INTRACELLULAR CHANGES IN IONS AND ORGANIC SOLUTES IN HALOTOLERANT BREVIBACTERIUM SP. STRAIN JCM-6894 AFTER EXPOSURE TO HYPEROSMOTIC SHOCK

In the present study we aimed to observe the intracellular responses w hen there was a hyperosmotic shock with a large shift in ionic strengt h in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halot olerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmo tic shock was induced by transferring cells in the late exponential ph ase of growth in a complex medium containing 0.5 M NaCl into either ol d or fresh culture medium containing 2 M NaCl. Changes in the growth r ate, in the pH of the medium, and in the internal cation or organic so lute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different respo nses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became mor e acidic, whereas the old culture medium repressed growth and the medi um became more alkaline. The intracellular free Na+ concentrations rem ained low (80 nmol mg of protein(-1)) after an upshock in fresh cultur e medium, although they quickly increased twofold in the old culture m edium. In contrast, K+ ions immediately accumulated in the cells in fr esh culture medium, whereas K+ ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upsho ck, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ec toine synthesis but was not observed after the shift to high-osmolarit y conditions in the old culture. In the fresh culture, however, ectoin e was vigorously synthesized in cells for more than 5 h after the upsh ock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein(-1) at 10 h, which corresponded to a ninefold increase com pared to the concentration before the shock These findings are consist ent with the results of an analysis of the extracellular medium compos ition before and after the upshock.