The role of methanotrophy in the microbial carbon metabolism of temperate lakes

Previous stable isotope and biomarker evidence has indicated that methanotrophy is an important pathway in the microbial loop of freshwater ecosystems, despite the low cell abundance of methane-oxidizing bacteria (MOB) and the low methane concentrations relative to the more abundant dissolved organic carbon (DOC). However, quantitative estimations of the relative contribution of methanotrophy to the microbial carbon metabolism of lakes are scarce, and the mechanism allowing methanotrophy to be of comparable importance to DOC-consuming heterotrophy remained elusive. Using incubation experiments, microscopy, and multiple water column profiles in six temperate lakes, we show that MOB play a much larger role than their abundances alone suggest because of their larger cell size and higher specific activity. MOB activity is tightly constrained by the local methane:oxygen ratio, with DOC-rich lakes with large hypolimnetic volume fraction showing a higher carbon consumption through methanotrophy than heterotrophy at the whole water column level. Our findings suggest that methanotrophy could be a critical microbial carbon consumption pathway in many temperate lakes, challenging the prevailing view of a DOC-centric microbial metabolism in these ecosystems.

Genomic Insights into Denitrifying Methane-Oxidizing Bacteria Gemmobacter fulva sp. Nov., Isolated from an Anabaena Culture

The genus Gemmobacter grows phototrophically, aerobically, or anaerobically, and utilizes methylated amine. Here, we present two high-quality complete genomes of the strains con4 and con5T isolated from a culture of Anabaena. The strains possess sMMO (soluble methane monooxygenase)-oxidizing alkanes to carbon dioxide. Functional genes for methane-oxidation (prmAC, mimBD, adh, gfa, fdh) were identified. The genome of strain con5T contains nirB, nirK, nirQ, norB, norC, and norG genes involved in dissimilatory nitrate reduction. The presence of nitrite reductase gene (nirK) and the nitric-oxide reductase gene (norB) indicates that it could potentially use nitrite as an electron acceptor in anoxic environments. Taxonomic investigations were also performed on two strains through polyphasic methods, proposing two isolates as a novel species of the genus Gemmobacter. The findings obtained through the whole genome analyses provide genome-based evidence of complete oxidation of methane to carbon dioxide. This study provides a genetic blueprint of Gemmobacter fulva con5T and its biochemical characteristics, which help us to understand the evolutionary biology of the genus Gemmobacter.

Anaerobic 3-methylhopanoid production by an acidophilic photosynthetic purple bacterium

Bacterial lipids are well-preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylhopanoids, pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-methylhopanoids and a suite of related hopanoids and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-methylhopanoids can be produced under anoxic conditions and challenges the use of 3-methylhopanoids as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.

Acetate excretion by a methanotroph, Methylocaldum marinum S8, under aerobic conditions

ABSTRACT Methane-oxidizing bacteria (methanotrophs) often coexist with methylotrophs that utilize methanol excreted by methanotrophs. Recently, we found that a facultative methylotroph, Methyloceanibacter caenitepidi Gela4T, possibly utilizes acetate rather than methanol in the coculture with a methanotroph, Methylocaldum marinum S8. Here, we examined the effects of oxygen concentrations on growth of and acetate excretion by M. marinum S8 in pure culture and the coculture with M. caenitepidi Gela4T. M. marinum S8 excreted acetate during the exponential growth phase not only under microaerobic (O2 concentrations of 3.5%-6%) but also under aerobic (O2 concentrations of 20%-31%) conditions. RNA-Seq analyses of M. marinum S8 cells grown under aerobic conditions suggested that phosphoketolase and acetate kinase were candidate genes involved in acetate production. Nonmethylotrophic bacteria, Cupriavidus necator NBRC 102504, could grow when cocultured with M. marinum S8, also supporting the existence of methanol-independent cross-feeding from M. marinum S8 under aerobic conditions.

USC γ Dominated Community Composition and Cooccurrence Network of Methanotrophs and Bacteria in Subterranean Karst Caves

Karst caves have recently been demonstrated to be a potential atmospheric methane sink, presumably due to consumption by methane-oxidizing bacteria. However, the sparse knowledge about the diversity, distribution, and community interactions of methanotrophs requires us to seek further understanding of the ecological significance of methane oxidation in these ecosystems.

Carbon sources of benthic fauna in temperate lakes across multiple trophic states

Previous studies have shown that microbially produced methane can be a dominant carbon source of lacustrine sedimentary macrofauna in eutrophic lakes, most likely through grazing on methane-oxidizing bacteria. Here we investigate the contributions of different carbon sources to macrofaunal biomass across five lakes in central Switzerland that range from oligotrophic to highly eutrophic. Macrofaunal communities change with trophic state, with chironomid larvae dominating oligotrophic and tubificid oligochaetes dominating eutrophic lake sediments. The 13C-isotopic data suggest that the average contribution of methane-derived carbon to the biomass of both macrofaunal groups is similar but consistently remains minor, ranging from only ∼1 % in the oligotrophic lake to at most 12 % in the eutrophic lakes. The remaining biomass can be explained by the assimilation of detritus-derived organic carbon. Low abundances of methane-cycling microorganisms in macrofaunal specimens, burrows, and surrounding sediment based on 16S ribosomal RNA (rRNA) gene sequences and copy numbers of genes involved in anaerobic and aerobic methane cycling (mcrA, pmoA) support the interpretation of isotopic data. Notably, 16S rRNA gene sequences of macrofauna, including macrofaunal guts, are highly divergent from those in tubes or sediments. Many macrofaunal specimens are dominated by a single 16S rRNA phylotype of Fusobacteria, α-, β-, γ-, or ε-Proteobacteria, Bacteroidetes, or Parcubacteria. This raises the question of whether dominant lake macrofauna live in so far uncharacterized relationships with detrital organic-matter-degrading bacterial endosymbionts.

Copper binding by a unique family of metalloproteins is dependent on kynurenine formation

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned ∼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.

A Toll-like receptor identified in Gigantidas platifrons and its potential role in the immune recognition of endosymbiotic methane oxidation bacteria

Symbiosis with chemosynthetic bacteria is an important ecological strategy for the deep-sea megafaunas including mollusks, tubeworms and crustacean to obtain nutrients in hydrothermal vents and cold seeps. How the megafaunas recognize symbionts and establish the symbiosis has attracted much attention. Bathymodiolinae mussels are endemic species in both hydrothermal vents and cold seeps while the immune recognition mechanism underlying the symbiosis is not well understood due to the nonculturable symbionts. In previous study, a lipopolysaccharide (LPS) pull-down assay was conducted in Gigantidas platifrons to screen the pattern recognition receptors potentially involved in the recognition of symbiotic methane-oxidizing bacteria (MOB). Consequently, a total of 208 proteins including GpTLR13 were identified. Here the molecular structure, expression pattern and immune function of GpTLR13 were further analyzed. It was found that GpTLR13 could bind intensively with the lipid A structure of LPS through surface plasmon resonance analysis. The expression alternations of GpTLR13 transcripts during a 28-day of symbiont-depletion assay were investigated by real-time qPCR. As a result, a robust decrease of GpTLR13 transcripts was observed accompanying with the loss of symbionts, implying its participation in symbiosis. In addition, GpTLR13 transcripts were found expressed exclusively in the bacteriocytes of gills of G. platifrons by in situ hybridization. It was therefore speculated that GpTLR13 may be involved in the immune recognition of symbiotic methane-oxidizing bacteria by specifically recognizing the lipid A structure of LPS. However, the interaction between GpTLR13 and symbiotic MOB was failed to be addressed due to the nonculturable symbionts. Nevertheless, the present result has provided with a promising candidate as well as a new approach for the identification of symbiont-related genes in Bathymodiolinae mussels.