The starvation-survival response of Staphylococcus aureus as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [35S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.

A Staphylococcus aureus mutant (SPW1) which is unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to the sodA family of manganese-dependent superoxide dismutases (SOD). Whole-cell lysates of the parental 8325-4 strain demonstrated three zones of SOD activity by nondenaturing gel electrophoresis. The activities of two of these zones were dependent on manganese for activity and were absent in SPW1. The levels of SOD activity and sodA expression were growth-phase dependent, occurring most during postexponential phase. This response was also dependent on the level of aeration of the culture, with highest activity and expression occurring only under high aeration. Expression of sodA and, consequently, SOD activity could be induced by methyl viologen but only during the transition from exponential- to postexponential-phase growth. SPW1 was less able to survive amino acid limitation and acid stress but showed no alteration in pathogenicity in a mouse abscess model of infection compared to the parental strain 8325-4.

A Staphylococcus aureus mutant (SPW3) apparently unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to ctaA of Bacillus subtilis which encodes a heme A synthase. Analysis of the cytochrome profiles of SPW3 revealed the absence of heme A-containing cytochromes compared to the parental 8325-4 strain. SPW3 demonstrated a 100-fold reduction in the ability to survive starvation induced by glucose limitation, under aerated conditions, compared to 8325-4. Analysis of starved cultures revealed that greater than 90% of the cells which demonstrated metabolism (as shown by rhodamine 123 accumulation) were unable to recover and form colonies on agar. Analysis of the lag phase and initial growth kinetics of those cells which could recover also showed a defect. This recovery defect could be partially alleviated by the inclusion of catalase in the recovery medium, indicating the probable involvement of oxidative stress. SPW3 also exhibited reduced colony size similar to that of a small-colony variant, increased resistance to aminoglycoside antibiotics, and reduced hemolysin and toxic shock syndrome toxin 1 production, but no alteration in the ability to form lesions in a subcutaneous mouse infection model. MATERIALS AND METHODSCloning and sequencing of ctaA. Plasmid pSPW3, which contained chromosomal DNA flanking the lacZ proximal region of the Tn insertion in S. aureus SPW3, was generated in a previous study (42). The insert, which contains 750 bp of chromosomal DNA, was excised, digoxigenin (DIG) labeled according to the protocol of Boehringer GmbH (Mannheim, Germany), and used to probe a ZAP Express library of a partial Sau3A digest (2 to 10 kb) of S. aureus 8325-4 genomic DNA (10). A clone containing a 3.5-kb genomic DNA fragment, spanning the pSPW3 insert, was identified, and the stable phagemid pSPW30 was excised from ZAP Express in Escherichia coli XLOLR (Stratagene). A primerwalking-based approach was used to sequence a 1,100-bp region.Strains and growth conditions. Strains and plasmids used in this study are listed in Table 1. S. aureus 8325-4 (23) and SPW3 (42) were grown in either a chemically defined media (CDM) containing 0.1% glucose (14, 43) or brain heart infusion (BHI) broth (Oxoid). Agar plates were prepared by the addition of 1% (wt/vol) agar to the above. Catalase plates were prepared by overlaying 4 ml of CDM agar (1% [wt/vol]) containing bovine catalase (5 mg ml Ϫ1 ; Sigma) onto CDM agar. Sheep blood agar plates were made by the supplementation of blood agar base (Difco) with 5% (vol/vol) sheep blood (TCS Biologicals, Buckingham, United Kingdom). Starvation cultures were prepared by the inoculation of CDM to an optical density at 600 nm (OD 600 ) of 0.01, which was then incubated at 37°C with shaking (250 rpm). Stationary phase was reached after about 8 to 10 h. After 18 h of growth, cultures were either incubated statically or shaken further. Starved cells were recovered by the addition of 1/10 volume of glucose (1% [wt/vol]) and a mixture of 18 amino ...