Syntrophus aciditrophicus (SB) degrades benzoate, cyclohexane-1-carboxylate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms and ferments crotonate in pure culture. ATP formation coupled to acetate production is the main mechanism of energy conservation by S. aciditrophicus. However, the method by which S. aciditrophicus synthesizes ATP from acetyl-CoA is unclear. The genome of S. aciditrophicus does not contain an annotated acetate kinase gene, but has two genes for butyrate kinase; several genes for AMP-forming, acetyl-CoA synthetases; and nine genes for archaeal ADP-forming, acetyl-CoA synthetases all of which could be used to synthesize ATP from acetyl-CoA. Two-dimensional gel electrophoresis and quantitative-real time-polymerase chain reaction detected peptides and transcripts, respectively, from AMP-forming, acetyl-CoA synthetase; ADP-forming, acetyl-CoA synthetases; and butyrate kinase genes. Acetyl-CoA synthetase activity was high (0.5 ± 0.01 to 7.4 ± 0.3 µmol min-1 mg-1 of protein ) in cell-free extracts of S. aciditrophicus grown in pure culture on crotonate or in coculture with Methanospirillum hungatei on crotonate, benzoate and cyclohexane-1-carboxylate. Acetate kinase, butyrate kinase and phosphotransacetylase activities were low (< 0.2 ± 0.03 µmol min-1 mg-1 of protein) and only detected in cell-free extracts of crotonate-grown pure and coculture cells. Only the acetyl-CoA synthetase activity was high enough to account for the acetate production rate (1.2 ± 0.2 µmol min-1 mg-1 of protein) during crotonate growth. Competitive inhibition of the native adenylate kinase showed that the dominant acetyl-CoA synthetase activity was an AMP-forming, acetyl-CoA synthetase; ADP-forming, acetyl-CoA synthetase activity was not detected. The acetyl-CoA synthetase activity was purified to homogeneity with an 80% recovery. The purified protein was an AMP-forming, acetyl-CoA synthetase encoded by gene SYN_02635 (acs1), which had a Vmax of 7.5 ± 1.2 µmol min-1 mg-1 of protein in the acetate-forming direction, sufficient to account for the acetate production rates under all growth conditions. A recombinant Acs1 had similar kinetic properties. Transcripts of acs1 represented 0.58 to 0.76% of the total transcriptome compared to 0 to 0.1% for the other possible candidates. Polypeptides of the Acs1 represented 1.3 to 4.4% of the total peptides detected compared to 0 to 0.1% for other possible candidates. The above analyses show that S. aciditrophicus uses Acs1 for ATP formation from acetyl-CoA. S. aciditrophicus has two candidate genes that could encode for acetate kinase, SYN_03090 and SYN_01210, which share 99% identity with each other at the nucleotide level, and both of which annotate as butyrate kinases. The nucleotide sequence of SYN_03090 was cloned heterologously expressed in Escherichia coli Bl21. The purified recombinant protein had acetate kinase activity but not propionate or butyrate kinase activity. ATP was the preferred nucleotide triphosphate with acetate as the substrate. The purified recombinant protein synthesized ATP from acetyl-phosphate. Phylogenetic analyses showed butyrate kinases clustered together and acetate kinases clustered together on a neighbor joining phylogenetic tree. The amino acid sequences of both SYN_03090 and SYN_01210 grouped with those of the butyrate kinases. These data support the conclusion that SYN_03090/SYN_01210 gene product is an acetate kinase.
Previous studies indicated that S. aciditrophicus uses AMP-forming, acyl-CoA synthetases (substrate:CoA ligase) rather than CoA transferases for substrate activation. Here, two crotonate/benzoate:CoA ligases were purified and characterized from cell-free extracts of S. aciditrophicus. Peptide analysis showed that these proteins were gene products of SYN_02896 and SYN_02898. The gene for SYN_03128, annotated as a long-chain fatty acid-CoA ligase, was cloned and heterologously expressed in Escherichia coli. The purified SYN_03128 gene product had high activity and affinity for cyclohexane-1-carboxylate (Vmax and Km of 15 ± 0.5 µmol min-1mg-1 and 0.04 ± 0.007 mM, respectively), showing that it is a cyclohexane-1-carboxylate:CoA ligase. S. aciditrophicus uses benzoate:CoA ligase for activation of both benzoate and crotonate, and uses a separate ligase for activation of cyclohexane-1-carboxylate. By coupling the activation of benzoate, crotonate, and cyclohexane-1-carboxylate to the AMP-forming, acetyl-CoA synthetase reaction for ATP synthesis the net reaction is functionally equivalent to a CoA transferase reaction.