CHARACTERIZATION OF AN NACL-SENSITIVE STAPHYLOCOCCUS-AUREUS MUTANT AND RESCUE OF THE NACL-SENSITIVE PHENOTYPE BY GLYCINE BETAINE BUT NOT BYOTHER COMPATIBLE SOLUTES

To further study mechanisms of coping with osmotic stress-low water ac tivity, mutants of Staphylococcus aureus with transposon Tn917-lacZ-in duced NaCl sensitivity were selected for impaired ability to grow on s olid defined medium containing 2 M NaCl. Southern hybridization experi ments showed that NaCl-sensitive mutants had a single copy of the tran sposon inserted into a DNA fragment of the same size in each mutant, T hese NaCl-sensitive mutants had an extremely long lag phase (60 to 70 h) in defined medium containing 2.5 M NaCI, The osmoprotectants glycin e betaine and choline (which is oxidized to glycine betaine) dramatica lly shortened the lag phase, whereas L-proline and proline betaine, wh ich are effective osmoprotectants for the wild type, were ineffective, Electron microscopic observations of the NaCl-sensitive mutant under NaCI stress conditions revealed large, pseudomulticellular cells simil ar to those observed previously in the wild type under the same condit ions, Glycine betaine, but not L-proline, corrected the morphological abnormalities, Studies of the uptake of L-[C-14]proline and [C-14]glyc ine betaine upon osmotic upshock revealed that the mutant was not defe ctive in the uptake of either osmoprotectant, Comparison of pool K+, a mino 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-stresse d cells beyond those of other osmoprotectants, The NaCl stress protein responses of the wild type and the NaCl-sensitive mutant were charact erized and compared by labeling with L-[S-35]methionine and two-dimens ional gel electrophoresis. The synthesis of 10 proteins increased in t he wild type in response to NaCl stress, whereas the synthesis of thes e 10 proteins plus 2 others increased in response to NaCl stress in th e NaCl-sensitive mutant, Five proteins, three of which were NaCI stres s proteins, were produced in elevated amounts in the NaCl-sensitive mu tant under unstressed conditions compared to the wild type, The presen ce of glycine betaine during NaCl stress decreased the production of t hree NaCl stress proteins in the mutant versus one in the wild type.