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The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance ¹³C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K⁺ and proline rose markedly after osmotic upshock. K⁺ influx from the medium was rapid (< 1 h) but proline synthesis was a slower process (5–9 h). Proline synthesis appeared to be dependent on the prior accumulation of K⁺ and it is possible that K⁺ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K⁺ was also …