The pha2 gene cluster involved in Na + resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter

China Agriculture University-East, Peping, Beijing, China
FEMS Microbiology Letters (Impact Factor: 2.12). 09/2006; 262(2):172-7. DOI: 10.1111/j.1574-6968.2006.00385.x
Source: PubMed


Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.

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    • "About ten families of single-gene-encoded Na + /H + antiporters including NhaA [3], NhaB [4], NhaC [5], NhaD [6], NapA [7], NhaP [8], NhaG [9] and NhaH [10] have been identified in many microorganisms. Another kind of Na + /H + antiporter consists of multiple subunits encoded by an operon or a gene cluster such as mnhABCDEFG gene cluster from Staphylococcus aureus [11], mrp operon from Bacillus subtilis [1] and phaA2B2C2D2E2F2G2 gene cluster from Sinorhizobium fredii [12] [13]. "
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    ABSTRACT: NhaH is a novel Na(+)/H(+)antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na(+)(Li(+))/H(+) antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na(+) and Li(+) without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na(+) and Li(+) but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H(+) binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na(+) and Li(+), indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li(+), without affecting that for Na(+), suggesting that E229 may be mainly responsible for Li(+)binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na(+) and Li(+), indicating that H87 and H88 are involved in H(+) binding or pH regulation.
    Preview · Article · Nov 2012 · Biochimica et Biophysica Acta
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    • "The pha2 cluster encodes proteins that demonstrate high similarity to the group 1 Mrp antiporters. Recently, Yang et al. (2006) suggested that the Pha2 system is a Na + (Li + , K "
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    ABSTRACT: The pha1 gene cluster (pha1A'-G) of Sinorhizobium meliloti has previously been characterized as a necessary component for proper invasion into plant root tissue. It has been suggested to encode a multi-subunit K(+)/H(+) antiporter, since mutations in the pha1 region rendered S. meliloti cells sensitive to K(+) and alkali, and because there is high amino acid sequence similarity to previously characterized multi-subunit cation/H(+) antiporters (Mrp antiporters). However, the detailed transport properties of the Pha1 system are yet to be determined. Interestingly, most of the Mrp antiporters are highly selective for Na(+), unlike the Pha1 system. Here, we report the functional expression of the Pha1 system in Escherichia coli and the measurement of cation/H(+) antiport activity. We showed that the Pha1 system is indeed a K(+)/H(+) antiporter with a pH optimum under mildly alkaline conditions. Moreover, we found that the Pha1 system can transport Na(+); this was unexpected based on previous phenotypic analyses of pha1 mutants. Furthermore, we demonstrated that the cation selectivity of the Pha1 system was altered when the pH was lowered from the optimum. The downregulation of Na(+)/H(+) and K(+)/H(+) antiport activities upon acidic shift appeared to occur via different processes, which might indicate the presence of distinct mechanisms for the regulation of the K(+)/H(+) and Na(+)/H(+) antiport activities of the Pha1 system.
    Full-text · Article · Jun 2009 · Microbiology
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    ABSTRACT: 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+, Ca2+, and Mg2+ did not support significant antiport by any of the test antiporters. All three Na+(Li+)/H+ Mrp antiporters had alkaline pH optima and apparent Km values for Na+ that are among the lowest reported for bacterial Na+/H+ antiporters. Using a fluorescent probe of the transmembrane electrical potential (ΔΨ), Mrp Na+/H+ antiport was shown to be ΔΨ consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from E. coli EP432 expressing Mrp antiporters generated higher ΔΨ 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, ΔΨ-consuming antiporters can elicit increased capacity for ΔΨ generation in a bacterial host.
    Full-text · Article · May 2007 · Journal of Bacteriology
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