrophica DFL-43 (ZP_02167710.1) 7e-94 Dihydroxyacid dehydratase Hoeflea phototrophica(TR:A9DBJ3_9RHIZ) 8e-76 Dihydroxy-acid dehydratase Silicibacter pomeroyi (sil:SPO2410) 1e-75 Dehydratase family 1.3e-38

ORF24 3953 - 4171 Dihydroxyacid dehydratase Mesorhizobium alhaqi (ZP_09297654.1) 6e-33 Dihydroxyacid dehydratase Xanthobacter autotrophicus (TR:A71GU9_XANP2) 4e-26 Dihydroxy-acid dehydratase Xanthobacter autotrophicus (xau:Xaut_1998) 4e-27 Dehydratase family 0.065

ORF25 4469 -5656 TIR protein Prosthecochloris aestuarii strain DSM 271 (YP_002014909.1) 6e-06 TIR protein Prosthecochloris aestuarii (TR:B4S3R6_PROA2) 4e-5 TIR protein Prosthecochloris aestuarii (paa:Paes_0204) 5e-5 TIR domain 3.9e-8

ORF26 5425 -5949 Leucine-rich repeat- containing protein Syntrophomonas wolfei(YP-754057.1) 9e-13 Leucine-rich repeat (LRR) protein-like protein Syntrophomonas wolfei (TR:Q0AX68_SYNWW) 7e-12 Leucine-rich repeat-containing protein Syntrophomonas wolfei (swo:Swol_1379) 1e-11 Leucine rich repeats (2 copies) 6.7e-9

149

ORF27 5969 -6922 NADH-dependent degydrogenase Ahrensia sp R2A130 (ZP_07375767.1) 9e-105 Inositol 2-dehydrogenase Rhizobium melioti (SP:M12D_RHIME) 4e-13 Oxidoreductase (Roseobacter litoralisrli:RLO149_c006530) 2e-67 Oxidoreductase family, NAD-binding Rossman fold 1.3e-14

ORF28 7003 -7416 SMP-30/ gluconolaconase/LRE-like region family protein Achromobacter xylosoxidans 5e-33 (YP_003980645.1) SMP-30/ gluconolaconase/LRE-like region family protein Achromobacter xylosoxidans 7e-27 (TR: E3H171_ACHXA) SMP-30/gluconolaconase/LRE-like region family protein Achromobacter xylosoxidans 2e-27 (axy:AXYL_04615) No hit -

ORF29 8412 - 8792 Molybdate ABC transporter nucleotide binding protein/ATPase Agrobacterium sp. (YP_004279798.1) 1e-39 Molybdate ABC transporter nucleotide binding /ATPase protein Agrobacterium sp. 2e-31 (TR:FOL2TU_AGRSH) Molybdate ABC transporter nucleotide-binding protein/ ATPase Agrobacterium sp. 9e-33 (agr:AGROH133_08706) ABC transporter 4.2e-13

ORF30 8789 -9109 Molybdate ABC transporter, ATP binding protein Oceanicola granulosus(ZP_01157787.1) 5e-23 Molybdate ABC transporter, ATP binding protein Oceanicola granulosus (TR:Q2CBP2_9RHOB) 7e-20 Molybdenum ABC transporter, ATP-binding protein Dehalococcoides ethenogenes (det:DET1159) 6e-14 No hit -

ORF31 9061 -9759 Molybdenum transport system permease protein ModB Photobacterium damselaesubspecies damselae 1e-85 CIP 102761 (ZP_06157227.1) Binding-protein-dependent transport system inner membrane Cronobacter 5e-71 turicensis(TR:C9XZ07_CROTZ) Molybdate ABC transporter inner membrane subunit Desulfatibacillum alkenivorans (dal:Dalk_3645) 3e-47 Binding-protein-dependent transport system inner membrane 2.3e-24 Pfam-B 3338 4e-7

ORF32 10013 -10348 Ribosomal protein S2 Rhodobacter sp. SW2 (ZP_05843092.1) 2e-66 30S ribosomal protein S2 Citreicella sp.(TR:D0D246_9RHOB) 6e-50 30S ribosomal protein S2Roseobacter denitrificans (rde:RD1_2653) 1e-44 Ribosomal protein S2 7.3e-41

150

ORF33 10702 - 10980 Ribosomal protein S2 Methylosinus trichosporium OB3b (ZP_06887428.1) 2e-41 30S ribosomal protein S2 Methylocystys sp. (TR:E8L2S7_9RHIZ) 6e-35 30S ribosomal protein S2Methylocella silvestris (msl:Msil_1694) 2e-29 Ribosomal protein S2 2.5e-24

ORF34 11095 -12030 Elongation factor Ts Agrobacterium vitis S4 (YP_002549818.1) 2e-134 Elongation factor Ts Polymorphum gilvum(TR;F21Y16_POLGS) 1e-110 Elongation factor TsPolymorphum gilvum (pgv:SL003B_2094) 1e-93 UBA/TS-N domain 5.8e-10 Elongation factor TS 2.2e-70

ORF35 12234 -12836 Superoxide dismutase, Fe Roseovarius nubinhibens(ZP_00961078.1) 6e-99 Superoxide dismutase, Fe Roseovarius nubinhibens (TR:A3SQA9_9RHOB) 4e-78 Superoxide dismutase Ruegeria sp. (sit:TM1040_0976) 1e-65 Iron/manganese superoxide dismutase, alpha -hairpin domain 2.2e-22 Iron/ manganese superoxide dismutase, C-terminal domain 5.6e-35

ORF36 12919 -13065 RarD protein Roseobacter sp. GAI101 (ZP_05099655.1) 3e-08 Chloramphenicol-sensitive protein RarDRuegeria sp. (sit:TM1040_0071) 5e-5 RarD protein Roseobacter sp.(TR:B7RLM4_9RHOB) 1e-6 No hit 1e-31

ORF37 14006 - 14470 RarD Roseovarius sp. 217 (ZP_01038290.1) 1e-29 RarD Silicibacter pomeroyi (TR:Q5LQZ4_SILPO) 9e-21 Chloramphenicol-sensitive protein Silicibacter pomeroyi (sil:SPO2338) 1.8e-6 EamA-like transporter family 4e-65

ORF38 14410 -14964 Decarboxylase family protein Sulfitobacter sp. (ZP_00962909.1 3e-50 Decarboxylase, putative Oceanibulbus indolifex(TR:A9E8B4_9RHOB) 2e-45 Hypothetical protein Rhodobacter sphaeroides (rsq:Rsph17025_1870) 2.3e-35 No hit -

ORF39 15022 - 15234 rhlE gene product Ruegeria pomeroyi (YP:166684.1) 1e-5 DEAD/DEAH box helicase domain protein Parvibaculum lavamentivorans (TR: A7HY74_PARL1) 1e-5 No hit - No hit -

151

Appendix 5. Annotation of shotgun sequencing of fosmid B6 via Blastp analysis compared to several databases.The data on column two (encoded protein and accession number) were resulted from annotation from several databases; the first was from nucleotide collection NCBI; the second was from UniProtKB/TrEMBL and UniProtKB/Swiss-Prot (EBI); the third was from KEGG genes (KEGG database) and the fourth was from Pfam database. The name of the protein was provided as it was written in UniProtKB, if necessary. A particular function was identified from protein sequences that had an e-value of less or similar to 1x 10-20, or had the same annotations under two or more databases (if the e-value resulted from UniProtKB was larger than 1x 10-20).

ORF Position Encoded protein and accession number E value Scaffold 12 ORF 1 210 -380 Ureidoglycolate lyase Leadbetterella byssophilia DSM 17132 (YP_003997993.1) 4e-16 2-keto-4-pentenoate hydratase/ 2-oxohepta-3-ene-17-dioic acid hydratase Rhodobacter sphaeroides 6e-13 (TR:Q3J118_RHOS4) 8e-13 Ureidoglycolate lyase Leadbetterella byssophilia (lby:Lbys_1938) 2.6e-11 Fumarylacetoacetate (FAA) hydrolase family

ORF 2 1746 - 2318 Hypothetical protein RL0149_c026590 Roseobacter litoralis Och 149 (YP_004691585.1) 1e-34 Putative uncharacterized protein Roseobacter litoralis (TR:F7ZEA9_ROSLO) 2e-28 Hypothetical protein Roseobacter litoralis (rli:RLO149_c026590) 2e-22 No hit -

ORF 3 2387 -2980 Dihydroxyacid dehydratase Hoeflea phototrophica DFL-43 (ZP_02167710.1) 7e-94 Dihydroxyacid dehydratase Hoeflea phototrophica (TR:A9DBJ3_9RHIZ) 8e-76 Dihydroxy-acid dehydratase Silicibacter pomeroyi (sil:SPO2410) 1e-75 Dehydratase family 1.3e-38

ORF 4 3953 - 4171 Dihydroxyacid dehydratase Mesorhizobium alhaqi(ZP_09297654.1) 6e-33 Dihydroxyacid dehydratase Xanthobacter autotrophicus(TR:A71GU9_XANP2) 4e-26 Dihydroxy-acid dehydratase Xanthobacter autotrophicus (xau:Xaut_1998) 4e-27 Dehydratase family 0.065

ORF 5 4469 -5656 TIR protein Prosthecochloris aestuarii strain DSM 271 (YP_002014909.1) 6e-05 TIR protein Prosthecochloris aestuarii (TR:B4S3R6_PROA2) 4e-5 TIR protein Prosthecochloris aestuarii (paa:Paes_0204) 5e-5 TIR domain 3.9e-8

152

ORF 6 5425 -5949 Leucine-rich repeat- containing protein Syntrophomonas wolfei(YP-754057.1) 9e-13 Leucine-rich repeat (LRR) protein-like protein Syntrophomonas wolfei (TR:Q0AX68_SYNWW) 7e-12 Leucine-rich repeat-containing protein Syntrophomonas wolfei (swo:Swol_1379) 1e-11 Leucine rich repeats (2 copies) 6.7e-9

ORF 7 5969 -6922 NADH-dependent degydrogenase Ahrensia sp R2A130 (ZP_07375767.1) 9e-105 Inositol 2-dehydrogenase Rhizobium melioti(SP:M12D_RHIME) 4e-13 Oxidoreductase Roseobacter litoralis (rli:RLO149_c006530) 2e-67 Oxidoreductase family, NAD-binding Rossman fold 1.3e-14

ORF 8 7003 -7416 SMP-30/ gluconolaconase/LRE-like region family protein Achromobacter xylosoxidans(YP_003980645.1) 5e-33 SMP-30/ gluconolaconase/LRE-like region family protein Achromobacter xylosoxidans 7e-27 (TR: E3H171_ACHXA) SMP-30/gluconolaconase/LRE-like region family protein Achromobacter xylosoxidans (axy:AXYL_04615) 2e-27 No hit -

ORF 9 8412 - 8792 Molybdate ABC transporter nucleotide binding protein/ATPase Agrobacterium sp. (YP_004279798.1) 1e-39 Molybdate ABC transporter nucleotide binding /ATPase protein Agrobacterium sp. (TR:FOL2TU_AGRSH) 2e-31 Molybdate ABC transporter nucleotide-binding protein/ ATPase Agrobacterium sp.(agr:AGROH133_08706) 9e-33 ABC transporter 4.2e-13

ORF 10 8789 -9109 Molybdate ABC transporter, ATP binding protein Oceanicola granulosus 5e-23 (ZP_01157787.1) Molybdate ABC transporter, ATP binding protein Oceanicola granulosus 7e-20 (TR:Q2CBP2_9RHOB) Molybdenum ABC transporter, ATP-binding protein Dehalococcoides ethenogenes (det:DET1159) 6e-14 No hit -

ORF 11 9061 -9759 Molybdenum transport system permease protein ModB Photobacterium damselae subspecies damselae CIP 102761 1e-85 (ZP_06157227.1) Binding-protein-dependent transport system inner membrane Cronobacter turicensis (TR:C9XZ07_CROTZ) 5e-71 Molybdate ABC transporter inner membrane subunit Desulfatibacillum alkenivorans (dal:Dalk_3645) 3e-47 Binding-protein-dependent transport system inner membrane ; Pfam-B 3338 2.3e-24; 4e-7

153

ORF 12 10013 -10348 Ribosomal protein S2 Rhodobacter sp. SW2 (ZP_05843092.1) 2e-66 30S ribosomal protein S2 Citreicella sp. (TR:D0D246_9RHOB) 6e-50 30S ribosomal protein S2 Roseobacter denitrificans (rde:RD1_2653) 1e-44 Ribosomal protein S2 7.3e-41

ORF 13 10702 - 10980 Ribosomal protein S2 Methylosinus trichosporium OB3b (ZP_06887428.1) 2e-41 30S ribosomal protein S2 Methylocystys sp. (TR:E8L2S7_9RHIZ) 6e-35 30S ribosomal protein S2 Methylocella silvestris (msl:Msil_1694) 2e-29 Ribosomal protein S2 2.5e-24

ORF 14 11095 -12030 Elongation factor Ts Agrobacterium vitis S4 (YP_002549818.1) 2e-134 Elongation factor Ts Polymorphum gilvum (TR:F21Y16_POLGS) 1e-110 Elongation factor Ts Polymorphum gilvum (pgv:SL003B_2094) 1e-93 UBA/TS-N domain 5.8e-10 Elongation factor TS 2.2e-70

ORF 15 12234 -12836 Superoxide dismutase, Fe Roseovarius nubinhibens (ZP_00961078.1) 6e-99 Superoxide dismutase, Fe Roseovarius nubinhibens (TR:A3SQA9_9RHOB) 4e-78 Superoxide dismutase Ruegeria sp. (sit:TM1040_0976) 1e-65 Iron/manganese superoxide dismutase, alpha -hairpin domain 2.2e-22 Iron/ manganese superoxide dismutase, C-terminal domain 5.6e-35

ORF 16 12919 -13065 RarD protein Roseobacter sp. GAI101 (ZP_05099655.1) 3e-08 Chloramphenicol-sensitive protein RarD Ruegeria sp. (sit:TM1040_0071) 5e-5 RarD protein Roseobacter sp.(TR:B7RLM4_9RHOB) 1e-6 No hit -

ORF 17 14006 - 14470 RarD Roseovarius sp. 217(ZP_01038290.1) 1e-31 RarD Silicibacter pomeroyi (TR:Q5LQZ4_SILPO) 1e-29 Chloramphenicol-sensitive protein Silicibacter pomeroyi (sil:SPO2338) 9e-21 EamA-like transporter family 1.8e-6

ORF 18 14410 -14964 Decarboxylase family protein Sulfitobacter sp. (ZP_00962909.1) 4e-65 Decarboxylase, putative Oceanibulbus indolifex(TR:A9E8B4_9RHOB) 3e-50 Hypothetical protein Rhodobacter sphaeroides (rsq:Rsph17025_1870) 2e-45 Possible lysine decarboxylase 2.3e-35

154

ORF 19 15022-15234 rhlE gene product Ruegeria pomeroyi(YP:166684.1) 1e-5 DEAD/DEAH box helicase domain protein Parvibaculum lavamentivorans (TR:A7HY74_PARL1) 1e-5 No hit - No hit -

155

Appendix 6. Annotation of shotgun sequencing of fosmid B7 via Blastp analysis compared to several databases.The data on column two (encoded protein and accession number) were resulted from annotation from several databases; the first was from nucleotide collection NCBI; the second was from UniProtKB/TrEMBL and UniProtKB/Swiss-Prot (EBI); the third was from KEGG genes (KEGG database) and the fourth was from Pfam database. The name of the protein was provided as it was written in UniProtKB, if necessary. A particular function was identified from protein sequences that had an e-value of less or similar to 1x 10-20, or had the same annotations under two or more databases (if the e-value resulted from UniProtKB was larger than 1x 10-20).

ORF Position Encoded protein and accession number E value

Scaffold 9 ORF 1 14 -535 1-deoxy-D-xylulose-5-phosphate synthaseMaribacter sp. HTCC2170 (YP_003861878.1) 2e-61 1-deoxy-D-xylulose-5-phosphate synthase Maribacter sp. (TR:A4ANK7_MARSH) 6e-50 1-deoxy-D-xylulose-5-phosphate synthase Maribacter sp.(fbc:FB2170_04810) 7e-52 Transketolase, C-terminal domain 2.9e-17

ORF 2 495 -1577 Unnamed protein product Cellulophaga algicola DSM 14237 (YP_004164557.1) 2e-154 Putative uncharacterized protein Cellulophaga algicola(TR:E6XCSU_CELAD) 1e-124 Hypothetical protein Cellulophaga algicola (cao:Celal_1758) 1e-118 Protein of unknown function DUF 3078 3.7e-30 Pfam-B 13797 5.4e-35

ORF 3 1587-2366 Deoxyguanosinetriphosphate triphosphohydrolase Muricauda ruestringensis DSM 13258 8e-131 (YP_004787999.1) Deoxyguanosinetriphosphate triphosphohydrolase Muricauda ruestringensis(TR:G2PQ12_9FLAO) 1e-102 Deoxyguanosinetriphosphate triphosphohydrolase Muricauda ruestringensis (mrs:Murru_1536) 1e-105 No hit -

ORF 4 2396 - 2635 No hit - No hit - No hit - No hit -

ORF 5 2657 - 3394 Ribonuclease 3 Helicobacter pullorum (ZP_04808409.1) 1e-56

Ribonuclease 3 Sediment metagenome (TR:D9PK52_9ZZZZ) 1e-56 Ribonuclease III Alkaliphilus oremlandii (aoe:Clos_1458) 7e-44 Double strands RNA binding motif Pfam-B 6419 9.2e-21

156

ORF 6 3513 -3977 NusB antitermination factor Thermaerobacter subterraneus DSM 13965 (ZP_07835044.1) 5e-29 N utilization substance protein B homolog Thermaerobacter subterraneus(TR:E4M0V9_9FIRM) 4e-22 NusB antitermination factor Thermaerobacter marianensis (tmr:Tmar_1173) 6e-23 NusB family 1e-35

ORF 7 4075 -4269 50S ribosomal protein L32 Anaeromyxobacter dehalogenans (YP_465959.1) 1e-11 50S ribosomal protein L32 Anaeromyxobacter dehalogenans(TR:B8JEX7_ANAD2) 1e-7 Ribosomal protein L32 Desulfarculus baarsii (dbr:Deba_2393) 6e-4 Ribosomal L32p protein family 2.4e-15

ORF 8 4435 -5310 Putative transposase Candidatus Nitrospira defluvii (YP_003799838.1) 4e-68 Putative transposase Candidatus Nitrospira defluvii (TR:D8P8T1_9BACT) 6e-52 Putative transposase Candidatus Nitrospira defluvii (vnde:NIDE4248) 4e-55 Winged helix-turn helix 3.8e-10 Integrase core domain 3.2e-17

ORF 9 5416 -6606 Glycosyltransferase Clostridium ljungdahlii DSM 13528 (YP_003778727.1) 1e-50 Predicted glycosyltransferase Clostridium ljungdahlii 1e-43 Glycosyl transferase Clostridium ljungdahlii (clj:CLJU_c05430) 1e-43 Glycosyl transferase group 1 1.5e-23

157

Appendix 7. Annotation of shotgun sequencing of fosmid B10 via Blastp analysis compared to several databases.The data on column two (encoded protein and accession number) were resulted from annotation from several databases; the first was from nucleotide collection NCBI; the second was from UniProtKB/TrEMBL and UniProtKB/Swiss-Prot (EBI); the third was from KEGG genes (KEGG database) and the fourth was from Pfam database. The name of the protein was provided as it was written in UniProtKB, if necessary. A particular function was identified from protein sequences that had an e-value of less or similar to 1x 10-20, or had the same annotations under two or more databases (if the e-value resulted from UniProtKB was larger than 1x 10-20).

ORF Position Encoded protein and accession number E value

Scaffold 9 ORF 1 14 -535 1-deoxy-D-xylulose-5-phosphate synthase Maribacter sp. HTCC2170 (YP_003861878.1) 2e-61 1-deoxy-D-xylulose-5-phosphate synthase Maribacter sp. (TR:A4ANK7_MARSH) 6e-50 1-deoxy-D-xylulose-5-phosphate synthase Maribacter sp. (fbc:FB2170_04810) 7e-52 Transketolase, C-terminal domain 2.9e-17

ORF 3 1587-2366 Deoxyguanosinetriphosphate triphosphohydrolase Muricauda ruestringensis DSM 13258 8e-131 (YP_004787999.1) Deoxyguanosinetriphosphate triphosphohydrolase Muricauda ruestringensis 1e-102 (TR:G2PQ12_9FLAO) Deoxyguanosinetriphosphate triphosphohydrolase Muricauda ruestringensis (mrs:Murru_1536) 1e-105 No hit -

ORF 4 2396 - 2635 No hit - No hit - No hit - No hit -

158

ORF 5 2657 - 3394 Ribonuclease 3 Helicobacter pullorum (ZP_04808409.1) 1e-56 Ribonuclease 3 Sediment metagenome (TR:D9PK52_9ZZZZ) 1e-56 Ribonuclease III Alkaliphilus oremlandii (aoe:Clos_1458) 7e-44 Double strands RNA binding motif 9.2e-21 Pfam-B 6419 5.6e-12

ORF 6 3513 -3977 NusB antitermination factor Thermaerobacter subterraneus DSM 13965 (ZP_07835044.1) 5e-29 N utilization substance protein B homolog Thermaerobacter 4e-22 subterraneus(TR:E4M0V9_9FIRM) NusB antitermination factor Thermaerobacter marianensis (tmr:Tmar_1173) 6e-23 NusB family 1e-35

ORF 8 4435 -5310 Putative transposase Candidatus Nitrospira defluvii (YP_003799838.1) 4e-68 Putative transposase Candidatus Nitrospira defluvii(TR:D8P8T1_9BACT) 6e-52 Putative transposase Candidatus Nitrospira defluvii (nde:NIDE4248) 4e-55 Winged helix-turn helix 3.8e-10 Integrase core domain 3.2e-17

ORF 9 5416 - 6606 Glycosyltransferase Clostridium ljungdahlii DSM 13528 (YP_003778727.1) 1e-50 Predicted glycosyltransferase Clostridium ljungdahlii 1e-43 Glycosyl transferase Clostridium ljungdahlii (clj:CLJU_c05430) 1e-43 Glycosyl transferase group 1 1.5e-23

Scaffold 11 ORF 10 79 - 1680 Alanyl tRNA synthetase Haliscomenobacter hydrossis (YP_004449404.1) 0.0 Alanyl tRNA synthetase Haliscomenobacter hydrossis (TR:F4KWP4_HALH1) 1e-175 Alanyl-tRNA synthetase Haliscomenobacter hydrossis (hhy:Halhy_4697) 1e-164 tRNA synthetases class IIA 8.9e-31 Threonyl ; alanyl tRNA synthetase second additional domain; 3.5e-18 DHHA1 domain 2.3e-5

159

ORF 11 2199 - 2426 Hypothetical protein L8106_19371 Lyngbya sp.(ZP_016218891.1) 2e-44 Putative uncharacterized protein Lyngbya sp. (TR:A0Y568_LYNSP) 1e-9 Hypothetical protein Cyanothece sp. (cyc:PCC7424_0840) 3e-9 Yia A/B two helix domain 6.1e-8

ORF 12 2892 - 4358 Aminoacyl-histidine dipeptidase Flavobacteriales bacterium(ZP_02180397.1) 0.0 Aminoacyl-histidine dipeptidase Flavobacteriales bacterium (TR:A8UG77_9FLAO) 1e-174 Aminoacyl-histidine dipeptidase Aeromonas veronii (avr:B565_3292 ) 1e-122 Peptidase family M20/M25/M40; Peptidase dimerisation domain 1.9e-20; 5.6e-8

ORF 13 4540 - 7653 Hypothetical protein SCB49_13515 Unidentified eubacterium SCB49 (ZP_01890174.1) 2e-87 Putative uncharacterized protein Unidentified eubacterium SCB49 (TR:A6EP04_9BACT) 2e-80 Hypothetical protein Krokinobacter sp. (kdi:Krodi_2730) 1e-71 Pfam-B 5685 3.7e-9

ORF 14 7661 - 7936 No hit - No hit - No hit - No hit -

ORF 15 8169- 9821 Secreted protein containing N-terminal Zinc-dependent carboxypeptidase 2e-158 Microscilla marina (ZP_01691349.1) Secreted protein containing N-terminal Zinc-dependent carboxypeptidase related domain 1e-132 Microscilla marina (TR:A1ZP4_9BACT) Peptidase M14 carboxypeptidase A Cyclobacterium marinum (cmr: Cycma_2036) 1e-119 Pfam-B 186 5.3e-9

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Appendix 8. Protein sequence alignment of Bel1

Salmonella_a MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Salmonella_bMSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Aeromonas MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Bacterium MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Vibrio MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Pseudomonas MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Streptococcus MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Proteus MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Capnocytophaga MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Bel1 MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Shigella MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Acinetobacter MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Enterobacter MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Acetobacter MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Klebsiella MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Raoultella -SIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Morganella -SIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Escherichia MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Providencia MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Serratia MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Neisseria MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Salmonella_cMSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Haemophilus MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP Salmonella_dMSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRP ***********************************************************

Box1

Salmonella_a EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Salmonella_b EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Aeromonas EERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Bacterium EERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Vibrio EERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Pseudomonas EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Streptococcus EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Proteus EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Capnocytophaga EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVKYSPVTEKHLTDGMTVREL Bel1 EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Shigella EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Acinetobacter EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Enterobacter EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Acetobacter EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Klebsiella EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Raoultella EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Morganella EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Escherichia EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Providencia EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Serratia EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Neisseria EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Salmonella_cEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Haemophilus EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL Salmonella_d EERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVREL *********************:*******************:******************

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Box 2

Salmonella_a CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Salmonella_b CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Aeromonas CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Bacterium CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Vibrio CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Pseudomonas CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGEHVTRLDRWEPELNEAIPNDERDTTM Streptococcus CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Proteus CSAAITMSDNTAANLLLTTIGGPKELTAFLHNIGDHVTRLDRWEPELNEAIPNDERDTTM Capnocytophaga CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Bel1 CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Shigella CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Acinetobacter CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Enterobacter CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Acetobacter CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Klebsiella CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Raoultella CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Morganella CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Escherichia CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Providencia CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Serratia CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Neisseria CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Salmonella_c CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Haemophilus CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM Salmonella_d CSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTM ********************************:*:*************************

Box 3

Salmonella_a PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Salmonella_b PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Aeromonas PVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Bacterium PVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Vibrio PVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Pseudomonas PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Streptococcus PAAIATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Proteus PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Capnocytophaga PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Bel1 PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Shigella PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Acinetobacter PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Enterobacter PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Acetobacter PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Klebsiella PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Raoultella PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Morganella PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Escherichia PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Providencia PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Serratia PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Neisseria PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Salmonella_c PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Haemophilus PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS Salmonella_d PAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGS *.*:********************************************************

Protein sequence alignment of the Bel1with sequences selected from the non-redundant protein of the National Centre for Biotechnology Information and from the curated/ non curated parts of Swiss protein databases. Salmonella_a: Salmonellaschwarzengrund .Salmonella_b: Salmonella infantis. Salmonella_c: Salmonella choleraesuis. Salmonella_d: Salmonella enterica.Symbols:”*”= column of the alignment contains identical amino acids residue in all sequences. “:”= column of alignment contains conserved substitutions of amino acids. “.”= column of alignment contains semi-conserved substitutions. “-“= gaps in the amino acid sequence. The box 1 indicates the active site (S*XXK motif). The box 2 indicates the motif S(Y) XN. The box 3 indicates the K (H) T (S)G motif.

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Appendix 9. Predicted phylogenetic origins of the fosmid insert DNA of antibiotic resistant clones using the Phylopythia algorithm.

Clone Domain Phylum Class Order B3 Bacteria Proteobacteria Alphaproteobacteria Rhodobacterales

B5 Bacteria Proteobacteria Alphaproteobacteria Rhodobacterales

B6 Bacteria Proteobacteria Alphaproteobacteria Rhodobacterales

B7 Bacteria Proteobacteria - -

B10 Bacteria - - -

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Appendix 10. Restriction digests of the randomly picked colonies.

M U D U D U D U D M U D U D U D U D U D M U D U D

M U D U D U D U D M U D U D M U D U D

M: 1 Kb DNA ladder (Fermentas Inc., Glen Burnie, MD, USA); U: undigested fosmid; D: digested fosmid.

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