The Mrp Na+/H+ Antiporter Increases the Activity of the Malate:Quinone Oxidoreductase of an Escherichia coli Respiratory Mutant (original) (raw)
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Journal of Bacteriology, 2008
Mrp antiporters catalyze secondary Na+(Li+)/H+ antiport and/or K+/H+ antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex o...
Journal of Biological Chemistry, 2010
Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires heterooligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na ؉ (Li ؉ )/H ؉ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na ؉ ] in . Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na ؉ (Li ؉ )/H ؉ antiport.
Journal of Bacteriology, 2007
Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from Escherichia coli transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient E. coli EP432 transformants produced higher levels of secondary Na ؉ (Li ؉ )/H ؉ antiport than previously reported. Assays were conducted on Mrps from alkaliphilic Bacillus pseudofirmus OF4 and Bacillus subtilis and the homologous antiporter of Staphylococcus aureus (Mnh), all of which exhibited Na ؉ (Li ؉ )/H ؉ antiport. A second paralogue of S. aureus (Mnh2) did not. K ؉ , Ca 2؉ , and Mg 2؉ did not support significant antiport by any of the test antiporters. All three Na ؉ (Li ؉ )/H ؉ Mrp antiporters had alkaline pH optima and apparent K m values for Na ؉ that are among the lowest reported for bacterial Na ؉ /H ؉ antiporters. Using a fluorescent probe of the transmembrane electrical potential (⌬⌿), Mrp Na ؉ /H ؉ antiport was shown to be ⌬⌿ consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher ⌬⌿ levels than control membranes, as did membranes from E. coli EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic E. coli antiporter. Assays of respiratory chain components in membranes from Mrp and control E. coli transformants led to a hypothesis explaining how activity of secondary, ⌬⌿-consuming antiporters can elicit increased capacity for ⌬⌿ generation in a bacterial host.
Journal of Biological Chemistry, 2004
The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na ؉ /H ؉ exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na ؉ /H ؉ antiport is discussed.
Journal of Bacteriology, 2005
Sha (also known as Mrp/Mnh/Pha) is a Na+/H+ antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H+, Na+, and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the sha homologue genes from the opportunistic pathogen Pseudomonas aeruginosa, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na+/H+ antiport activity of the Escherichia coli TO114 mutant lacking the three major Na+/H+ antiporters, indicating that genes PA1054 to PA1059 are responsible for Na+/H+ antiport activity. We disrupted PA1054 (a shaA homologue gene) and determined its effect on Na+ tolerance during growth, Na+ efflux, and pathogenicity in mice. Disrupti...
Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli
1998
Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal N a + /H + antiporters, the result was a Li + sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na + /H + antiporters. These cells became more resistant to external LiCl, but only in Na + containing media. In the absence of external Na + , the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na + /H + antiporter in conjunction with different housekeeping systems of E. coli host cells.
Biochemistry, 2009
Vibrio cholerae and many other marine and pathogenic bacteria posses a unique respiratory complex, the Na +-pumping NADH: quinone oxidoreductase (Na +-NQR)1, which pumps Na + across the cell membrane using the energy released by the redox reaction between NADH and ubiquinone. In order to function as a selective sodium pump, Na +-NQR must contain structures that: 1) allow the sodium ion to pass through the hydrophobic core of the membrane, and 2) provide cation specificity to the translocation system. In other sodium transporting proteins, the structures that carry out these roles frequently include aspartate and glutamate residues. The negative charge of these residues facilitates binding and translocation of sodium. In this study we have analyzed mutants of acid residues located in the transmembrane helices of subunits B, D and E of Na +-NQR. The results are consistent with the participation of seven of these residues in the translocation process of sodium. Mutations at NqrB-D397, NqrD-D133 and NqrE-E95 produced a decrease of approximately ten times or more in the apparent affinity of the enzyme for sodium (Km app), which suggests that these residues may form part of a sodium-binding site. Mutation at other residues, including NqrB-E28, NqrB-E144, NqrB-E346 and NqrD-D88, had a large effect on the quinone reductase activity of the enzyme and its sodium sensitivity, but less effect on the apparent sodium affinity, consistent with a possible role in sodium conductance pathways. The sodium pumping NADH:quinone oxidoreductase (Na +-NQR) is a unique prokaryotic respiratory enzyme capable of sustaining a sodium gradient across the plasma membrane, using the free energy released in the coupled oxidation of NADH and reduction of ubiquinone (1-3). Na +-NQR is composed of six subunits (NqrA-F) and contains five cofactors involved in the internal electron transfer: a non-covalently bound FAD and a 2Fe-2S center located in NqrF (4-8), two covalently-bound FMN's in NqrB and NqrC (9-11), which have been shown to give rise to two anionic flavosemiquinone radicals, observed in the partially and fully reduced forms of the enzyme (12), and a non-covalently bound riboflavin molecule that is found as a stable neutral flavosemiquinone radical in the oxidized state of the enzyme (13,14). This is notable because it is the only known instance in which riboflavin is present as a bona fide redox cofactor in any enzyme.