The Impact of ackA, pta, and ackA-pta Mutations on Growth, Gene Expression and Protein Acetylation in Escherichia coli K-12
The Impact of ackA, pta, and ackA-pta Mutations on Growth, Gene Expression and Protein Acetylation in Escherichia coli K-12
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DOI：
10.3389/fmicb.2020.00233
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发表时间：
2020-02-21
影响因子：
5.2
通讯作者：
Bettenbrock, Katja
中科院分区：
文献类型：
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作者：
Schuetze, Andrea;Benndorf, Dirk;Bettenbrock, Katja
醋酸盐是大肠杆菌K-12在分批培养中与葡萄糖一起生长的一种特征副产物，在好氧和厌氧条件下都是如此。虽然好氧产生醋酸酯的原因仍在讨论中，但在厌氧生长过程中，通过底物水平的磷酸化产生醋酸盐对ATP的产生是重要的。在这两种条件下，乙酸酯的生成途径包括从乙酰辅酶A产生乙酰磷酸的磷酸乙酰转移酶(PTA)和从乙酰磷酸产生醋酸酯的醋酸激酶(AckA)，该反应与三磷酸腺苷的产生相耦合。AckA-PTA途径中的突变体在产生和积累乙酰磷酸的潜力方面各不相同。在手头的出版物中，我们研究了乙酸酯途径中的不同突变体，包括在有氧和无氧条件下。在好氧条件下，所有醋酸酯突变株的生长速率变化不大，但在厌氧生长过程中，所有乙酸酯突变株的生长速率和副产物模式都发生了严重的变化。AckA(-)突变体表现出最严重的生长缺陷。该菌株的葡萄糖摄取速率和ATP浓度显著降低。该突变体还表现出基因表达的变化。在该菌株中，toDAEB操纵子在厌氧条件下显著上调，提示乙酰乙酸酯的产生。在厌氧生长过程中，ackA突变体的蛋白质乙酰化显著增加。糖酵解和中枢代谢的几种酶，天冬氨酸氨基甲酰转移酶，蛋氨酸合成酶，过氧化氢酶和参与翻译的蛋白质的乙酰化增加。蛋氨酸和尿嘧啶的补充消除了ackA突变体的额外生长缺陷。数据显示，AckA-PTA途径中突变株的厌氧、发酵生长有所减少，但仍有可能。生长减慢的原因可以解释为缺乏一条重要的混酸发酵生成ATP的途径。AckA缺失突变体比pta或ackA-pta缺失突变体损伤更严重。这很可能是由于ackA突变体中产生了乙酰基-P，导致了蛋白质乙酰化的增加。
Acetate is a characteristic by-product of Escherichia coli K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP. Mutants in the AckA-Pta pathway differ from each other in the potential to produce and accumulate acetyl-phosphate. In the publication at hand, we investigated different mutants in the acetate pathway, both under aerobic as well as anaerobic conditions. While under aerobic conditions only small changes in growth rate were observed, all acetate mutants showed severe reduction in growth rate and changes in the by-product pattern during anaerobic growth. The AckA(-) mutant showed the most severe growth defect. The glucose uptake rate and the ATP concentration were strongly reduced in this strain. This mutant exhibited also changes in gene expression. In this strain, the atoDAEB operon was significantly upregulated under anaerobic conditions hinting to the production of acetoacetate. During anaerobic growth, protein acetylation increased significantly in the ackA mutant. Acetylation of several enzymes of glycolysis and central metabolism, of aspartate carbamoyl transferase, methionine synthase, catalase and of proteins involved in translation was increased. Supplementation of methionine and uracil eliminated the additional growth defect of the ackA mutant. The data show that anaerobic, fermentative growth of mutants in the AckA-Pta pathway is reduced but still possible. Growth reduction can be explained by the lack of an important ATP generating pathway of mixed acid fermentation. An ackA deletion mutant is more severely impaired than pta or ackA-pta deletion mutants. This is most probably due to the production of acetyl-P in the ackA mutant, leading to increased protein acetylation.