Data_Sheet_1_The Impact of ackA, pta, and ackA-pta Mutations on Growth, Gene Expression and Protein Acetylation in Escherichia coli K-12.pdf

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.

乙酸是大肠杆菌K-12（Escherichia coli K-12）在以葡萄糖为底物的分批培养体系中，于有氧与厌氧条件下产生的特征性副产物。尽管有氧条件下乙酸生成的具体机制仍存在争议，但厌氧生长时乙酸生成对通过底物水平磷酸化（substrate level phosphorylation）产生ATP至关重要。在两种条件下，乙酸均通过由磷酸乙酰转移酶（phosphate acetyltransferase, Pta）与乙酸激酶（acetate kinase, AckA）组成的代谢通路生成：前者催化乙酰辅酶A（acetyl-coenzyme A）生成乙酰磷酸（acetyl-phosphate），后者则以乙酰磷酸为底物合成乙酸，该反应伴随ATP的生成。AckA-Pta通路的突变株在乙酰磷酸的产生与积累能力上存在差异。本研究针对乙酸代谢通路中的不同突变株，分别在有氧与厌氧条件下开展了探究。有氧条件下仅观察到生长速率的小幅变化，而所有乙酸代谢通路突变株在厌氧生长时均出现了生长速率的显著下降及副产物谱的改变。其中，AckA缺失突变株的生长缺陷最为严重。该菌株的葡萄糖摄取速率与ATP浓度均大幅降低，同时伴随基因表达的改变。在厌氧条件下，该菌株的atoDAEB操纵子（atoDAEB operon）显著上调，提示存在乙酰乙酸的生成。厌氧生长过程中，ackA突变株的蛋白质乙酰化水平显著升高，糖酵解与中心代谢通路的多种酶、天冬氨酸氨甲酰转移酶（aspartate carbamoyl transferase）、甲硫氨酸合酶（methionine synthase）、过氧化氢酶（catalase）以及参与翻译过程的蛋白质的乙酰化水平均有所提升。添加甲硫氨酸与尿嘧啶可消除ackA突变株额外的生长缺陷。研究数据表明，AckA-Pta通路突变株的厌氧发酵生长能力有所下降，但仍可维持生长。生长抑制可归因于混合酸发酵（mixed acid fermentation）中一条重要的ATP生成通路的缺失。ackA缺失突变株的损伤程度较pta单突变株或ackA-pta双缺失突变株更为严重，这大概率是由于ackA突变株仍可产生乙酰磷酸，进而导致蛋白质乙酰化水平升高所致。