Environmental metagenomics, transcriptomics and proteomics of methane seeps and vents

Methane seeps harbour a diverse array of bacteria and archaea that have no known cultured representatives. The most prominent are the anaerobic methanotrophic archaea (ANME) and the sulfate reducing bacteria (SRB). These two groups of organisms form a syntrophic partnership that together oxidize methane to carbon dioxide and reduce sulfate to sulfide. Due to their slow growth and lack of pure culture representatives we have employed environmental microbiology techniques such as metagenomics to characterize the metabolic potential of methane seep microorganisms. The power of metagenomics that DNA from all members of the community can be analyzed, which has enabled us to hypothesize the metabolic function of many members of the methane seep community.


Current Metagenomic Efforts

Methane Seep Microcosm Metagenomes

We have currently sequenced three laboratory microcosm sediment samples and recovered 31 different genomic bins from a range of organisms. These include the ANME archaea, SRB bacteria related to deltaproteobacteria, members of the bacteroidetes, representatives from the gamma- and epsilon-proteobacteria, as well as members of the Firmicutes and Tenericutes. Metabolic inferences based on the genomes of these organisms showed that organisms could be grouped into three types: involved in anaerobic oxidation of methane, generalists capable of many anaerobic and aerobic carbon and nitrogen transformations, and more specialized anaerobic fermenting organisms. and SRB organisms that are involved in the anaerobic oxidation of methane and other organisms that appear to contain metabolisms for sulfur oxidation, denitrification and fermentation of sugars and other organic matter.


Methane oxidation Sulfate reduction Nitrogen fixation Dentrification Fermentation Aerobic respiration
ANME X X
Deltaproteobacteria
(SRB)
X O
Gammaproteobacteria X X X
Epsilonproteobacteria X X
Tenericutes X
Clostridia X
Anaerolinea X
Bacteroidetes X X

Table 1: Possible metabolisms of methane seep microorganisms based on the presence of genes in their genomes. The letter "X" indicates that all members of the methane seep community contain these genes, while the letter "O" indicates that only some of the genome bins from the metagenome contain genes for this function.


Phylogenomic Analysis of Novel Tenericutes from Methane Seeps

From our initial metagenomic sequencing efforts of laboratory microcosms, two genomes phylogenetically associated with Tenericutes. Tenericutes are characterized by their lack of a cell wall and are often associated with plants or animals as parasites or commensals. Environmental 16S rDNA surveys have identified Tenericutes in ocean sediments and a range of other habitats however the physiology of these organisms has yet to be determined. The two reconstructed genomes from our metagenomic data were found to be part of a clade currently known as ‘NB1-n’ (SILVA taxonomy) or ‘RF3’ (Greengenes taxonomy). Metabolic reconstruction revealed that these ‘NB1-n’ have a simple carbon metabolism that relies on anaerobic fermentation of simple sugars for substrate level phosphorylation. However there were a number of unique metabolic features not found in any other tenericute genome, including hydrogenases and a simplified electron transport chain. The role of an electron transport chain requires further biochemical characterization but its presence in both of these genomes suggests that these Tenericutes may perform more than substrate-level phosphorylation. An enrichment media devised to select for Tenericutes resulted in these organisms dominating the community (~60% of cells) and allowed for visual characterization. Visual identification by fluorescence in situ hybridization and cryo-electron tomography confirmed the absence of a cell wall, the primary phenotypic property of Tenericutes. Based on their unique gene content and phylogenetic placement, we propose the names Candidatus ‘Izimaplasma sp. HR1’ and Candidatus ‘Izimaplasma sp. HR2’ for the two genome representatives, which may represent a novel class of Tenericutes.


Data Availability

Izimaplasma sp. HR1 and HR2 are available on NCBI as CP009415 and JRFF01000000, respectively. The metagenomic data is available on NCBI under the bioproject accession PRJNA290197.


Relevant Publication

In Preparation: Skennerton et. al Phylogenomic Analysis of Candidatus ‘Izimaplasma’ species: Representatives from a Tenericutes Clade found in Methane Seeps


Deep-sea Hydrothermal Vent Metagenomes

Physiology of Novel Methylothermaceae from Lau Basin Hydrothermal Vents

Hydrothermal vents are an important contributor to marine biogeochemistry, producing large volumes of reduced fluids, gasses, and metals and housing unique, productive microbial and animal communities fueled by chemosynthesis. Methane is a common constitute of hydrothermal vent fluid and is frequently consumed at vent sites by methanotrophic bacteria that serve to control escape of this greenhouse gas into the atmosphere. Despite their ecological and geochemical importance, little is known about the ecophysiology of uncultured hydrothermal vent-associated methanotrophic bacteria. Using metagenomic binning techniques, we recovered and analyzed a near-complete genome from a novel gammaproteobacterial methanotroph (B42) associated with a white smoker chimney in the Southern Lau basin. B42 was the dominant methanotroph in the community, at ~80x coverage, with only four others detected in the metagenome, all on low coverage contigs (7x - 12x). Phylogenetic placement of this genome showed it is a member of the Methylothermaceae, a family that contains only one previously sequenced genome. Metabolic inferences based on the presence of known pathways in the genome showed that B42 possesses a branched respiratory chain with A- and B-family heme copper oxidases, cytochrome bd oxidase and a partial denitrification pathway. These genes could allow B42 to respire over a wide range of oxygen concentrations within the highly dynamic vent environment. Phylogenies of the denitrification genes revealed they are the result of separate horizontal gene transfer from other proteobacteria and suggest that denitrification is a selective advantage in conditions where extremely low oxygen concentrations require all oxygen to be used for methane activation.


Data Availability

B42 genome is in Integrated Microbial Genomes (IMG) database under the accession 2623620619


Relevant Publications

In Review: Skennerton et al. Genomic reconstruction of an uncultured hydrothermal vent gammaproteobacterial methanotroph (family Methylothermaceae) indicates diverse nitrogen utilization potential. Frontiers in Microbiology


Associated Personnel

Connor Skennerton, Postdoctoral Scholar, Division of Geological and Planetary Sciences, California Institute of Technology


Collaborators

Dr. Gene Tyson, University of Queensland
Dr. Viviana Gradinaru, Caltech