Tet(L) and Tet(K) Tetracycline-Divalent Metal/H
            <sup>+</sup>
            Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

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

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“…Interestingly, in both cases the drug resistance and the alkaline pH tolerance functions are linked (this study and ref. 40).…”

Section: Discussionmentioning

confidence: 99%

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

Lewinson

Padan

Bibi

2004

Proc. Natl. Acad. Sci. U.S.A.

111

122

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“…This suggested that these mutant Tet(L) proteins promote leakage of K + into the vesicles in response to the generation of the transmembrane potential, ΔΨ, while such a leak is not observed in wild type Tet(L) vesicles or with other Tet(L) mutants (e.g. see Figure 3) (40,41,49). The activity in the complete assay, with both electron donor and intravesicular K + , was lower than wild type in each Pro 156 mutant ( Figure 4).…”

Section: Mutants Of Pro 156 In the Motif C Gp Dipeptidementioning

confidence: 99%

“…In this assay, 86 Rb + uptake into the RSO vesicles by wild type Tet(L) depends upon both the addition of the electron donor and on the presence of an efflux substrate for Tet(L) inside the vesicles; no Tet(L)-dependent 86 Rb + uptake was observed in the absence of added D-lactate (the electron donor) and or intravesicular K + (the efflux substrate loaded inside the vesicles in this experiment) (Figure 4). Earlier work showed that the net Rb + -K + uptake in this assay is a mode of the antiport that is electrogenic and it could be supported by the presence of either K + or Tc-Co 2+ inside the vesicles (40,42). It was therefore notable that 86 Rb + uptake by the mutant Tet(L) P156A,G or C forms was as high or higher under conditions in which the electron donor was present but the intravesicular K + was absent.…”

Section: Mutants Of Pro 156 In the Motif C Gp Dipeptidementioning

confidence: 99%

“…Everted vesicles were prepared from E. coli transformants in 10 mM BTP at pH 7.5 using a French Pressure cell method of Rosen (51) as described previously (40). RSO membrane vesicles were prepared by the lysozyme-method of Kaback (52) and were pre-loaded with either 100 μM choline chloride or KCl (42).…”

Section: Preparation Of Membrane Vesiclesmentioning

confidence: 99%

“…Both Tet(L) and Tet(K) can also substitute K + for part of the H + coupling ion complement, especially at high pH, thus catalyzing both net K + and H + uptake under particular conditions of pH and K + status (40)(41)(42). This use of K + makes it possible to track the movement of the coupling ion (via inward fluxes of 86 Rb + ) in a direct manner that is not possible when only H + is used as coupling ion (40).…”

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confidence: 99%

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Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

et al. 2005

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Proline and glycine residues are well represented among functionally important residues in hydrophobic domains of membrane transport proteins and several critical roles have been suggested for them. Here, the effects of mutational changes in membrane-embedded proline and glycine residues of Tet(L) were examined, with a focus on the conserved GP 155,156 dipeptide of Motif C, a putative "antiporter motif". Mutation of Gly 155 to cysteine resulted in a mutant Tet(L) that bound its tetracycline-divalent metal (Tc-Me 2+ ) substrate but did not catalyze efflux or exchange of TcMe 2+ or catalyze uptake or exchange of Rb + which was used to monitor the coupling ion. These results support suggestions that this region is involved in the conformational changes required for translocation. Mutations in Pro 156 resulted in reduction (P156G) or loss (P156A or C) of Tc-Me 2+ efflux capacity. All three Pro 156 mutants exhibited a K + leak (monitored by 86 Rb + fluxes) that was not observed in wild type Tet(L). A similar leak was observed in a mutant in a membrane-embedded proline residue elsewhere in the Tet(L) protein (P175C) as well as in a P156C mutant of related antiporter Tet(K). These findings are consistent with roles proposed for membrane-embedded prolines in tight helix packing. Patterns of Tc-resistance conferred by additional Tet(L) mutants indicate important roles for another GP dipeptide in transmembrane segment (TMS) X as well as for membrane-embedded glycine residues in TMS XIII.Almost all transport proteins have membrane-embedded proline residues in some of the multiple α-helices that are a general feature of transporters (1). These residues are preferentially paired with glycine residues that are also found in abundance in TMS 1 of transporters (2,3). Membrane-embedded proline and glycine residues of transporters are hypothesized to promote helix kinks and swivels that play crucial roles in the conformational flexibility required for transport mechanisms (2-6). They are further hypothesized to have roles in helix packing and association (7-9). These hypotheses have been stunningly supported for the largest family of membrane transport proteins, the MFS, a family of structurally related transporters of prokaryotes and eukaryotes that includes uniporters, antiporters and symporters. The MFS comprises about a quarter of all transport proteins (10,11). The first three high resolution

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Biological Aspects of Metal Enolates

Ming

2010

Patai's Chemistry of Functional Groups

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Tet(L) and Tet(K) Tetracycline-Divalent Metal/H ⁺ Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

Cited by 42 publications

References 51 publications

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

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

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

Biological Aspects of Metal Enolates

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Tet(L) and Tet(K) Tetracycline-Divalent Metal/H + Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

Cited by 42 publications

References 51 publications

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

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

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

Biological Aspects of Metal Enolates

Contact Info

Product

Resources

About

Tet(L) and Tet(K) Tetracycline-Divalent Metal/H ⁺ Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences