Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

Abstract

MdfA is an Escherichia coli multidrug-resistance transporter. Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds. In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH. MdfA-deleted E. coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid. This activity of MdfA requires Na ؉ or K ؉ . Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na ؉ -or K ؉ -dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon. Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related. Together, the results demonstrate that a single protein has an unprecedented capacity to turn E. coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.MdfA ͉ multidrug transport ͉ sodium proton antiporter ͉ alkaline pH tolerance ͉ Escherichia coli

Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

“…This host is deficient in K ϩ /H ϩ as well as Na ϩ (Li ϩ )/H ϩ and Ca 2ϩ /H ϩ antiport (31)(32)(33). Tris⅐HCl was used instead of the Tris-Hepes used in earlier assays (11,12), and higher concentrations of K ϩ were tested in view of recent reports of several bacterial K ϩ /H ϩ antiporters with low affinity for cation (32,34). In assays of the B. subtilis YhaTU system, neither the transporter protein YhaU nor the ancillary protein YhaT exhibited K ϩ /H ϩ antiport activity at pH 8.5 when expressed individually.…”

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

“…Transport experiments have shown that MdfA is driven by the proton electrochemical potential (6,9,10), and in addition to its drug/proton exchange activity, it may also function as a (Na + ) (K + )/proton antiporter (11). Clearly, therefore, proton translocation by MdfA is crucial for all of its known biological activities.…”

A Role of thecspAGene Encoding a Mycolyltransferase in the Growth under Alkaline Conditions ofCorynebacterium glutamicum

“…The C. glutamicum strains were grown at 31. 24) For the antimicrobial susceptibility assay, LB medium consisting of 10 g/l of tryptone, 5 g/l of yeast extract, and 10 g/l of NaCl (pH 7.2) was used.…”