UCLA

Post-translational modification (PTM) of proteins is a conserved strategy used by organisms to efficiently control biological mechanisms, allowing rapid, adaptive cellular responses. PTMs can be categorized into two classes based on their biochemical origin: enzymatic and non-enzymatic. Non-enzymatic modifications arise from reactions between reactive electrophilic metabolites and nucleophilic or redox-sensitive amino acid side chains. Lysine acylation, a type of PTM, can occur spontaneously by intrinsically reactive metabolites, reactive acyl-CoA species (RACS). Metabolic pathways generate a variety of RACS that, in turn, produce equally diverse acyl-modifications. These PTMs regulate diverse biological mechanisms, deeply intersecting with cellular metabolism. Characterizing these PTMs comprehensively is important to understand their impact on metabolic regulation. While proteomic investigations have identified PTMs in bacteria, a majority of these studies focus on acetylation and not on other acylations formed by RACS.

Syntrophic bacteria are key players in microbial syntrophy, an important component of the global carbon cycle where different microbial species cooperate to decompose biopolymers into smaller molecules; e.g., hydrogen and methane. Syntrophic bacteria degrade fatty acid intermediates to hydrogen, formate, and CO2 and must cooperate with hydrogen consuming partners, methanogens, due to the reactions’ energetically unfavorable thermodynamics. As syntrophs live under thermodynamically challenging environments and its metabolic intermediates largely comprise of RACS, we hypothesized that these reactive metabolites lead to acyl-lysine modification and impact microbial metabolism. We utilized mass spectrometry (MS)-based proteomics to identify acyl-PTMs in syntrophic bacteria and developed MS methods to incorporate diagnostic marker ions for more confident identification of acyl-lysine. We characterized 10 types of acyl-modifications found amongst Syntrophus aciditrophicus and Syntrophomonas wolfei, including novel PTMs not reported in any other system. All modification types correspond directly to RACS produced in degradation pathways and a majority of modified proteins are involved in metabolism. The type and abundance of acyl-PTMs change with cultivation in different carbon substrates, linking protein acylation by RACS to shifts in cellular metabolism. We also characterized the proteomes of bacteria from other phyla, proteobacteria, identifying similar acyl-PTMs and raising the compelling possibility that non-enzymatic lysine acylation may be prevalent in microbial metabolism.