23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli.

23Na NMR spectroscopy was used to determine free Na+ concentrations in a halotolerant bacterium, Brevibacterium sp., and Escherichia coli. The internal Na+ concentration of both strains depended little on the growth phases and was unchanged after 5 d storage at 2 degrees C. In Brevibacterium sp. the level of intracellular sodium increased gradually at higher extracellular NaCl concentrations in both the presence and absence of yeast extract in the growth medium. E. coli cells accumulated a higher concentration of free Na+ than those of Brevibacterium sp. The change of Na+ concentration in both strains was inverse to that of growth rate. When appropriate amounts of osmoprotectants (proline, glycine betaine, or gamma-aminobutyrate) were added with the NaCl, internal free Na+ levels in Brevibacterium sp. were lowered, but those of E. coli were unchanged. While addition of KCl to medium containing NaCl increased the intracellular level of free Na+, the total sodium concentration in the cells remained unchanged, indicating that sodium that had been bound or attached was made free in the cytosol. In Brevibacterium sp. grown in the presence of 0.5 M NaCl, free and bound sodium concentrations in the cytosol were estimated to be 0.14 and 0.23 mumol (mg protein)-1, respectively. As a result, visibility by 23Na NMR was 38%.