Abstract
BackgroundIn neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis.ResultsAssays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress.ConclusionsTaken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.
Highlights
IntroductionMonovalent metal cation/H+ antiporters play a key role in pH homeostasis
In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis
We show through a combination of cell growth studies, transport assays using whole cells and inverted vesicles, and measurements of intracellular pH, that MdtM is required for adaptation of E. coli to alkaline environments and that the observed alkalitolerance is due to a monovalent metal cation/H+ antiport activity of MdtM that functions to maintain a cytoplasm that is acidic relative to the outside of the cell; this activity is only apparent at distinct alkaline pH values of between pH 9 and pH 10, and in the presence of Na+ or K+ ions in the growth medium
Summary
Monovalent metal cation/H+ antiporters play a key role in pH homeostasis. The capacity to survive at pH values outside their normal growth range is a prominent feature of many pathogenic bacteria [1] During their life cycles the neutralophilic enterobacteria Escherichia coli and Vibrio cholerae can be released into alkaline marine and estuarine environments where they can remain viable and sustain a threat to public health for periods of up to weeks [2,3]. Such alkalitolerance requires neutralophilic bacteria to maintain a stable cytoplasmic pH, in the narrow range of pH 7.4 to 7.8, that is acidic relative to that of the external environment [4]; to achieve this they employ diverse strategies, all designed to contribute to the maintenance of cytoplasmic proton concentration. Active inward transport of protons by cytoplasmic membrane cation/H+ antiporters is crucial to the latter strategy and often plays a dominant role in alkaline pH homeostasis in bacteria [6,7].
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