Title: Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

Marine methane seep habitats represent an important control on the global flux of methane between the subsurface and water column reservoirs. Meta-omics studies have begun to outline community-wide metabolic potential, but expression patterns of proteins that enact sulfate-mediated anaerobic methane oxidation in seeps are poorly characterized. Proteomic stable isotope probing (proteomic SIP) offers an additional layer of information for characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track the protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 proteins were identified, 21% of which were 15N-labeled. We observed active synthesis (15N enrichment) of all proteins believed to be involved in sulfate reduction and reverse methanogenesis including methylenetetrahydromethanopterin reductase (Mer). The abundance and phylogenetic range of methyl-coenzyme M reductase (Mcr) orthologs produced during incubation experiments suggests that seeps provide sufficient niches for multiple organisms performing analogous metabolisms. Twenty-eight previously unreported post-translational modifications of McrA were measured, indicating dynamic enzymatic machinery and offering a dimension of functional diversity beyond gene-dictated sequence. RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and aerobic Gammaproteobacteria were more abundant among pre-incubation proteins, suggesting diminished metabolic activity in long-term anoxic, sulfidic experimental incubations. Twenty-six proteins of unknown function were detected in all proteomic experiments and actively expressed in labeled experiments, suggesting that they play important roles in methane seep ecosystems. The addition of stable isotope probing to environmental proteomics experiments provides a mechanism to begin to assess the degree to which diagnostic meatbolic proteins are long-lived or acively synthesized in complex, slow-growing microbial communities. Our work here demonstrates that sediment-hosted microbial assemblages in marine methane seeps are dynamic, heterogeneous systems with broad functional diversity.