Characterization of an NaCl-sensitive Staphylococcus aureus mutant and rescue of the NaCl-sensitive phenotype by glycine betaine but not by other compatible solutes

Abstract

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.

Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus

“…We hypothesize that under these conditions, ClpP is required as part of a ClpXP proteolytic complex to control induction of specific stress responses. In the case of osmotic stress S. aureus adapt by accumulating non-ionic compatible solutes such as betaine, proline, trehalose or glycerol to restore turgor pressure, and we propose that this response may be regulated by ClpXP (Towsend and Wilkinson, 1992;Vijaranakul et al, 1997). Based on our results, the functional contribution of the individual ATPases or putative protease complexes to environmental stress tolerance has been summarized in Fig.…”

Osmoprotectants and cryoprotectants for Listeria monocytogenes

“…A possible explanation for the lack of osmoprotective activity of certain compounds for L. monocytogenes may be an inability of the organism to accumulate them in sufficient concentrations. However, glycine betaine was clearly a more potent osmoprotectant for an NaCl-sensitive S. aureus mutant than proline, even though both compounds were accumulated to similar concentrations upon NaCl stress (Vijaranakul et al 1997).…”

Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach

“…Approximately equal amounts of the biomass from the control and oxacillin treated culture adjusted via A 600 determinations, were collected by centrifugation (10 000Ug, 10 min, 4³C) and washed once with 50 mM Tris^HCl bu¡er, pH 7.5 containing 0.145 M NaCl. Cells were lysed by using lysostaphin and cellular proteins were prepared and examined by two-dimensional gel electrophoresis essentially as described [7]. Following electrophoresis the gels were stained with Coomassie brilliant blue R250.…”