Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesize that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC-MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant to analyze post-translational modifications (PTMs) without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3-hydroxybutyryl-, crotonyl-, valeryl-, and hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the potential impact of these acyl-protein modifications. The extensive changes in acylation-type, abundance, and modification sites with carbon substrate suggest that protein acylation by RACS may be an important regulator of syntrophy.

Unscheduled PRM-targeted MS analysis (Peterson et al., 2012) was utilized to quantify acyl-peptides of interest using the list of target peptide ions identified in HILIC DDA experiments. Desalted peptides from the eFASP procedure were analyzed without offline fractionation. Liquid chromatography was performed identically to the shotgun and HILIC-fractionated experiments. The inclusion list used for PRMs is shown in Supplemental Table 1. The MS2 scans were obtained at a 17500 resolution with a normalized AGC target 100% and an isolation window of m/z 1.2 using HCD fragmentation at a normalized collision energy of 30%.

Syntrophomonas wolfei subspecies wolfei strain Göttingen (DSM 2245B) (Lorowitz, Zhao & Bryant, 1989) (hereafter referred to as S. wolfei Göttingen) cells were grown axenically with crotonate as the carbon substrate (McInerney et al., 1981), or in coculture with methanogenic partner, Methanospirillum hungatei JF1 (DSM864), on butyrate as previously described (McInerney, Bryant & Pfennig, 1979). Cells were harvested anaerobically and then frozen and stored at -70°C until processed (Muroski et al., 2022). Syntrophomonas wolfei subspecies methylbutyratica strain 4J5T (JCM 14075) was grown in the presence of methanogenic partner M. hungatei as described above but using the substrates, butyrate, crotonate, 2-methylbutyrate, valerate or hexanoate. Analyses were performed on three biological replicates from each culture condition.