Effects of pH and repellent tactic stimuli on protein methylation levels in Escherichia coli

Abstract

Intracellular pH (pH(int)) and extracellular pH (pH(ext)) of Escherichia coli were measured at 12-s time resolution by (31)P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH(ext) (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH(int) 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 pH(ext) also caused a lowering of pH(int); at high concentrations it generated an inverted transmembrane pH gradient (DeltapH). 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 pH(int) was lowered by weak acid addition or when pH(ext) 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 pH(ext) 7.0). The weak-acid concentration needed to reach that limit was dependent on pH(ext), as would be expected on the basis of DeltapH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH(int), 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 pH(int) and pH(ext). Evidence is presented that pH(int) may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-(3)H- and (35)S-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.