Attractants, in the presence of respiration and ATPase inhibitors, stimulate a hyperpolarization in Escherichia coli [Eisenbach, M. (1982) Biochemistry 21, 6818-6825]. In order to examine whether this hyperpolarization is correlated with chemotaxis, the effect of the attractant D-galactose and its analogues on the membrane potential of wild-type E. coli strains and some of their mutants was studied. The main observations were the following: (i) Wild-type cells became hyperpolarized by either galactose or its nonmetabolizable analogues, D-fucose and L-sorbose. (ii) A mutant defective in galactose metabolism became hyperpolarized by galactose. (iii) Inhibiting the galactose permease system did not prevent the hyperpolarization, rather it facilitated the observation of the hyperpolarization. (iv) Mutants unable to transport galactose via the methyl beta-galactoside (Mgl) transport system but having normal chemotaxis to galactose became normally hyperpolarized by D-fucose. (v) Mutants which cannot bind galactose were not hyperpolarized by galactose. (vi) The hyperpolarization in flaI mutants, in which the whole chemotaxis machinery is repressed, was reduced to 12-15% of the hyperpolarization in the parent strains. (vii) Nonattractant sugars did not stimulate hyperpolarization. It is concluded that the hyperpolarization is the consequence of neither galactose metabolism nor transport but rather is correlated with galactose taxis.
Galactose and other chemotactic attractants have been shown to trigger an apparent hyperpolarization in Escherichia coli (Eisenbach, M., 1982, Biochemistry, 21:6818-6825). The probe used to measure membrane potential in that study, tetraphenylphosphonium (TPP+), may respond also to surface-charge changes in the membrane. The distinction between true changes in membrane potential and changes in the surface charge of the membrane is crucial for the study of this phenomenon in bacterial chemotaxis. To distinguish between these parameters, we compared the response to galactose with different techniques: K+ distribution in the presence of valinomycin (measured with a K+-selective electrode), TPP+ distribution (measured with a TPP+-selective electrode) at different ionic strengths, absorbance changes of bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V), and fluorescence changes of three probes with different mechanisms of response. All the techniques revealed stimulation by galactose of transient hyperpolarization, of comparable magnitude. This indicates the involvement of ion currents rather than alterations of local surface properties.
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