Summary: Internal solutes in a halotolerant sp. (strain JCM 6894) were analysed by H NMR spectroscopy. At the lower osmolarity of complex medium, the main compatible solutes in the cells were glutamate, glycine betaine and hydroxyproline. At higher external salt concentrations, ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) levels increased and this became the predominant compatible solute at <1.5 M NaCl or KCI. When sp. was grown in the absence of yeast extract, glycine betaine levels were reduced to about half and a new solute, glycine, was detected. Irrespective of whether the medium contained yeast extract, sp. accumulated hydroxyproline, which has not been previously reported as an osmolyte in halophilic eubacteria. Trehalose and glutamate were the main solutes in cells grown in chemically defined medium containing 0-0.5 M NaCl. When grown in the presence of > 1.5 M NaCl, both ectoine and hydroxyectoine became dominant, as was also the case in complex medium. Additional compatible solutes detected were γ-aminobutyrate and proline, neither of which were detected in cells grown in complex medium. Regardless of the medium composition and incubation time, ectoine levels were high at high osmolarity, indicating that the halotolerant nature of sp. is closely related to its accumulation of ectoine. From its accumulation of compatible solutes, we suggest that sp. JCM 6894 is a different species from other members of the genus


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