Characterizing aerobic methanotrophs

We are interested in more fully describing bacterial methane oxidation in the oceans. We've used a variety of approaches towards this end, from molecular surveys to cultivation efforts of marine methanotrophs to controlled experimentation on select methanotorph cultivars.

Analyzing changes in the alkane oxidizing microbial community during a large gas leak

The 2015/2016 Porter Ranch gas leak was the largest such leak in US history. Dr. Tavormina has been analyzing shifts in the microbial community structure by sampling and analyzing soils and gas concentrations throughout 2016. A member of Sphingobium may have been a key lineage that responded by blooming during this event. This organism encodes two diverged methane monooxygenase enzymes; such enzymes have not been demonstrated in Sphingobium previously. Experiments underway are delineating the ability of this lineage to consume a variety of alkane gases, and determining the end products (complete oxidation or carbon assimilation) of these pathways. Of interest, members of Methylococcaceae and Methylocystaceae were detected throughout the region, but were not significantly enriched in any soils analyzed to date, suggesting that they are optimized to normal atmospheric levels of methane.

Soil core from Porter Ranch soils

Sphingobium spp isolated in Porter Ranch soils, which encodes 2 divergent alkane monooxygenase enzymes

Funding for this work is provided by the National Science Foundation: NSF RAPID 1632329.

Methane oxidizing bacteria through oxygen minimum zones

Past work from our group has demonstrated cosmopolitan distributions of two lineages of aerobic methabnotrophs (OPU1, OPU3), in the oceans. More recent work has shown that these lineages occupy unique ecological niches, and that a major determinant of niche specialization is likely to be oxygen availability. These findings imply that aerobic methanotrophs will shift in distribution and abundance as anthropogenically-influenced OMZs continue to expand. These organisms are uncultivated, and the effect that such shifts may have on methane flux to the atmosphere is unknown. Additionally, the seeming ability of lineage OPU3 to flourish in the absence of significant oxygen raises the possibility that this lineage may encode unique pathways for the bacterial oxidation of methane.

Distributions of lineages OPU1 and OPU3 through water depth in the Costa Rica OMZ. Lineage OPU1 shows elevated abundances near the seafloor where both methane and oxygen increase in concentration. In contrast, lineage OPU3 is substantially more abundant in the core of the OMZ, where methane, but not oxygen, is measured.

Relevant Publications:

1. Patricia L. Tavormina, William Ussler III, and Victoria J. Orphan. 2008. Applied and Environmental Microbiology, 74(13), 3985–3995. Planktonic and Sediment-Associated Aerobic Methanotrophs in Two Seep Systems along the North American Margin.

2. Patricia L Tavormina, William Ussler III, Samantha B Joye, Benjamin K Harrison and Victoria J Orphan. 2010. The ISME Journal 4, 700–710. Distributions of putative aerobic methanotrophs in diverse pelagic marine environments

3. Patricia L. Tavormina, William Ussler III, Joshua A. Steele, Stephanie A. Connon, Martin G. Klotz and Victoria J. Orphan (2013).
Environmental Microbiology Reports 5(3) 414-423. Abundance and distribution of diverse membrane-bound monooxygenase (Cu-MMO) genes within the Costa Rica oxygen minimum zone.

Cultivation of a new methanotrophic genus from marine sediments

Over the past two years, we have focused efforts on characterizing a new marine methanotrophic isolate (Methyloprofundus sedimenti, strain WF1), which has close phylogenetic relatedness to the methanotrophic endosymbionts of deep - sea mussels. This isolate represents one of only a handful of marine methanotrophs in pure culture. As such, it provides a much needed resource to conduct controlled laboratory experimentation into oceanic methane cycling. M. sedimenti displays several characteristic biological capabilities and cellular structures of a typical gammaproteobacterial methanotroph, evidencing stacked intracytoplasmic membranes, a Gram negative cell wall, and a dependance on the presence of methane and oxygen for growth.

Morphology of strain WF1T. (a) Negative stain (3⁄41000), showing the presence of capsules in stationary-phase cells. (b) Fluorescence microscopy. DAPI (blue), EUB338 I-III (green) and MetI-444 (red) FISH illustrating the purity of the culture as well as stain-recalcitrant intracellular areas. (c) Thin section of cells of strain WF1T, illustrating typical type I stacked intracytoplasmic membranes (ICM), storage granules (SG) and a typical Gram-negative cell wall (GNCW). Bar, 0.2 mm.

Relevant publications:

1. Tavormina, P. L., Hatzenpichler, R., McGlynn, S., Chadwick, G., Dawson, K. S., Connon, S. A., & Orphan, V. J. (2015). Methyloprofundus sedimenti gen. nov., sp. nov., an obligate methanotroph from ocean sediment belonging to the ‘deep sea-1’clade of marine methanotrophs. International journal of systematic and evolutionary microbiology, 65(Pt 1), 251-259.

Type strain deposited at DMSZ
Additional information on M. sedimenti WF1:

Response of cultured marine methanotrophs to a common environmental cue: Methane flux

Currently, we continue to study M. sedimenti, in tandem with our ongoing work cultivating additional marine methanotrophs. M. sedimenti has now been imaged at higher resolution using electron cryotomography, under conditions of high methane concentration and methane starvation. The strain rapidly shifts cellular resources, within days of carbon starvation. Electron cryotomography work was done in collaboration with Drs. Elitza Tocheva and Grant Jensen, Caltech.

Effect of starvation on cell morphology of M. sedimenti. Upon carbon starvation (96h), many cellular features are altered. The cell loses volume as evidenced by greater periplasmic space, glycogen granules disappear, and ribosomes become more evident. These observations suggest that the cell has fairly rapidly consumed its carbon stores, yet has retained the essential machinery to translate proteins, a necessary step to reenter growth. Credit: Tocheva and Tavormina

Additionally, its transcriptional response under starvation has been analyzed in some depth. Surprisingly, we find that genes central to methane oxidation are present in higher numbers during methane starvation than during conditions where methane is plentiful. This finding has implications for the correct interpretation of environmental transcriptomic datasets, as well as for our understanding of the competitive strategies utilized by these organisms in situ.

Effect of starvation on transcription of key genes in M. sedimenti. Upon carbon starvation (96h), many housekeeping and metabolic genes reduce in abundance. Ribosomal transcripts increase moderately, and genes pertinent to methane utilization increase substantially.

In our current manuscript (in preparation), we describe these responses of M. sedimenti to the common environmental stress of starvation, and integrate the structural and physiological features of M. sedimenti under active growth and starvation states, with genomic content. Near term plans include similar experimentation on a second marine methanotroph isolate (Methylococcaceae strain GO1) to determine how broadly these starvation-induced trends apply across marine methanotrophic genera, towards a fuller description of bacterial methane cycling in ocean environments.

Relevant manuscript in preparation:

1. Tavormina, P. L., & Orphan, V. J. et al. (in prep). Methyloprofundus sediment enters a state of poised persistence during carbon starvation.

Additional work for future publications

A second lineage of marine methanotroph has been highly enriched by our group. This isolate (Methylococcaceae strain GO1) was obtained from the Del Mar seep system near San Diego, California. The isolate has been difficult to obtain in pure culture, and genomic sequencing indicates that a low level of contamination (spindle shaped cells) exists from a member of Actinobacteria.

Fluorescent In Situ Hybridization (FISH) imaging of strain GO1. Strain GO1 cells are fat rods, and large by bacterial standards. The contaminating cells, members of Actinobacteria, appear as spindle shaped cells.

Both Methylococcaceae strain GO1 and M. sedimenti strain WF1 genomes encode genes predicted to fix nitrogen gas, and both occur within methane seep systems. We are currently planning experiments to ask how marine methanotrophs contribute to the methane seep nitrogen cycle, using these organisms as experimental models. Additionally, a close relative of Methylococcaceae strain GO1 (same species, different strain) has been isolated from a mussel bed in the Gulf of Mexico. We are designing experiments to ask how the growth characteristics of these two close relatives, isolated from unique ecological and geographical niches, compare to one another.