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This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology
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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 constituent 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 B42 showed it is a member of the
Deep-sea hydrothermal vent systems are a significant contributor to the marine methane cycle, considered both a global source and sink of this potent greenhouse gas. Hydrothermal vent systems are common along mid-ocean ridges, back-arc spreading centers, and other subaqueous divergent plate boundaries (
Methanotrophic bacteria utilize methane to provide cellular carbon and energy through oxidation via methane monooxygenase (MMO). Carbon assimilation proceeds by either the ribulose monophosphate (RuMP) pathway found in Type I methanotrophs, or the serine pathway found in Type II methanotrophs (
The role of aerobic methanotrophs in global nitrogen cycling is also of growing interest. Some methanotrophs have been shown to contribute to nitrogen fixation (
Recently, additional overlap between the methane and nitrogen cycling has been demonstrated. Denitrifying microbes have been implicated in nitrate reduction coupled to methane oxidation using either the reverse methanogenesis pathway in
In this study, metagenomic sequencing was used to sample the microbial community and associated metabolic potential with a white smoker chimney from the Tu’i Malila vent field in the Lau Basin. From this metagenomic dataset, we successfully reconstructed a draft genome from an aerobic methanotroph that, like
The Lau Basin in the western Pacific is a back-arc basin that consists of several ridge segments arranged approximately North-South, aligned subparallel to the convergent Pacific-Australian plate margin to the east. A large (∼15 cm) piece of a white smoker chimney, sample #2044C, was collected during cruise tuim06mv on Dive J2_144 (May 21, 2005) from the Tu’i Malila vent field (176° 34.060′ W, 21°59.350′ S; depth 1876 m), located on the Valu Fa Ridge at the southern end of the Lau Basin in the western Pacific Ocean. Fluids emanating from this white smoker were measured at 260°C, with 10–12°C temperatures measured on the exterior surface of the chimney where #2044C was sampled. Upon recovery shipboard, a sterile chisel and hammer were used to subsample the exterior chimney material, followed by storage at -80°C until genomic DNA extraction in 2015.
Mineralogical analysis of sample #2044C by XRD (B. Harrison personal communication) indicates a composition dominated by sphalerite, anhydrite, chalcopyrite (copper iron sulfide), and galena (lead sulfide) with lower contributions of wurtzite (zinc iron sulfide), barite (barium sulfate), pyrite (iron sulfide), and molybdenum.
DNA from sample #2044C was extracted using the MO BIO PowerSoil® DNA isolation kit, following the manufacturer’s instructions, and sequenced using the Illumina HiSeq2500 platform. Raw metagenomic sequencing reads were assembled using megahit 0.1.2 (
Gene phylogenies for NirK and NarG were constructed by identifying genes in the IMG database (
The draft genome sequence is available in the Integrated Microbial Genomes (IMG) database under the accession 2623620619.
Metagenomic sequencing, assembly and binning resulted in a number of high quality genome bins from the hydrothermal vent metagenome. Analysis of the metabolisms of these genome bins identified only a single draft genome that contained the enzymes for aerobic methane oxidation. This genome bin, referred to hereafter as B42, was assembled into 39 scaffolds containing 3.04 Mbp of total sequence at an average coverage of 80x. B42 was estimated to be 97% complete and 1% contaminated based on the presence of single-copy marker genes. The genome bin contained a single complete rRNA operon that allowed for phylogenetic classification as a member of the
B42 contained all of the necessary RuMP pathway genes (type I methanotroph) for assimilation of methane-derived carbon that has been found in all previously sequenced gammaproteobacterial methanotrophs (
The pMMO catalyzes the production of methanol, which is further oxidized to formaldehyde by a periplasmic methanol dehydrogenase (MDH;
B42 has the ability to store excess carbon in the form of glycogen. The genome contains genes involved in glyconeogenesis (glycogen formation) including glycogen synthase, and branching enzymes and glycogen utilizing enzymes such as debranching enzymes, glycogen phosphorylase, and alpha-amylase. Methanotrophs have been observed to use either glycogen or PHB, with a preference for type I methanotrophs to produce glycogen exclusively (
Copper is the metal cofactor for pMMO, and as such, the copper requirement of methanotrophs is estimated to be 10 times that of other organisms (
As an aerobic methanotroph, B42 requires oxygen for the oxidation of methane. Perhaps to survive oxygen limitation, B42 encodes a number of respiratory complexes that allow for respiration over a wide range of oxygen concentrations (
The genome of B42 contains all of the subunits of two members of the heme-copper oxidase (HCO) superfamily that encode an A-family and a B-family O2 reductase. The B-family O2 reductase enzymes are adapted to lower concentrations of oxygen than the A-family, having converted a conserved proton channel into an O2 channel. This result in a higher affinity for O2 but fewer protons pumped per electron (
B42 also possesses a cytochrome
Multiple pathways suggest that B42 is capable of generating or consuming either a H+ or Na+ gradient for energy. Two separate and complete ATP synthase operons were detected in the genome, one of which was annotated as Na+-translocating. Precise understanding of the sequence-level differences between proton and sodium translocating ATP synthases is lacking in most organisms other than
B42 may be able to utilize nitrate as an alternative electron acceptor under extreme oxygen limitation (
An incomplete denitrification pathway is present in the B42 genome to convert nitrate to nitrous oxide (
The complement of terminal electron accepting reactions possessed by B42 implies an organism capable of thriving under a range of oxygen concentrations. While it would conserve the most energy utilizing its A-family HCO under high oxygen conditions, B42 appears to be capable of continuing to respire under low-O2 conditions using the B-family HCO or the cytochrome
It remains unclear how a transition of oxygen to nitrate utilization would be accomplished, and what electron donors would be utilized under denitrifying conditions. An alternative, though perhaps not mutually exclusive hypothesis, is that denitrification and aerobic respiration are run simultaneously. As aerobic respiration and denitrification share many components of the electron transport chain, differing only in the terminal electron acceptor, it has been proposed that a “hybrid” of the two pathways can be run to maximize energy conservation under low-O2 conditions and to minimize response times when oxygen levels fluctuate (
Metagenomic sequencing of a deep-sea hydrothermal vent has recovered the genome of a novel methanotroph, B42, from the family
VO and VG conceived the initial study of the hydrothermal vent systems. KC performed laboratory work to prepare samples for sequencing. CS performed initial assembly and binning. VO and CS conceived the analysis of the metagenome bin. CS, LW, AM, KM, CV, SM performed analysis of the metagenome bin. CS, LW, AM, KM, CV, SM, VO wrote and revised the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The metagenomic analysis and annotation for B42 was done in part by the students of the GeBI 246 Molecular Geobiology course at Caltech. We thank Ben Harrison for XRD analysis on sample #2044C and Patty Tavormina for assistance during the course and critical reading of this manuscript. We also thank Chief Scientist Robert Vrijenhoek (Monterey Bay Aquarium Research Institute) for providing the opportunity to collect samples during the 2005 tuim06mv cruise. We thank Igor Antoshechkin of the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech for his services and input during sequencing of the genomic library.