Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1
Authors: Helgerson S. L. , Cramer W. A. 
Published: May 2005

AbstractIt is of interest to inquire whether agents that uncouple or deenergize membranes cause concomitant structural changes. The agents considered here are the uncoupler carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone and the bacteriocidal protein colicin E1, agents for which there is some precedent for believing that they interact with membranes.In intact E. coli ML 308‐225 cells the inhibition of [14C]‐proline active transport by FCCP increases with uncoupler concentration from ∼ 20% at 2 μM to ∼100% at 5 μM. The increase in the rotational relaxation time (ρ) of the cell‐bound fluorescent probe N‐phenyl‐1‐naphthylamine (PhNap)Abbreviations: FCCP – carbonyl cyanide p‐trifluoromethoxyphenylhydrazone; ANS – 8‐anilino‐1‐naphthalenesulfonate; PhNap, N‐phenyl‐1‐naphthylamine; EDTA – ethylenediaminetetraacetate.
and 8‐anilino‐1‐naphthalene‐sulfonate (ANS) under these conditions shows the same dependence on FCCP concentration. For cells treated with EDTA to remove part of the outer lipopolysaccharide layer, inhibition of proline transport and the increase in ρ value of ANS show the same dependence on FCCP concentration with saturation at 0.3 μM. EDTA treatment causes a large increase in the binding and rotational relaxation time of PhNap, the latter quantity approaching a value obtained with purified inner membrane. Similar effects are produced in untreated cells by 5 μM FCCP. It is concluded that (a) EDTA treatment removes a permeability barrier t o FCCP and PhNap in the outer membrane; (b) uncoupling by FCCP removes a similar permeability barrier to PhNap; (c) binding of amphiphilic ANS, assumed to be located in the outer membrane, is hardly changed by these treatments; (d) deenergization of the inner membrane by FCCP thus causes a structural change in the outer membrane as measured by the permeability change to hydrophobic PhNap and the increase in ρ values of the amphiphilic ANS; (e) The binding sites reached by PhNap within the permeability barrier at or near the inner membrane are changed by FCCP from their initial state. This is inferred from an increase in PhNap quantum yield extrapolated to infinite cell concentration, and from removal by FCCP of an apparent phase transition sensed by the PhNap rotational relaxation time. Thus, uncoupling and deenergization by FCCP appears to cause structural change both in the outer membrane and inside the permeability barrier of the outer membrane.Transmission of the colicin E1 response in the envelope of intact and EDTA‐treated cells can also be monitored by an increase in ANS and PhNap fluorescence intensity, a smaller fractional increase in dye binding, and a large increase in probe rotational relaxation time. The fluorescence changes of ANS again imply structural effects in the outer membrane caused by colicin. The binding and fluorescence changes of PhNap caused by colicin E1 acting on intact cells again imply an effect of deenergization on the permeability barrier of the outer membrane. Fluorescence changes with PhNap in intact and EDTA‐treated cells show that the dye binding sites are altered in the presence of colicin E1. It is also shown that the PhNap intensity change can be blocked by low concentrations of vitamin B12, which competes for the colicin E1 receptor.Some properties are presented of the probe chlorotetracycline, which has been proposed by others to be an indicator of magnesium. The probe appears to reside in an environment somewhat similar to that of ANS, but the colicin‐induced changes in its fluorescence parameters appear to be small under our conditions.