Abstract
Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to 15 and CH4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were 15N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis—including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)—were all observed to be actively synthesized (15N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of 15N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.
Highlights
Marine methane seep sediments harbor complex microbial communities that play significant roles in the global carbon and sulfur biogeochemical cycles (Jørgensen and Kasten, 2006; Reeburgh, 2007)
Samples of methane seep sediment from Hydrate Ridge were each allocated into two anaerobic microcosm incubations overpressured with methane treatment) or a1n5dNHam+4 en(dlaebdelewdithtreeaitthmeernt1;4NFHig+4ur(eu1n)l.abeCleedll aggregate abundance and sulfide concentrations, both of which correlate positively with sulfate-based anaerobic oxidation of methane (AOM) activity (Iversen and Jorgensen, 1985), were monitored throughout the incubation period
The detection of multiple functionally relevant orthologs provides a broad sense of in situ active metabolic pathways, while the incorporation of stable isotope probing methods reveals the subset of proteins actively produced under laboratory-based methanotrophic conditions
Summary
Marine methane seep sediments harbor complex microbial communities that play significant roles in the global carbon and sulfur biogeochemical cycles (Jørgensen and Kasten, 2006; Reeburgh, 2007). Proteomic stable isotope probing (SIP) couples these experimental objectives by offering a functionally- and phylogenetically-constrained enzymatic profile of constituent organisms as well as a temporal reporter of protein synthesis and metabolic response to distinct conditions (Pan et al, 2011; Seifert et al, 2012; Justice et al, 2014; Mohr et al, 2014) In environments such as anoxic methane seep sediment and an array of subsurface habitats, where energy can become limiting, microbes frequently exhibit extremely slow growth rates and are recalcitrant to activitybased analyses. Challenges remain— surrounding protein extraction, peptide quantification, database collation, and the interpretation of non-detections—proteomic SIP offers a promising method for assessing the in vivo activity and catalytic function of microbial communities
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