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.

FIG 1

Inactivation of the Pta-AckA pathway affects growth characteristics of B. anthracis. (A) Growth curves of the wild-type (wt) strain V770-NP1-R and mutant strains V770-ackA and V770-pta grown aerobically in TSB containing 0.25% glucose. The OD₆₀₀ and the pH of the culture medium were determined at the indicated times. (B) Growth rate of the wild-type strain V770-NP1-R and mutant strains V770-ackA and V770-pta grown aerobically in TSB containing 0.25% glucose, determined between 0 and 3 h of growth. (C) Temporal accumulation and depletion of acetic acid in the culture medium of strains V770-NP1-R, V770-ackA, and V770-pta. (D) Acetate excretion rate determined for strains V770-NP1-R, V770-ackA, and V770-pta between 0 and 3 h of growth. (E) Temporal depletion of glucose from the culture medium of strains V770-NP1-R, V770-ackA, and V770-pta. (F) Glucose consumption rate determined for strains V770-NP1-R, V770-ackA, and V770-pta between 0 and 3 h of growth. For panels A, C, and E, the results are representative of at least three independent experiments. For panels B, D, and F, the results are presented as the means plus standard errors of the means of duplicate determinations for at least three independent experiments. Statistical significance between the wild-type strain and the pta and ackA mutants was determined by using Student's t test. *, P <0.005.

FIG 2

Inactivation of the Pta-AckA pathway alters carbon flux at the pyruvate and acetyl-CoA nodes and affects the energy status of B. anthracis. (A) Intracellular acetyl-CoA (Ac-CoA) concentrations determined for strains V770-NP1-R, V770-ackA, and V770-pta after 3 h of aerobic growth in TSB containing 0.25% glucose. wt, wild-type. (B) Intracellular pyruvate concentrations determined for strains V770-NP1-R, V770-ackA, and V770-pta after 3 h of aerobic growth in TSB containing 0.25% glucose. (C) Concentrations of pyruvate in the culture medium determined for strains V770-NP1-R, V770-ackA, and V770-pta after 3 h of aerobic growth in TSB containing 0.25% glucose. (D) Intracellular ATP concentrations determined for strains V770-NP1-R, V770-ackA, and V770-pta after 3 h of aerobic growth in TSB containing 0.25% glucose. (E) Intracellular NAD⁺ and NADH concentrations determined for strains V770-NP1-R, V770-ackA, and V770-pta after 3 h of aerobic growth in TSB containing 0.25% glucose. The results are presented as the means plus standard errors of the means of duplicate determinations for at least three independent experiments. Statistical significance between the wild-type strain and the pta and ackA mutants was determined by using Student's t test. *, P <0.01.

FIG 3

Inactivation of the Pta-AckA pathway in B. anthracis directs carbon into the TCA cycle, fatty acid biosynthesis, and PHB production. (A) Relative transcript levels of the pfkA, citZ, citC, aceB, sucA, fabH, and fabI genes determined by quantitative RT‐PCR after 3 h of aerobic growth in TSB containing 0.25% glucose. Transcript levels in the V770-ackA and V770-pta mutants are presented as a fold difference, compared to those in the wild‐type (wt) strain. (B) Intracellular citrate concentrations determined for strains V770-NP1-R, V770-ackA, and V770-pta after 3 h of aerobic growth in TSB containing 0.25% glucose. (C) Visualization of PHB granules for strains V770-NP1-R, V770-ackA, and V770-pta after 3 and 6 h of aerobic growth in TSB supplemented with 0.25% glucose by confocal laser scanning microscopy using the fluorescent dye Nile red. For panels A and B, the results are presented as the means plus standard errors of the means of duplicate determinations for at least three independent experiments. Statistical significance between the wild-type strain and the pta and ackA mutants was determined by using Student's t test. *, P <0.01.

FIG 4

Overexpression of the ptb gene restores growth of the pta mutant in S. aureus. (A) Growth curves of the wild-type (wt) strain UAMS-1, the mutant strain UAMS-1-pta, and UAMS-1-pta with the plasmid pMRS183, containing the ptb gene from B. anthracis, grown aerobically in TSB containing 0.25% glucose. The OD₆₀₀ and the pH of the culture medium were determined at the indicated times. (B) Temporal depletion of glucose from the culture medium of strains UAMS-1, UAMS-1-pta, and UAMS-1-pta with the plasmid pMRS183. (C) Temporal accumulation and depletion of acetic acid in the culture medium of strains UAMS-1, UAMS-1-pta, and UAMS-1-pta with the plasmid pMRS183. In the experiments, the wild-type strain and the pta mutant contain pCN51 (empty vector) plasmid. The results are representative of at least three independent experiments.