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Cycling Single-Carbon Compounds: from Omics to Novel Concepts

Formerly obscure microbial species emerge as major players in environmental cycling of carbon and nitrogen

Ludmila Chistoserdova, Marina G. Kalyuzhnaya, and Mary E. Lidstrom

➤ Probing with stable isotopes followed by high-throughput DNA sequencing provides a means for identifying and characterizing specific functional species types within complex microbial communities.
➤ By analyzing novel Methylophilaceae species, we uncovered modes of methylotrophy that do not rely on the hallmark enzyme methanol dehydrogenase; instead, xoxF genes appear to play a key role in metabolism of methanol and nitrate.
➤ Community responses to changes in oxygen and nitrate levels suggest that several species cooperate to metabolize methane.

We study the bacteria that oxidize methane and potentially other single-carbon compounds in Lake Washington in Seattle. These organisms typically are found in the top layers of the sediment, where they capture the methane that forms following methanogenic degradation of organic matter in the anoxic layers of the sediment, or where they consume other similar single-carbon compounds.

Our former collaborator Ann Auman, currently of Pacifıc Lutheran University, Tacoma, Wash., identifıed two major groups of methanotrophs from sediment samples taken from Lake Washington: Alphaproteobacteria of the family Methylocystaceae and Gammaproteobacteria of the family Methylococcaceae. She also amplifıed 16S rRNA as well as functional genes involved in methane oxidation and nitrate fıxation from those samples to confırm their presence. Representatives of each type were cultivated and identifıed as Methylosinus, Methylocystis, Methylomonas, or Methylosarcina species.

Further analysis with PCR primers to amplify genes for tetrahydromethanopterin-linked formaldehyde oxidation indicate that other microbial species without obvious roles in methane oxidation are also active in sediments from this lake. Their presence suggests that either single-carbon substrates other than methane support these communities or that methane oxidation to carbon dioxide, instead of being carried out exclusively by methanotrophs, may be a community affair. Follow-up analysis in which stable isotope probing was combined with whole-genome shotgun sequencing led us to identify previously unrecognized microbial communities that play a major role in cycling methane and other singlecarbon compounds. These studies also provide insights into the relationship between methylotrophy and nitrogen metabolism.

Uncovering Dominant Methylotrophs and Identifying Nitrogen Metabolic Strategies

To determine how specifıc methylotrophs metabolize single-carbon compounds, we used stable isotope probing (SIP) to label the DNA of active species in sediment samples from Lake Washington. We exposed sediment samples separately to 13C-labeled methane, methanol, methylamine, formaldehyde, and formate to target microbial populations actively using each of these single-carbon compounds. We then extracted DNA from each microcosm and separated the 13C-labeled fractions from unlabeled DNA by isopycnic centrifugation. Shotgun libraries were generated from these DNA samples and their Schematic for functional metagenomics approach combining stable isotope probing and metagenomics. For details see Kalyuzhnaya et al., 2008.sequences determined (Fig. 1).

Analysis of taxonomic and functional gene signatures in the respective metagenomes reveals shifts toward specifıc functional guilds in each community, highlighting roles of specifıc guilds in using specifıc single-carbon compounds. From these analyses, we learned that the methylamine microcosm is one of the least complex in terms of species richness, and it is dominated by a group of closely related strains from the genus Methylotenera, until recently a relatively understudied guild of bacteria.

High coverage ofDNArepresenting these species allowed us to extract a composite genome of Methylotenera from the metagenomic sequence, whose metabolism we reconstructed and then compared with that of close relatives. One notable element that was missing from the composite genome of Methylotenera is the methanol dehydrogenase- encoding gene cluster thought to be highly conserved in methylotrophs.

Another distinctive feature of the Methylotenera genome is the genes needed for denitrifıcation, suggesting a role for Methylotenera species in nitrate metabolism. Further, the Methylotenera species are also highly enriched in microcosms fed with methane or methanol, creating a paradox: the reconstructed genomes of these organisms do not contain enzymes for metabolizing these substrates. That result left us wondering how they could effıciently label their DNA.

The other highly covered genomewe retrieved from metagenomic sequences is the composite genome of a Methylobacter species that is closely related to Methylobacter tundripaludum, especially well represented in the methane microcosm dataset. This suggested that uncultivated, Methylobacter- type methanotrophs and not the previously cultivated methanotroph species are the major functional types in the sediment.

Thus, metagenomics enables the detailed analysis of environmentally relevant microbes, bypassing isolation and pure culture experiments. In this case, the specifıc enrichment step, i.e. separation of DNA labeled via SIP, proved key to increasing resolution, focusing the metagenomic sequencing effort on specifıc functional types. Overall, this study uncovered dynamic and diverse microbial populations responding to single- carbon substrates, pointing toward the existence of a complex, multi-tiered microbial food web involved in environmental carbon and nitrogen cycling in Lake Washington sediment and likely other freshwater lake sediments.

Active Methylotenera Ecotypes Are Methylophilaceae with Novel Physiology

We again employed the SIP approach on Lake Washington sediment populations—this time to better understand the Methylotenera paradox and to target nitrate-stimulated populations. We fed natural communities with labeled methanol while varying oxygen tensions and supplementing them with nitrate. These experiments indicate that as many as seven different major Methylophilaceae phylotypes respond to methanol.

Meanwhile, the response to nitrate differs among different phylotypes, further suggesting differences in the modes of carbon and nitrogen metabolism for taxonomically closely related bacteria. These results further highlight the complexity of metabolic schemes employed for methylotrophy in this ecological niche.

In the laboratory, we experimented with model strain Methylotenera mobilis JLW8, which was isolated from Lake Washington and whose physiology (i.e., growth on methylamine and poor growth on methanol) match the predicted phenotype for active Methylotenera ecotypes detected through SIP-based metagenomics.

We tested whether M. mobilis JLW8 generates nitrous oxide from nitrate as a product of a truncated denitrifıcation pathway that we predicted from the composite SIP-enriched Methylotenera genome. Indeed, nitrous oxide (N2O) forms when this strain grows using either methanol or methylamine as electron donors.

We later obtained the genomic sequence of strain JLW8, along with transcriptomic and proteomic data, and these data further confırmed that, metabolically, this organism closely resembles the major strain from the methylamine microcosm. It encodes a truncated denitrifıcation pathway, possesses a methylamine dehydrogenase, and lacks a true methanol dehydrogenase. Further analyses indicated that two XoxF proteins that are homologs of the large subunit of methanol dehydrogenase are highly expressed. Moreover, methanol induced at least one of them, suggesting it functions in methylotrophy.

The denitrifıcation pathway components that we deduced from the genome include a single-subunit periplasmic nitrate reductase, a copper-containing nitrite reductase, a NAD(P)H-linked nitrite reductase, and a nitric oxide reductase. Knockout mutants yielded negative growth phenotypes only for those mutants lacking the nitrate reductase and NAD(P)H-linked nitrite reductase and only when nitrate was supplemented as a nitrogen source, suggesting that nitrate respiration was not essential for methylotrophy in this organism, according to our former collaborator Ildar Mustakhimov (currently of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences). However, mutants with deletions in each of these denitrifıcation enzymes also could not produce N2O from NO3-, confırming their roles in the denitrifıcation pathway.

Meanwhile, mutants in the two xoxF genes reveal a similar phenotype, suggesting their involvement in nitrate metabolism, likely as donors of electrons from methanol and/or methylamine. Adouble xoxF mutant, in addition to a defıciency in nitrate reduction, lost its ability to grow on methanol, suggesting that the two xoxF enzymes serve as methanol dehydrogenases in this organism.

Methane Metabolism Metagenomics Suggest Cooperative Behavior

The SIP-enabled functional metagenomics approach is helping us to determine which methylotrophic species are aerobic versus microaerobic when consuming methane, whether nitrate stimulates this process, and what species potentially link the carbon and nitrogen cycles in freshwater environments. We began by sequencing metagenomes of Lake Washington populations that were enriched in 13C label from 13C-methane. These experiments involved populations in microcosms that were exposed to labeled methane under conditions in which oxygen tension was varied while nitrate was added.

Under aerobic conditions, two major guilds of bacteria respond to 13C-labeled methane, which quickly accumulates in their DNA: the Methylococcaceae, with the major type being Methylobacter species, and the Methylophilaceae, with the major type being Methylotenera species. Their responses are more pronounced, and the labeled methane accumulates more rapidly in the presence of nitrate. The dominance of these species and their coordinated Identification of major active types through taxonomic profiling of metagenomes. Rows A and B, pyrotag analysis; rows C and D, genome recruitment analysis. A, distribution of pyrotag sequences among major phyla. Clockwise starting with blue: Proteobacteria, Bacteroidetes, Chloroplast, Acidobacteria, Chloroflexi, Actinobacteria, Planctomycetes, Gemmatimonadetes, Firmicutes, Chlorobi, Nitrospirae, Verrucomicrobia, Other (phyla making up to less than 1% of total). B, Methylococcaceae are in blue, Methylophilaceae are in red, all others are in yellow. C, sequences classified at 90% identity level. Gammaproteobacteria are in blue, Betaproteobacteria are in red, Alphaproteobacteria are in green, all others are in purple. D, Methylococcaceae are in blue, Methylophilaceae are in red, Methylocystaceae are in green, all others are in purple. For details see Beck et al., 2013.responses to both methane and nitrate (Fig. 2) suggest that the Methylococcaceae and the Methylophilaceae species engage in cooperative behavior under aerobic conditions.

The community response differs markedly in microaerobic conditions. For one thing, the proportion of the Methylococcaceae as part of the labeled DNA pool is much smaller in these conditions (Fig. 2). Further, the Methylococcaceae and predominantly Methylobacter remain the only active methanotroph species, and no alternative methane-oxidizing species (such as the anaerobic methanotrophs of the NC10 phylum) appear active. However, the community complexity remains very high and the 13C label distributes evenly among a variety of nonmethylotrophic heterotroph species, suggesting that, in low-oxygen conditions, a different type of cooperative behavior may be taking place. Moreover, this cooperation appears not to select for a special functional type.

Schematic of potential relationships between methane-oxidizing species and non-methane utilizing heterotrophs. The factors involved remain unknown.The mechanistic details of either of these types of cooperation are not known (Fig. 3). For the Methylobacter/Methylotenera partnership, a simple scenario of methanol being the cross-feeding substrate is likely not the case, as the predominant Methylotenera species are poor methanol oxidizers. For the Methylobacter/generalist heterotroph partnership, in one proposed scenario, the methanotroph releases organic compounds that then serve as electron donors for denitrifıcation by the nonmethylotrophic heterotrophs, according to Oskar Modin, now of Chalmers University of Technology in Göteborg, Sweden, Kazuo Yamomoto of the University of Tokyo in Tokyo, Japan, and their collaborators, or, alternatively, as carbon sources, according to David van der Ha, Nico Boon, and their colleagues at Ghent University in Belgium. However, the nature of these excreted organics, the role of nitrate in such cooperative behavior, and whether the relationships are mutualistic or saprophytic are not known.

One approach toward learning more about these microbial communities would involve manipulating controlled consortia of methanotrophs and nonmethanotroph microorganisms, followed by analyzing their transcripts, metabolites, and other factors. The time is ripe to initiate such studies, because we have access to numerous prototype organisms in pure cultures and to some of their genomic sequences, to genetic tools with which to manipulate these organisms, and to an array of -omics technologies. Future discoveries will shed light on methylotrophy and nitrogen metabolism, possibly revealing novel metabolic strategies involved in carbon and nitrogen cycling in freshwater environments.

Ludmila Chistoserdova is a Senior Scientist at the Department of Chemical Engineering, University of Washington, Marina G. Kalyuzhnaya is a Research Associate Professor at the Department of Microbiology, University of Washington, and Mary E. Lidstrom is Frank Jungers Chair in Chemical Engineering, Professor of Microbiology, and Vice-Provost for Research at the University of Washington.


The authors acknowledge support from the National Science Foundation (Grant MCB-0950183) and from the Department of Energy (Grant DE-SC0005154).

Suggested Reading

Beck, D. A. C., M. G. Kalyuzhnaya, S. Malfatti, S. Tringe, T. Glavina del Rio, N. Ivanova, M. E. Lidstrom, and L. Chistoserdova. 2013. A metagenomic insight into freshwater methane-utilizing communities and evidence for cooperation between the Methylococcaceae and the Methylophilaceae. PeerJ 1:e23.

Beck, D. A., E. L. Hendrickson, A. Vorobev, T. Wang, S. Lim, M. G. Kalyuzhnaya, M. E. Lidstrom, M. Hackett, and L. Chistoserdova. 2011. An integrated proteomics/ transcriptomics approach points to oxygen as the main electron sink for methanol metabolism in Methylotenera mobilis. J. Bacteriol. 193:4758–4765.

Chistoserdova, L. 2011. Methylotrophy in a lake: from metagenomics to single organism physiology. Appl. Environ. Microbiol. 77:4705–4711.

Chistoserdova, L., and M. E. Lidstrom. 2013. Aerobic methylotrophic prokaryotes, p. 267–285. In E. Rosenberg, E. F. DeLong, F. Thompson, S. Lory, and E. Stackebrandt (ed.), The prokaryotes, fourth edition. Springer, New York, p. 267–285.

Dumont, M. G., and J. C. Murrell. 2005. Stable isotope probing-linking microbial identity to function. Nature Rev. Microbiol. 3:499–504.

Kalyuzhnaya, M. G., A. Lapidus, N. Ivanova, A. C. Copeland, A. C. McHardy, E. Szeto, A. Salamov, I. V. Grigoriev, D. Suciu, S. R. Levine, V. M. Markowitz, I. Rigoutsos, S. G. Tringe, D. C. Bruce, P. M. Richardson, M. E. Lidstrom, and L. Chistoserdova. 2008. High resolution metagenomics targets major functional types in complex microbial communities. Nature Biotechnol. 26:1029–1034.

Lapidus, A., A. Clum, K. LaButti, M. G. Kalyuzhnaya, S. Lim, D. A. C. Beck, T. Glavina del Rio, M. Nolan, K. Mavromatis, M. Huntemann, S. Lucas, M. E. Lidstrom, N. Ivanova, and L. Chistoserdova. 2011. Genomes of three methylotrophs from a single niche uncover the genetic and metabolic divergence of the Methylophilaceae. J. Bacteriol. 193:3757–3764.

Modin, O., K. Fukushi, and K. Yamamoto. 2007. Denitrifıcation with methane as external carbon source. Water Res. 41:2726–2738.

Mustakhimov, I., M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova. 2013. Insights into denitrifıcation in Methylotenera mobilis from denitrifıcation pathway and methanol metabolism mutants. J. Bacteriol. 195:2207–2211.

van der Ha, D., I. Vanwonterghem, S. Hoefman, P. De Vos, and N. Boon. 2013. Selection of associated heterotrophs by methane-oxidizing bacteria at different copper concentrations. Antonie Van Leeuwenhoek 103

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