It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.

The amount of F 1 Fo-ATPase in Enterococcus hirae (formerly Streptococcus faecalis) increases when the cytoplasmic pH is lowered below 7.6, and protons are extruded to maintain the cytoplasmic pH at around 7.6. In the present study, we found that the transcriptional activity of the F 1 Fo-ATPase operon was not regulated by pH. The synthesis of F 1 subunits was increased 1.65^0.12-fold by the acidification of medium from pH 8.0 to pH 5.3. Western-blot analysis showed that there were F 1 subunits in the cytoplasm, and the number of alpha plus beta subunits in the cytoplasm was 50% of the total number of the subunits in cells growing at pH 8.0. This decreased to 22% after shifting the medium pH to 5.3, with a concomitant 5.1-fold increase in the level of membrane-bound F 1 Fo-ATPase. The cytoplasmic F 1 subunits were shown to be degraded, and Fo subunits not assembled into the intact F 1 Fo complex were suggested to be digested. These data suggest that regulation of the enzyme level of F 1 FoATPase by the intracellular pH takes place mainly at the step of enzyme assembly from its subunits.Keywords: Enterococcus hirae; F 1 Fo-ATPase; assembly; pH regulation.Since the first demonstration in the anaerobic bacterium Enterococcus hirae (formerly Streptococcus faecalis) that cytoplasmic pH is regulated by proton extrusion mediated by a proton-translocating ATPase [1], many reports have shown that the H + -ATPase is involved in pH homeostasis in prokaryotes and eukaryotes [2,3]. In eukaryotes, a V-type H + -ATPase regulates the internal pH, while the regulator of the enterococcal cytoplasmic pH is an F-type enzyme. Because V-type and F-type H + -ATPases are very similar in structure and catalytic properties, the basic mechanism for pH regulation should be the same.In prokaryotes, it was already known by 1984 that the amount of the enterococcal F 1 Fo-ATPase was increased by lowering the cytoplasmic pH [4,5]. Both experimental [6] and theoretical [7] data have shown that internal pH homeostasis is maintained by changes in the amount of this enzyme. However, how the enzyme level is regulated is still unclear.Smith et al.[8] have shown no stimulation of the transcription of the H + -ATPase operon at low pH in S. mutans, in which the enzyme level is regulated by pH. In the present study, we found that the transcriptional activity of the F 1 Fo-ATPase operon was not affected by pH in E. hirae, while its translation was stimulated at low pH, but the stimulation was too low to account for the increase in the level of membrane-bound F 1 FoATPase. Our present data suggest that the enzyme level is regulated mainly during the step in which the enzyme is assembled from its subunits. MATERIALS AND METHODS Bacterial strains, plasmids and culture mediaEnterococcus hirae was grown at 378C in either medium 2KTY [1] or the following media without shaking. Medium A was a synthetic medium described previously [9] with the following modifications. Solution A contained 50 mm ammonium sulfate, 8 mm magnesium sulfate, 0.5 mm manganese sulfate, 0...