The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0·2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7·8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew over a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7·8 to 8·0 at external pH (pHo) values of 7·2 and 8·2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca2+-containing medium, and cell division were all strongly repressed under alkaline conditions.
The marine bacterium Vibrio alginolyticus, containing 470 mM-K+ and 70 mM-Na+ inside its cells, was able to regulate the cytoplasmic pH (pHin) in the narrow range 7·6–7·8 over the external pH (pHout) range 6·0–9·0 in the presence of 400 mM-Na+ and 10 mM-K+. In the absence of external K+, however, pHin was regulated only at alkaline pHout values above 7·6. When the cells were incubated in the presence of unusually high K+ (400 mM) and 4 mM Na+, the pHin was regulated only at acidic pHout values below 7·6. These results could be explained by postulating a K+/H+ antiporter as the regulator of pHin over the pHout range 6·0–9·0. When Na+-loaded/K+-depleted cells were incubated in 400 mM-Na+ in the absence of K+, an inside acidic ΔpH was generated at pHout values above 7·0. After addition of diethanolamine the inside acidic ΔpH collapsed transiently and then returned to the original value concomitant with the extrusion of Na+, suggesting the participation of a Na+/H+ antiporter for the generation of an inside acidic ΔpH. In the presence of 400 mM-K+, at least 5 mM-Na+ was required to support cell growth at pHout below 7·5. An increase in Na+ concentration allowed the cells to grow at a more alkaline pHout. Furthermore, cells containing more Na+ inside could more easily adapt to grow at alkaline pHout. These results indicated the importance of Na+ in acidification of the cell interior via a Na+/H+ antiporter in order to support cell growth at alkaline pHout under conditions where the activity of a K+/H+ antiporter is marginal.