Dynamic changes in lysine acetylation and succinylation of the elongation factor Tu in Bacillus subtilis.

N^ε-lysine acetylation and succinylation are ubiquitous post-translational modifications in eukaryotes and bacteria. In the present study, we showed a dynamic change in acetylation and succinylation of TufA, the translation elongation factor Tu, from Bacillus subtilis. Increased acetylation of TufA was observed during the exponential growth phase in LB and minimal glucose conditions, and its acetylation level decreased upon entering the stationary phase, while its succinylation increased during the late stationary phase. TufA was also succinylated during vegetative growth under minimal citrate or succinate conditions. Mutational analysis showed that triple succinylation mimic mutations at Lys306, Lys308 and Lys316 in domain-3 of TufA had a negative effect on B. subtilis growth, whereas the non-acylation mimic mutations at these three lysine residues did not. Consistent with the growth phenotypes, the triple succinylation mimic mutant showed 67 % decreased translation activity in vitro, suggesting a possibility that succinylation at the lysine residues in domain-3 decreases the translation activity. TufA, including Lys308, was non-enzymatically succinylated by physiological concentrations of succinyl-CoA. Lys42 in the G-domain was identified as the most frequently modified acetylation site, though its acetylation was likely dispensable for TufA translation activity and growth. Determination of the intracellular levels of acetylating substrates and TufA acetylation revealed that acetyl phosphate was responsible for acetylation at several lysine sites of TufA, but not for Lys42 acetylation. It was speculated that acetyl-CoA was likely responsible for Lys42 acetylation, though AcuA acetyltransferase was not involved. Zn²⁺-dependent AcuC and NAD⁺-dependent SrtN deacetylases were responsible for deacetylation of TufA, including Lys42. These findings suggest the potential regulatory roles of acetylation and succinylation in controlling TufA function and translation in response to nutrient environments in B. subtilis.

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