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Signature Redacted Signature of Author: Dep Rtn, Nt of Miobiology August, 29Th 2013 Examination of the Nematostella vectensis Holobiont by Comparative Bacterial Genomics and Metatranscriptomics by Timothy J. Helbig B.S. Biological Sciences Carnegie Mellon University, 2010 SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE AT THE MASSACHUSETTS INSfT E TFCHNOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY OCT 0 3 2013 September 2013 @2013 Timothy J. Helbig. All rights reserved. LIBRARIES The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature redacted Signature of Author: Dep rtn, nt of Miobiology August, 29th 2013 Certified by: Signature redacted-, Janelle R. Thompson Doherty Assis ant Professor in Ocean Utilization Thesispupervisor Signature redacted Accepted by Michael T. Laub Whitehead Career Development Associate Professor of Biology Chairman, Committee for Microbiology Graduate Students Examination of the Nematostella vectensis Holobiont by Comparative Bacterial Genomics and Metatranscriptomics by Timothy J. Helbig Submitted to the Department of Microbiology on August 30, 2013 in Partial Fulfillment of the Requirements for the Degree of Master of Science ABSTRACT Previous work has shown that similar microbial populations are associated with the starlet sea anemone Nematostella vectensis over distinct temporal and geographic locations; however, the functions these bacteria may be performing within their anemone hosts and the mechanisms with which the bacteria may be using to adapt are unknown. To address these issues comparative genomic analysis of ten newly sequenced bacterial isolates from four bacterial populations (Pseudomonas oleovorans,Agrobacterium tumefaciens, Limnobacter thiooxidans and Stappia stellulata) that are associated with Nematostella in the laboratory and/or its natural salt marsh habitat was performed and whole metatranscriptomes of lab-raised N. vectensis were sequenced and analyzed. Comparative genomic analysis revealed the isolates from these bacterial populations to likely be non-clonal, with no evidence that holobiont-specific orthologous groups (i.e. gene orthologs found only in N. vectensis-associated bacterial genomes and absent in closely related genomes of the same genus/family) were shared across the populations examined. Further, no evidence of lateral gene transfer or shared phage or mobile elements among the isolates was observed. Isolate genomes did, however, reveal conserved holobiont specific orthologs within members of the same bacterial population that could be reflective of the ecology of the anemone holobiont; for instance, 3 of the four P. oleovorans isolate genomes showed evidence of holobiont specific antibiotic production, the three A. tumefaciens isolates all shared common ion scavenging proteins and both L. thiooxidans had a holobiont specific antibiotic resistance protein. Whole anemone metatranscriptomic analysis based on BLASTx annotation of sequenced transcripts revealed bacterial expression of housekeeping genes such as those for replication, ribosomal structure and ATP-synthesis dominated by Proteobacteria, in particular Gammaproteobacteria. Further recruitment of the transcripts to sequenced Nematostella associates revealed an active and foraging Limnobacter population expressing genes for signaling, movement, iron scavenging and carbon storage in the form of PHA granules. The similarity of high Limnobacter and host anemone expression for iron regulators suggest iron may be a source of structuring within the anemone holobiont and a good area of further study. Thesis Supervisor: Janelle R. Thompson Title: Doherty Assistant Professor in Ocean Utilization 3 4 Acknowledgements Firstly I would like to thank the members of the Thompson Lab and everyone in Parsons for creating a wonderful and jubilant work environment. Particularly, I'd like to thank Sonia Timberlake who provided both invaluable programming and bioinformatics advice and great life advice in general when I needed it. Further, I would like to thank my advisor Janelle Thompson for patience and the ability to talk my life out of dark places in my times of most need. She was a wonderful mentor and one of the most thoughtful and caring people I have come across in my life. I would also like to thank my parental unit, who has provided a constant beam of unconditional love throughout my time in grad school. Finally, I would like to thank and give love to my crucial support factor in this interesting past year and a half, my boyfriend Adam. Adam, you are a true raraavis and the most special person in my life. Here's to more of the happiest times of my life together now that this thesis is over. 5 6 Table of Contents F ig u re K ey ........................................................................................................................... 9 Introduction .....................................................................................................................11 M e th o d s ............................................................................................................................1 5 Com parative Genom ics Results and Discussion ............................................................ 23 M etatranscriptom ics Results and Discussion ................................................................41 Conclusion ........................................................................................................................61 References ........................................................................................................................63 Appendices .......................................................................................................................73 7 8 Figure Key Comparative Genomics of Nematostella vectensis Associated Bacteria 1. Map and distribution of microbial diversity in field and lab-raised N. vectensis as determined through 16S clone libraries (Har, MS Thesis) - p. 23 2. 16S rRNA Tree of Symbiont Phylogenetic Relatedness - p. 25 3. Shared Gene Contents of Nematostella Associated Isolates - p. 31 4. Holobiont-specific orthologous groups found within multiple members of the Nematostella isolated populations - p. 33 5. MEGAN Visualization of N. vectensis Isolate Operational Core Genomes - p. 35 6. MEGAN Visualization of N. vectensis Isolate Operational Flexible Genomes - p. 36 7. Analysis of N. vectensis Genome Scaffolds containing and not containing PseudomonasDNA - p. 37 8. Shared Phage Elements of N. vectensis Isolates within and among Populations - p. 38 Nematostella vectensis Metatranscriptome Analysis 1. Ribosomal and Contaminant Read Breakdown of Initial Read Pairs - p. 42 2. MEGAN Taxonomic Breakdown of Filtered Reads - p. 44 3. MEGAN Analysis of N. vectensis Metatranscriptome Diversity - p. 45 4. Top 15 most highly represented orthologous groups among transcripts of Bacterial and Cnidarian binned reads - p. 47 5. Read mapping to second highest annotated "Cnidarian" Read Category, opiNOG08261 - p. 48 6. Read mapping to representative sequence of highest expressed "Bacterial" orthologous group NOG323497 - p. 49 7. Genus level assignment of SSU rRNA sequences from the unprocessed control sample - p. 51 9 8. Species Level assignment of SSU rRNA sequences from all Metatranscriptome samples - p. 52 9. COG Category Distribution of Reads Binned By MEGAN as Proteobacteria and Reads Mapped to Sequenced Limnobacter Genomes - p. 57 10. Orthologous Group comparison of those present in "Bacterial" reads as determined through MEGAN analysis and those present in the symbiont genome mapping analysis - p. 58 10 Introduction Significance of host-associated microbial communities: Emerging evidence Multicellular life emerged in a world teeming with microorganisms. Rather than something to overcome, this was, however, an opportunity for each type of life to make use of the other's unique physiological and enzymatic capabilities in order to help themselves better adapt to their surrounding environment. The success of these multicellular-microorganism partnerships is well illustrated in the fact that all studied mammals, lower vertebrates, invertebrates and plants are each distinctly colonized with unique and active microbial communities (Bosch, 2013; Nyholm et al., 2012; Hooper et al., 2001). Microbial inhabitants of multicellular creatures have recently come to light as powerful contributors to the well-being and success of their hosts. They are critical in the digestion and absorption of nutrients as they have been found to breakdown complex plant-polymers and polysaccharides in ruminants, termites and humans (Warnecke et al., 2007; Xu et al., 2003; Mahowald et al. 2009), synthesize essential amino acids in insects such as aphids (Moran, 2007) and produce vitamins in mice and humans (Chaucheyras-Durand et al., 2010). They have also been found to be imperative in the development of particular organs and systems within animals such the immune system of mice and humans (Dobber et al. 1992; O'Hara et al. 2004), the gut of zebrafish (Rawls et al., 2004) and the light creating organ of the Hawaiian Bobtail Squid (Rader et al., 2012). Further, they are known to have effects on complex physiological processes such as obesity in humans and mice (Backhed et al., 2004; Ley at al., 2005; Ley at al., 2006). Finally, the microbial constituents are known to be a passive and active deterrent of pathogens within
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