Deciphering the in situ activities of microorganisms is essential for understanding the biogeochemical processes occurring in complex environments. Here, we used environmental metaproteomics to obtain information about the identity of subsurface microbial populations in coal tar-contaminated groundwater and the metabolic processes they catalyze. Metaproteomic libraries (two shotgun and seven slices from one SDS-PAGE gel) were generated from replicate samples of microbial biomass. Peptide fragment analysis using nano-liquid chromatography (LC)-mass spectrometry (MS)/MS of the three protein pools generated a total of 95,725 mass spectra. When analyzed using mascot v.2.3.02 and searched against the NCBInr bacterial database [confidence interval 99% (P < 0.01)], a total of 1,270 proteins had at least two peptide matches. Replication of identified proteins across the three libraries was low (3.3%); however, in each library, the most frequently identified protein host was Candidatus Methylomirabilis oxyfera (15, 12 and 62 proteins for each shotgun and the gel-slice library respectively). Remarkably, eight of the nine proteins in the nitrite-dependent anaerobic methane oxidation pathway were found. Additionally, 39 proteins were matched to known anammox bacteria including hydroxylamine and hydrazine oxidase. Metaproteomics thus revealed a microbial population, closely related to Ca. Methylomirabilis oxyfera, actively engaged in nitrite-dependent anaerobic methane oxidation and likely competing for nitrite with anammox bacteria.
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