METAGENOMIC AND GENOMIC ANALYSES OF MODERN FRESHWATER MICROBIALITES: UNMASKING A COMMUNITY OF COMPLEX METABOLIC POTENTIAL by RICHARD ALLEN WHITE III Bachelors of Science, California State University East Bay, Hayward, California USA, 2007 Masters of Science, California State University East Bay, Hayward, California USA, 2009 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2014 © Richard Allen White III, 2014 Abstract Microbialites represent the oldest known persistent ecosystems and potentially the earliest evidence of life on the planet, having existed for ~85% of the geologic history of Earth (Dupraz et al., 2009). Despite over one hundred years of active research, little is known about modern freshwater microbialite ecosystems with regards to metabolic potential and microbialite- specific community structure. We performed metagenomic analysis of freshwater thrombolithic clotted microbialites from Pavilion Lake (British Columbia, Canada) and Clinton Creek (Yukon, Canada). In addition, metagenomes were obtained from the surrounding water and sediments to sort out which members of the microbial community were microbialite-specific. Pavilion Lake microbialites are distinct from the surrounding environments in microbial community structure and metabolic potential. The microbialites are dominated by heterotrophic processes with high abundances of heavy metal, antibiotic resistance, and alcohol fermentation pathways from the numerically dominant Proteobacteria. Clinton Creek houses the northern-most and fastest growing microbialites, which have a high proportion of photosynthetic genes, supporting isotopic data that photosynthesis drives microbialite formation. Clinton Creek has distinct communities, with microbialites dominated by Alphaproteobacteria (photoheterotrophs) and sediments dominated by Gammaproteobacteria (mainly heterotrophic nitrogen-fixers). To complement the metagenomic study of Pavilion Lake, a culturing based study was performed that yielded over one hundred new bacterial isolates. The new bacterial isolates were further screened for pigment containing strains that were non-photosynthetic. Amongst these pigment containing bacteria two new isolates were found and designated as an Exiguobacterium and an Agrococcus. Polyphasic analysis revealed that both are new species, which were named Agrococcus pavilionensis strain RW1 and Exiguobacterium pavilionensis strain RW2. Genome sequencing of both strain RW1 and ii RW2 was completed and a comparative genomic and phylogenetic study was performed to evaluate their evolutionary placement and metabolic potential. Both isolates have low abundance in the Pavilion Lake microbialites, although they contribute heavy metal resistance genes that are found amongst the microbialite metagenomes. Hypothetical carotenoid biosynthesis pathways are also described which may be responsible for the coloration in Agrococcus and Exiguobacterium and may be related to photo-protection. iii Preface Chapter 2 is based on a manuscript in preparation: White III RA, Suttle CA. Modern freshwater microbialites reveals distinct microbial community structure and metabolic potential that differs from the surrounding environment. CAS, RAWIII and AMC designed the project. RAWIII, AMC and CAS conducted field work. RAWIII did the laboratory work and data analysis. RAWIII and CAS wrote the manuscript. Chapter 3 is based on a manuscript in preparation: White III RA, Power IM, Hanson NW, Dipple GM, Southam G, Hallam SJ and Suttle CA. Comparative analysis of microbiome of rapidly forming modern microbialites from Canadian Yukon. CAS, GMD, RAWIII and IMP envisioned the project. RAWIII and IMP designed the experimental approach. IMP did the water chemistry and field sampling. RAWIII processed the genomic samples and did the data analysis with help from NWH and SJH. RAWIII, IMP and CAS wrote the paper. Chapter 4 is based on a published manuscript and manuscript in preparation: White III RA, Grassa CJ, Suttle CA. (2013). First draft genome sequence from a member of the genus Agrococcus, isolated from modern microbialites. Genome Announc. 1, e00391-13 iv White III RA, Soles SA, Gavelis G, Gosselin E, Slater GF, and Suttle CA. The genome of Agrococcus sp. strain RW1 isolated from modern microbialites reveals genetic promiscuity and elements responsive to environmental stresses. (In preparation). RAWIII and CAS initiated the project. RAWIII designed and conducted the experiments. EG contributed to experiments on growth and characterization. CJG completed the first draft assembly of the genome (published) and advised on de novo assembly and genome finishing. RAWIII revised the assembly, finished the genome and completed annotation. SAS and GFS completed all PLFA experiments and data analysis. GG did the electron microscopy of the isolate. RAWIII, CAS, GS, SAS and GG contributed to writing the manuscript in preparation. Chapter 5 is based on a published manuscript and manuscript in preparation: White III RA, Grassa CJ, Suttle CA. (2013). Draft genome sequence of Exiguobacterium pavilionensis strain RW-2, with wide thermal, salinity, and pH tolerance, isolated from modern freshwater microbialites. Genome Announc. 1, e00597-13 White III RA, Soles SA, Gavelis G, Gosselin E, Slater GF, and Suttle CA. Illumination of an Exiguobacterium through the comparative lenses of classical characterization and comparative genomic analysis. (In preparation). RAWIII and CAS initiated the project. RAWIII designed and conducted the experiments. EG contributed to experiments on growth and characterization. CJG completed the first draft assembly of the genome (published) and advised on de novo assembly and genome finishing. RAWIII revised the assembly, finished the genome and completed annotation. SAS and v GFS completed all PLFA experiments and data analysis. GG did the electron microscopy of the isolate. RAWIII, CAS, GS, SAS and GG contributed to writing the manuscript in preparation. vi Table of Contents Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iv Table of Contents .......................................................................................................................... vii List of Tables ............................................................................................................................... xiv List of Figures ............................................................................................................................... xv List of Abbreviations ................................................................................................................... xix List of Keywords.......................................................................................................................... xxi Acknowledgments...................................................................................................................... xxiii Dedication .................................................................................................................................. xxiv Chapter 1: Introduction ............................................................................................................... 1 1.1 What is a microbialite? ..................................................................................................... 1 Physical classification and brief history .................................................................... 1 1.2 Microbialites as the oldest persistent ecosystems ............................................................. 2 1.3 Global distribution of microbialites .................................................................................. 2 1.4 Proposed microbialite formation hypotheses .................................................................... 5 Abiotic (spectator hypothesis) ................................................................................... 5 Abiotic combined biotic hypothesis........................................................................... 6 vii 1.5 Major microbial taxa and their metabolic pathways that contribute to microbialite formation ................................................................................................................................. 8 Alphaproteobacteria .................................................................................................. 8 Cyanobacteria ............................................................................................................ 9 1.5.2.1 Planktonic cyanobacteria .................................................................................... 9 1.5.2.2 Filamentous cyanobacteria, mat-builders ........................................................... 9 Sulfur-oxidizing bacteria (SOBs) ............................................................................ 10 Sulfate-reducing bacteria (SRBs) ............................................................................ 11 Firmicutes ................................................................................................................ 13 Actinobacteria .........................................................................................................