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Inactivation of the Pta-AckA pathway impairs fitness of Bacillus anthracis during overflow metabolism.

ABSTRACT: Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux towards acetate fermentation, a phenomenon known as overflow metabolism or the bacterial 'Crabtree effect'. Numerous studies of the major acetate-generating pathway, the Pta-AckA, revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to fitness of the spore-forming bacterium, Bacillus anthracis We demonstrate that disruption of the Pta-AckA pathway causes a drastic growth reduction in the mutants and alters the metabolic and energy status of the cells. Our results revealed that inactivation of the Pta-AckA pathway increases the glucose consumption rate, affects intracellular ATP, NAD⁺ and NADH levels and leads to a metabolic block at the pyruvate and acetyl-CoA nodes. Consequently, accumulation of intracellular acetyl-CoA and pyruvate forces bacteria to direct carbon into the TCA and/or glyoxylate cycles as well as fatty acid and poly(3-hydroxybutyrate) (PHB) biosynthesis pathways. Notably, the presence of phosphate butyryltransferase in B. anthracis partially compensates for the loss of phosphotransacetylase activity. Furthermore, overexpression of the ptb gene not only eliminates the negative impact of the pta mutation on B. anthracis fitness, but also restores normal growth in the pta mutant of the non-butyrate-producing bacterium, Staphylococcus aureus Taken together, the results of this study demonstrate the importance of the Pta-AckA pathway for B. anthracis fitness by revealing its critical contribution to the maintenance of metabolic homeostasis during aerobic growth under conditions of carbon overflow.IMPORTANCE B. anthracis, the etiologic agent of anthrax, is a highly pathogenic, spore-forming bacterium that causes acute, life-threatening disease in both humans and livestock. A greater understanding of the metabolic determinants governing fitness of B. anthracis is essential for the development of successful therapeutic and vaccination strategies aimed at lessening the potential impact of this important biodefense pathogen. This study is the first to demonstrate the vital role of the Pta-AckA pathway in preserving energy and metabolic homeostasis in B. anthracis under conditions of carbon overflow, therefore, highlighting this pathway as a potential therapeutic target for drug discovery. Overall, the results of this study provide important insight into understanding the metabolic processes and requirements driving rapid B. anthracis proliferation during vegetative growth.

SUBMITTER: Won HI

PROVIDER: S-EPMC8092162 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Inactivation of the Pta-AckA pathway impairs fitness of Bacillus anthracis during overflow metabolism.

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