In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated ␥-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.Since bacterial habitats encompass a wide range of environmental conditions, bacteria are useful species for investigating the environmental adaptation of living things. Osmolarity of growth medium is one of the important conditions for bacterial growth, and there have been numerous reports concerning bacterial adaptation to changes in external osmolarity (1).Many materials have been identified as being accumulated after hyperosmotic shock in order to increase the cytoplasmic osmolarity or to protect cellular metabolism under high osmotic pressure. For example, potassium ion, proline, betaine, trehalose, glutamate, glutamine, glutamyl peptide, and taurine, etc., have been reported to function as compatible solutes or osmoprotectants (1, 5-8). Some of them are probably not absolutely essential for osmotic adaptation, but they may increase the growth rate of hyperosmosis-stressed cells. Thus, in order to clarify the mechanism of bacterial osmotic adaptation, examination of the minimum requirement for the adaptation would be required.Potassium glutamate is known to be a major osmolyte of Escherichia coli growing in hyperosmotic minimal medium (1, 7). In the present study, we investigated the accumulation of glutamate at various pH values of the minimal medium and found that the internal level of glutamate was increased at a pH above 8. ␥-Aminobutyrate was accumulated when the glutamate accumulation was low upon moderate hyperosmotic shock at a pH below 8. Thus, E. coli seems to use different osmolytes at different pH levels. MATERIALS AND METHODSBacterial strains and growth media. E. coli W3110 and TK2642 [F Ϫ thi lac rha trkA405 trkD1 del(kdpABC)54 nagA malT] were generously supplied by Y. Anraku (Tokyo University) and W. Epstein (Chicago University), respectively. E. coli was grown at 37ЊC in minimal medium containing 5 mM K 2 PO 4 , 20 mM (NH 4 ) 2 SO 4 , 1 mM MgCl 2 , 0.1 mM CaCl 2 , 1% glucose, and thiamine (2 g/ml). Tricine [tris(hydroxymethyl)methylglycine] (60 mM) or HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid) was used as a buffer, and the medium pH was adjusted by the addition of KO...
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