| Abstract: | Intracellular pH (pHint) and extracellular pH (pHext) of Escherichia coli were measured at 12-s time resolution by 31P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pHext (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pHint decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s−1 cm−2 pH unit−1. Addition of the membrane-permeant weak acid benzoate at constant pHext also caused a lowering of pHint; at high concentrations it generated an inverted transmembrane pH gradient (ΔpH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pHint was lowered by weak acid addition or when pHext was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pHext 7.0). The weak-acid concentration needed to reach that limit was dependent on pHext, as would be expected on the basis of ΔpH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pHint, demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pHint and pHext. Evidence is presented that pHint may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-3H- and 35S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli. |
