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Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

 

Abstract The formation and fate of pyrite (FeS 2 ) modulates global iron, sulfur, carbon, and oxygen biogeochemical cycles and has done so since early in Earth’s geological history. A longstanding paradigm is that FeS 2 is stable at low temperature and is unavailable to microorganisms in the absence of oxygen and oxidative weathering. Here, we show that methanogens can catalyze the reductive dissolution of FeS 2 at low temperature (≤38 °C) and utilize dissolution products to meet cellular iron and sulfur demands associated with the biosynthesis of simple and complex co-factors. Direct access to FeS 2 is required to catalyze its reduction and/or to assimilate iron monosulfide that likely forms through coupled reductive dissolution and precipitation, consistent with close associations observed between cells and FeS 2 . These findings demonstrate that FeS 2 is bioavailable to anaerobic methanogens and can be mobilized in low temperature anoxic environments. Given that methanogens evolved at least 3.46 Gya, these data indicate that the microbial contribution to the iron and sulfur cycles in ancient and contemporary anoxic environments may be more complex and robust than previously recognized, with impacts on the sources and sinks of iron and sulfur and other bio-essential and thiophilic elements such as nickel and cobalt. 

Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community’s metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach. 

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

 

A hallmark of the SUP05 clade of marineGammaproteobacteriais the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism inCandidatusThioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 μm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05.