Water column Methanotrophs

Diversity of planktonic methanotrophs associated with deep-sea methane vents (post-doc Patricia Tavormina)

Methane within, and released from, ocean sediments is metabolized anaerobically by members of Archaea, and aerobically by members of Bacteria. My work focuses on characterizing the contributions of aerobic methanotrophs in and above marine methane seeps. Over the past eighteen months, we have assayed the diversity of aerobic methane-consuming bacteria in methane seep sediments and the overlying water column via the functional gene marker pmoA as a proxy. Two deep branches in the pmoA phylogeny have been identified by our group, and these deep branches promise to provide insight into the diversity of organisms capable of methane oxidation as well as the evolutionary history of genes relevant to methane metabolism.

The first deep branch in the pmoA phylogeny corresponds to an organism which we believe is a member of the Beta proteobacteria. This organism would represent the first example of a betaproteobacteria capable of metabolizing methane via pmoA-mediated pathways. In one methane-rich environment, this organism may represent as much as 10% of the resident methanotrophic community. Enrichment cultures, and fosmid library construction, are planned in order to more fully describe this unique lineage and its distribution and relevance to methane oxidation in additional environments.

The second deep branch in the pmoA phylogeny identified in our work appears to be the result of an ancient gene duplication event, within gamma-proteobacterial methanotrophic cultivars. This duplication event has resulted in a second pmoA-like locus that has diverged significantly from the established pmoA locus. We have determined that the diverged locus is expressed, and that it is maintained in the environment. Analysis of the predicted proteins encoded by this second locus illustrate that the corresponding proteins have maintained key residues and membrane-spanning features. The presence of this locus implicates the potential for increased metabolic capabilities of gamma-proteobacteria, and experiments are underway to identify potential inducers and substrates corresponding to the diverged locus.

Finally, we are currently developing a rapid means of fingerprinting aerobic methanotroph diversity in environmental samples. The method is similar in concept to the ARISA (automated ribosomal intergenic spacer analysis) method and depends on differences in spacer length between two genes (pmoC and pmoA) within the pmo operon. We have successfully fingerprinted diversity at the DNA level (indicating resident organisms) and at the RNA level (indicating metabolically active groups within a population) from several environments.

ASTEP PROGRAM: "An environmental sample processor for deep-seep and hydrothermal vent applications".
In collaboration with Christopher Scholin, Christina Preston, Bill Ussler (Monterey Bay Aquarium Research Institute).

Project Summary:
Differences between planktonic and sediment-associated methanotrophs in two seep systems along the North American margin

Methane vents are of significant geochemical and ecological importance. Notable progress has been made towards understanding methane oxidation in anoxic sediments, mediated by a consortium of archaea and bacteria; however, the diversity and distribution of aerobic methanotrophs in the water column is poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and bacterial 16S rRNA genes from two methane vent environments along the California continental margin was characterized. Comparison of pmoA in methane-rich sediments and overlying water column revealed differences in diversity between these habitats. Sediments harbored the greatest number of unique pmoA clades broadly affiliated with the Methylococcaceae family, whereas planktonic phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs, and distantly related to established methanotrophic clades. These planktonic phylotypes were highly similar between sites, suggesting that methanotroph diversity in the water column depends on environmental factors (habitat) rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and Pmo. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.

Tavormina, P. L., W. Ussler III, S.M. Joye and V. J. Orphan (2010) Distributions of putative aerobic methanotrophs in diverse pelagic marine environments. ISME J. 4:700-710

Tavormina, P. L., W. Ussler III, and V. J. Orphan (2008) Diversity and stratification of aerobic methylotrophs in methane seeps along the North American continental margin. Appl. Environ. Microbiol. 74: 3985–3995

Project Summary:

A diverged pmo-like operon family among Type I methanotrophs

Gene duplication is an important mechanism that can increase the metabolic capabilities of an organism. Methanotrophs affiliated with the Alphaproteobacteria and Verrucomicrobiales are known to encode two diverged copies (paralogs) of the pmo operon within their genomes. Here, we provide evidence that members of Type I Gammaproteobacterial methanotrophs also encode a pmo paralog (para-mo). The open reading frames recovered in this work (para-moA and para-moB) predict proteins that are ~74% similar to Amo subunits A and B, and ~73% similar to Type I Pmo subunits A and B. Invariant residues in Amo/Pmo alignments are maintained in the predicted Para-moA and Para-moB proteins, and membrane-spanning segments are also maintained. RNA transcripts corresponding to the newly-identified sequences are recoverable from Methylomonas cultures, demonstrating that the sequences are expressed. Transcript levels are comparable to those of the housekeeping gene rpoD, and are low relative to those of pmo under all tested conditions. Primers were developed that allow the specific amplification of this monooxygenase clade from environmental samples. This targeted primer set was used to assess environmental sequence diversity of the novel clade in freshwater sediment, verifying its maintenance in situ.

Tavormina, P.L., V.J. Orphan, Marina K,  M. Jetten, M. Klotz (2010) A novel family of functional operons encoding methane/ammonia monooxygenase-related proteins in gamma-proteobacterial methanotrophs. Environ. Microbiol. Reports. doi:10.1111/j.1758-2229.2010.00192.x