Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions

Abstract

Recently, we reported that the prototypical Staphylococcus aureus strain RN6390 (a derivative of NCTC 8325) had significantly reduced aconitase activity relative to a diverse group of S. aureus isolates, leading to the hypothesis that strain RN6390 has impaired tricarboxylic acid (TCA) cycle-mediated acetate catabolism. Analysis of the culture supernatant from RN6390 confirmed that acetate was incompletely catabolized, suggesting that the ability to catabolize acetate can be lost by S. aureus. To test this hypothesis, we examined the carbon catabolism of the S. aureus strains whose genome sequences are publicly available. All strains catabolized glucose and excreted acetate into the culture medium. However, strains NCTC 8325 and N315 failed to catabolize acetate during the postexponential growth phase, resulting in significantly lower growth yields relative to strains that catabolized acetate. Strains NCTC 8325 and RN6390 contained an 11-bp deletion in rsbU, the gene encoding a positive regulator of the alternative sigma factor sigma(B) encoded by sigB. An isogenic derivative strain of RN6390 containing the wild-type rsbU gene had significantly increased acetate catabolism, demonstrating that sigma(B) is required for acetate catabolism. Taken together, the data suggest that naturally occurring mutations can alter the ability of S. aureus to catabolize acetate, a surprising discovery, as TCA cycle function has been demonstrated to be involved in the virulence, survival, and persistence of several pathogenic organisms. Additionally, these mutations decrease the fitness of S. aureus by reducing the number of progeny placed into subsequent generations, suggesting that in certain situations a decreased growth yield is advantageous.

FIG. 1.

Schematic representation of glucose catabolism by S. aureus. Green arrows, reactions or pathways used primarily during the exponential phase of growth; red arrows, reactions or pathways used in the postexponential growth phase; black arrows, reactions for which there are insufficient data to determine if the reaction occurs.

FIG. 2.

Growth characteristics of S. aureus strains RN6390 and SH1000. Strains (A) RN6390 (rsbU mutant) and (B) SH1000 were grown in TSB. At 1-h intervals, an aliquot (1.5 ml) was removed, the absorbance at 600 nm was measured, and the glucose, acetate, and ammonia concentrations in the culture supernatants were determined. The results presented are representative of at least two independent experiments.

FIG. 3.

Growth characteristics of eight S. aureus strains. The growth (A₆₀₀) and concentrations of ammonia, glucose, and acetate in culture supernatants were measured at 1-h intervals for strains (A) NCTC 8325, (B) Mu50, (C) MRSA-252, (D) MSSA-476, (E) N315, (F) COL, (G) RF122, and (H) MW2. The results presented are representative of at least two independent experiments.

FIG. 4.

RNAIII Northern blot. Total RNA was isolated from strains MW2, RF122, MSSA-476, N315, MRSA-252, Mu50, and NCTC 8325 after 7 h of growth, transferred to a charged nylon membrane, and probed with an RNAIII-specific probe. Total RNA was isolated from strain COL after 9 h of growth.

FIG. 5.

Stationary-phase survival of S. aureus strains NCTC 8325, Mu50, MRSA-252, MSSA-476, N315, COL, RF122, and MW2. Single colonies were inoculated into TSB, grown at 37°C, and aerated by shaking at 225 rpm for 8 d. At 24-h intervals, aliquots were removed, and CFU were determined in quadruplicate. Time zero on the graph represents the point at which the cultures were inoculated. The data are presented as the average and standard deviation.

FIG. 6.

Confirmation of α-ketoglutarate dehydrogenase mutation in S. aureus strain N315. (A) Schematic representation of the odhAB locus of strain N315, including the flanking genes. (B) PCR confirmation of the 66-bp deletion in the odhA gene of N315.