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

Abstract

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.

Keywords: acetate metabolism; acetyl-P; fermentation; overflow; protein acetylation.

FIGURE 1

Acetate pathway in E. coli. Shown are the reactions connecting pyruvate, acetyl-CoA and acetate during growth with glucose under aerobic and anaerobic conditions, respectively. Blue text denotes enzymes PDH, pyruvate dehydrogenase complex; AckA, acetate kinase; Pta, phosphotransacetylase; LDH, lactate dehydrogenase; PFL, pyruvate-formate lyase; ADH, alcohol dehydrogenase; POX, Pyruvate Oxidase.

FIGURE 2

Anaerobic growth of MG1655, KBM1081, KBM1082, and KBM1084. Cells were grown anaerobically in defined medium with 4 g/l glucose. Shown are exemplary time course data for the OD₄₂₀ from batch cultures incubated under N₂ atmosphere. The growth assays were performed in at least three independent experiments. Variation of these repeats and further data concerning substrate consumption and production of fermentation products can be seen from Table 2.

FIGURE 3

Gene expression data of acetate mutants during anaerobic growth with glucose. Strains were grown anaerobically in MM with 4 g/l glucose. RNA was extracted during exponential growth phase and gene expression was analyzed by qRT-PCR as described. Relative changes in gene expression were calculated by the ΔΔCt method using MG1655 grown under the same conditions as reference sample. Three housekeeping genes rpoD, yhbc and recA were used for normalization. Shown are only selected genes.

FIGURE 4

Protein lysine acetylation in MG1655, KBM1081, KBM1082, and KBM1084. Shown are Western Blots of gels probed with Acetylated Lysine Multi MAB with extracts from exponentially growing aerobic cultures (A) and anaerobic cultures (B). (C) Shows the Coomassie stained loading control for the anaerobic extracts used in (B). For the Western Blots shown in (A,B) 15 μl of the respective extracts were loaded per lane. For the loading control in (C) only 5 μl of the extracts were loaded. As markers MagicMark XP Western Protein Standard (Thermo Fisher Scientific) was used (A) and Precision Plus Unstained Protein (BioRad) for (B,C). Culture growth and extract preparation were performed as described in Section “Materials and Methods.”

FIGURE 5

Anaerobic growth of the ackA mutant KBM1081 in defined medium supplemented with uracil and methionine. Shown are time course data from representative growth assays for growth of the ackA mutant KBM1081 in defined medium with 4 g/l glucose as carbon source without any further growth supplements (black) and with addition of 30 μg/ml uracil (red) and with addition of 30 μg/ml uracil plus 40 mg/l L-methionine (green). Growth of the ackA-pta mutant KBM1084 with supplementation of uracil and methionine is shown for comparison (gray).