1Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
2Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Microbiology (Reading). 2019 Jan;165(1):65-77. doi: 10.1099/mic.0.000737. Epub 2018 Nov 5.
N-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. Zn-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.
N-赖氨酸乙酰化和琥珀酰化是真核生物和细菌中普遍存在的翻译后修饰。在本研究中，我们展示了枯草芽孢杆菌翻译延伸因子 Tu 的 TufA 的乙酰化和琥珀酰化的动态变化。在 LB 和最小葡萄糖条件下的指数生长阶段观察到 TufA 的乙酰化增加，当其进入静止期时其乙酰化水平下降，而在晚期静止期其琥珀酰化增加。在最小柠檬酸或琥珀酸盐条件下的营养生长过程中，TufA 也发生琥珀酰化。突变分析表明，TufA 结构域 3 中的 Lys306、Lys308 和 Lys316 的三琥珀酰化模拟突变对枯草芽孢杆菌的生长有负面影响，而这些三个赖氨酸残基的非乙酰化模拟突变则没有。与生长表型一致，三琥珀酰化模拟突变体在体外显示出 67%的翻译活性降低，表明结构域 3 中赖氨酸残基的琥珀酰化降低了翻译活性。包括 Lys308 在内的 TufA 被生理浓度的琥珀酰辅酶 A 非酶促琥珀酰化。G 结构域中的 Lys42 被鉴定为最常被修饰的乙酰化位点，尽管其乙酰化可能对 TufA 翻译活性和生长不是必需的。乙酰化底物和 TufA 乙酰化的细胞内水平的测定表明，乙酰磷酸负责 TufA 几个赖氨酸位点的乙酰化，但不负责 Lys42 的乙酰化。推测乙酰辅酶 A 可能负责 Lys42 的乙酰化，尽管 AcuA 乙酰转移酶不参与。Zn 依赖性 AcuC 和 NAD 依赖性 SrtN 去乙酰化酶负责 TufA 的去乙酰化，包括 Lys42。这些发现表明，在枯草芽孢杆菌中，乙酰化和琥珀酰化可能在调节 TufA 功能和翻译以响应营养环境方面发挥潜在的调控作用。
