Uncoupler Resistance in Escherichia coli: the Role of Cellular Respiration

Abstract

Bioenergetic properties of a mutant strain of Escherichia coli K12 designated TUV, which is resistant to the protonophoric uncoupling agent 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidiazole (TTFB) have been compared with those of its non-resistant parent, E. coli K12 Doc-S. Strain TUV grew and respired some 20-30% faster than strain Doc-S, and was cross-resistant to carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and triphenyltin, but not to 2,4-dinitrophenol. Phosphorus nuclear magnetic resonance demonstrated the TTFB-mediated collapse of the transmembrane pH gradient at identical rates in starved cells of both strains, indicating that uncoupler access and function were unimpaired in the mutant under these conditions. Strain TUV displayed enhanced uncoupler resistance and maintained intracellular pH and ATP levels only when respiring. On the other hand, strain TUV also showed increased resistance to novobiocin, implying that its outer wall permeability had been lowered. We suggest that the active resistance of strain TUV results from the exclusion of uncoupler by the interaction of inner and outer membrane components in a manner modulated by the degree of cellular energization.

Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP

“…One potential explanation for the phenomenology of phase V growth would be development of resistance to the uncoupling agent CCCP. Uncoupler-resistant mutants have been described in both E. coli and Bacillus species (11,18,30,41). At least some of the E. coli mutants described are thought to result in exclusion of uncoupling agents from the energized cytoplasmic membrane via alterations in outer membrane barrier function (30, 41); in Bacillus spp., there are also mutations that remain sensitive to uncoupling agents but whose membranes are somehow altered to function at reduced (but still measurable) levels of membrane potential (11,18).…”

Uncoupler-resistant mutants of bacteria.

“…In general, however, there have been few measurements of relevant energetic parameters. The /pH component of ApIH+ appears to be resistant to dissipation in all cases when nonstarved cells were used (45,46,86,99). As noted above, TTFB collapsed the ApH of starved cells of strain TUV and its nonresistant parent at identical rates (86).…”

“…The /pH component of ApIH+ appears to be resistant to dissipation in all cases when nonstarved cells were used (45,46,86,99). As noted above, TTFB collapsed the ApH of starved cells of strain TUV and its nonresistant parent at identical rates (86). Although the collapse was slow, possibly a consequence of the very concentrated cell suspensions used, this is perhaps the clearest evidence for an unimpaired permeation of uncoupler in any uncoupler-resistant strain of E. coli.…”

“…Uncoupler-resistant mutants of E. coli show pleiotropic phenotypic changes; this is particularly apparent in the best-characterized strains, UV6 (98, 99) and TUV (47,86). Energized cells of both strains exhibit many similarities: multiple cross-resistance to membrane-active agents but retention of sensitivity to DNP; uncoupler-resistant active transport in whole cells; enhanced maintenance of a ApH in the presence of uncouplers; and impaired uptake of TPP+.…”

“…Analysis of strain UV6 was complicated by the apparent uncoupler effects involving the outer membrane (99). The use of nuclear magnetic resonance spectroscopy (86) obviated some of the possible effects of this kind with strain TUV, since the small periplasmic volume could not contribute significantly to the spectra. In general, however, there have been few measurements of relevant energetic parameters.…”