Staphylococcus aureus cells grown in a defined medium under conditions of high ionic stress (2.5 M NaCl) were significantly larger than cells grown under unstressed conditions, even though the cells grew much more slowly under stressed conditions. Analysis of the structure of peptidoglycan from stressed cells showed a shorter interpeptide bridge than in peptidoglycan from unstressed cells. Glycine betaine inclusion in the high-NaCl medium resulted in cells with sizes and interpeptide bridges similar to those of cells grown under unstressed conditions.The gram-positive coccus Staphylococcus aureus is one of the most halotolerant eubacteria. The organism can grow in medium containing up to 3.5 M NaCl (28). During an investigation of the effects of growth in the presence of NaCl on the production of penicillin-binding protein 2a and autolytic activity (20), it was observed that cells grown in complex medium containing 0.85 M NaCl were larger than cells grown in complex medium alone (21). Similar observations of larger cells in complex medium containing NaCl than in complex medium alone have also been reported by others (13,17).When bacterial cells such as Escherichia coli are exposed to a sudden increase in external osmotic pressure, there is initially a fast efflux of water, resulting in decreased turgor pressure, plasmolysis, and cell shrinkage (1,5,18). Mitchell and Moyle (25) have shown a similar response for S. aureus cells transferred from dilute to concentrated NaCl solutions. Bacteria respond to a decrease in turgor by accumulating high concentrations of osmotically active, compatible solutes in order to restore turgor, either by transport from the medium or by biosynthesis (5). This allows the restoration of growth of the organism, and a new steady state is established.There have been several recent studies of osmoregulation in S. aureus (11,19,24), and choline, glycine betaine, L-proline, and taurine have been shown to act as osmoprotectants. Because complex media contain the osmoprotectants L-proline and glycine betaine (10), cells of S. aureus osmotically stressed by growth in complex media containing high concentrations of NaCl accumulate proline and glycine betaine to high levels (19,24). Thus, complex media are not well suited to study of the effects of NaCl on cell size because of potential modifying effects of osmoprotectants present in such media. In this paper, we report a careful examination of the effects of ionic and osmotic stress on the cell morphology and structure of peptidoglycan of S. aureus grown in defined medium in the presence and absence of the osmoprotectant glycine betaine. MATERIALS AND METHODSBacterial strains and culture conditions. S. aureus RN450 (NCTC 8325-4), a derivative of strain NCTC 8325, was used in this study. All experiments were carried out with cells grown in a defined medium that contained glycerol as the primary carbon source (31). The osmotic strength of this medium is approximately 0.20 osmol (kg of water) Ϫ1 (11). The ionic and/or osmotic strength of the mediu...

To further study mechanisms of coping with osmotic stress-low water activity, mutants of Staphylococcus aureus with transposon Tn917-lacZ-induced NaCl sensitivity were selected for impaired ability to grow on solid defined medium containing 2 M NaCl. Southern hybridization experiments showed that NaCl-sensitive mutants had a single copy of the transposon inserted into a DNA fragment of the same size in each mutant. These NaCl-sensitive mutants had an extremely long lag phase (60 to 70 h) in defined medium containing 2.5 M NaCl. The osmoprotectants glycine betaine and choline (which is oxidized to glycine betaine) dramatically shortened the lag phase, whereas L-proline and proline betaine, which are effective osmoprotectants for the wild type, were ineffective. Electron microscopic observations of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudomulticellular cells similar to those observed previously in the wild type under the same conditions. Glycine betaine, but not L-proline, corrected the morphological abnormalities. Studies of the uptake of L-[ 14 C]proline and [ 14 C]glycine betaine upon osmotic upshock revealed that the mutant was not defective in the uptake of either osmoprotectant. Comparison of pool K ؉ , amino acid, and glycine betaine levels under NaCl stress conditions in the mutant and the wild type revealed no striking differences. Glycine betaine appears to have additional beneficial effects on NaCl-stressed cells beyond those of other osmoprotectants. The NaCl stress protein responses of the wild type and the NaCl-sensitive mutant were characterized and compared by labeling with L-[ 35 S]methionine and two-dimensional gel electrophoresis. The synthesis of 10 proteins increased in the wild type in response to NaCl stress, whereas the synthesis of these 10 proteins plus 2 others increased in response to NaCl stress in the NaCl-sensitive mutant. Five proteins, three of which were NaCl stress proteins, were produced in elevated amounts in the NaCl-sensitive mutant under unstressed conditions compared to the wild type. The presence of glycine betaine during NaCl stress decreased the production of three NaCl stress proteins in the mutant versus one in the wild type.

In an earlier report, we had described the isolation and characterization of autolysis-defective mutants of Staphylococcus aureus (N. Mani, P. Tobin, and R.K. Jayaswal, J. Bacteriol. 175:1493-1499, 1993). In the study reported here, an autolysis-defective mutant showed attenuated virulence in a rat model of experimental endocarditis, supporting the role of autolysins in pathogenicity. Transmission electron micrographs of the mutant cells revealed a rough outermost surface as compared with the parent strain, ISP2018. Treatment of mutant cells with lysozyme, proteases, and lipase failed to alter this rough appearance. Physical and genetic data locate the site of mutation between the omega 1100 and ilv loci on the S. aureus chromosome.

We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889–1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureuschromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated α-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.