Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain α-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus . Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12°C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus . Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

Heat-shock proteins are essential for stress tolerance and allowing organisms to survive conditions that cause protein unfolding. The role of the Staphylococcus aureus DnaK system in tolerance of various stresses was studied by disruption of dnaK by partial deletion and insertion of a kanamycin gene cassette. Deletion of dnaK in S. aureus strain COL resulted in poor growth at temperatures of 37 °C and above, and reduced carotenoid production. The mutant strain also exhibited increased susceptibility to oxidative and cell-wall-active antibiotic stress conditions. In addition, the mutant strain had slower rates of autolysis, suggesting a correlation between DnaK and functional expression of staphylococcal autolysins. Deletion of dnaK also resulted in a decrease in the ability of the organism to survive in a mouse host during a systemic infection. In summary, the DnaK system in S. aureus plays a significant role in the survival of S. aureus under various stress conditions.

Methionine sulfoxide reductases (MSRA1 and MSRB) are proteins overproduced in Staphylococcus aureus during exposure with cell wall-active antibiotics. Later studies identified the presence of two additional MSRA proteins (MSRA2 and MSRA3) in S. aureus. These MSR proteins have been characterized in many other bacteria as well. This review provides the current knowledge about the conditions and regulatory network that mimic the expression of these MSR encoding genes and their role in defense from oxidative stress and virulence.

The bacterium Staphylococcus aureus utilizes a variety of different mechanisms to survive unfavorable stress conditions that are critical for its persistence in the environment and for pathogenicity. The staphylococcal DnaK heat shock system functions as a major protein folding machine under stress conditions that cause aggregation and un-folding of proteins. In prior studies, S. aureus cells with a non-functional DnaK system showed reduced tolerance to heat, oxidative and antibiotic stresses, a lowered carotenoid production, and decreased survival in a murine host. This study provides insights that the altered phenotypes of the dnaK mutant cells are not due to decreased SigB activity in the mutant cell. Transcriptomic profiling studies provide evidence that a large number of genes encoding proteins involved in cell wall biogenesis, virulence and general stress tolerance, and genes encoding proteins involved in metabolic processes are differentially regulated in dnaK mutant cells relative to wild-type S. aureus. It was also determined that loss of functional DnaK caused a reduction in the ability of S. aureus to make biofilms and its adherence to eukaryotic cells. This study provides evidence of a global significance of DnaK heat shock system in S. aureus.

Staphylococcus aureus is a major human pathogen and emergence of antibiotic resistance in clinical staphylococcal isolates raises concerns about our ability to control these infections. Cell wall-active antibiotics cause elevated synthesis of methionine sulfoxide reductases (Msrs: MsrA1 and MsrB) in S. aureus. MsrA and MsrB enzymes reduce S-epimers and R-epimers of methionine sulfoxide, respectively, that are generated under oxidative stress. In the S. aureus chromosome, there are three msrA genes (msrA1, msrA2 and msrA3) and one msrB gene. To understand the precise physiological roles of Msr proteins in S. aureus, mutations in msrA1, msrA2 and msrA3 and msrB genes were created by site-directed mutagenesis. These mutants were combined to create a triple msrA (msrA1, msrA2 and msrA3) and a quadruple msrAB (msrA1, msrA2, msrA3, msrB) mutant. These mutants were used to determine the roles of Msr proteins in staphylococcal growth, antibiotic resistance, adherence to human lung epithelial cells, pigment production, and survival in mice relative to the wild-type strains. MsrA1-deficient strains were sensitive to oxidative stress conditions, less pigmented and less adherent to human lung epithelial cells, and showed reduced survival in mouse tissues. In contrast, MsrB-deficient strains were resistant to oxidants and were highly pigmented. Lack of MsrA2 and MsrA3 caused no apparent growth defect in S. aureus. In complementation experiments with the triple and quadruple mutants, it was MsrA1 and not MsrB that was determined to be critical for adherence and phagocytic resistance of S. aureus. Overall, the data suggests that MsrA1 may be an important virulence factor and MsrB probably plays a balancing act to counter the effect of MsrA1 in S. aureus.