Abstract
DNA libraries from alkaliphilic Bacillus firmus OF4 had been screened earlier (Ivey, D.M., Guffanti, A.A., Bossewitch, J. S., Padan, E., and Krulwich, T. A. (1991) J. Biol. Chem. 266, 23483-23489) for clones that would functionally complement a strain of Escherichia coli (NM81) with a deletion in one of its Na+/H+ antiporter genes. During those studies, an alkaliphile antiporter gene was hypothesized to have been incorporated into the chromosome of strain NM81, producing Na(+)-resistant NM8191. After introduction of a deletion in the second known E. coli Na+/H+ antiporter gene, libraries were prepared from NM8191 and screened for complementation of Na+/H+ antiporter-deficient mutants of E. coli. Instead of retrieving an alkaliphile gene, an unexpected E. coli gene was identified on the basis of its ability to restore Na+ resistance and membrane Na+/H+ antiporter activity to such mutant strains. The active open reading frame in the clone maps at 27 min on the E. coli chromosome and is identical in sequence to a wild type counterpart. It would be predicted to encode an extremely hydrophobic protein with multiple membrane-spanning regions and a molecular weight of 39,200. A region in one of the predicted hydrophilic loops in the gene product structure possesses striking sequence similarity to calsequestrin. The Ca2+/H+ antiporter activity of membranes from an E. coli transformant with a clone possessing only this open reading frame was indeed found to have enhanced pH-independent Ca2+/H+ antiporter activity. The Ca2+/H+ and Na+/H+ antiporter activities conferred by the clone were both inhibited by Mg2+. The gene was designated chaA and is proposed to be the structural gene for a Ca2+/H+ antiporter whose overexpression leads to resistance to growth inhibition by both calcium and sodium.
Highlights
After introduction of a deletion in the second known E. coli Na+/H+antiporter gene, libraries were prepared from NM8191 and screened for complementation of Na+/H+ anti
The nhaA gene is regulated by a lysR-type regulatory protein called NhaR ( 5 ),its transcription being stimulated by high Na+ concentrations, with porter-deficient mutants of E. coli
The nhaA gene product, which has identified on the basis of its ability to restore Na+ been purified and studied in proteoliposomes, catalyzes elecresistance and membranNea+/H+antiporter activity to trogenic antiport that is activated at alkaline pH values [7]
Summary
The membrane antiport activity only by about 20%, and thepresence of the inducer on plates did not enhance the phenotype conferred by Bacteria and Plasmids-The following strains of E. coli were employed DH5aMCR (Gibco BRL) was grown in SOB medium (SOC medium minus glucose) [12] and was usedfor preparation of libraries and initialpreparation of subclones; DH5aF'IQ (Gibco Bethesda Research Laboratories), NM81 (AnhaA, kanR) (IO), and EP432 (AnhaAAnhaB kanRcamR)(4) were used for enrichments and screening of clones that conferred Na+ resistance; NM8191, the Na+(Li+)resistant derivative of NM81 described previously (Il),and a mutant of NM8191, designated NM8191AB, in which a deletion of nhaB was achieved with the introduction of a chloramphenicol resistance marker, as described in connection with the construction of EP432 [4]. The membrane vesicleswere prepared by the method of Rosen and colleagues [17, 18].The conditions described by Goldberg et al [19] were employed for the assays. This entailed use of the MgCIZ-containingassay buffer whose composition was 10 mM Tris/Hepes, pH 8 or 8.5, 140 mM choline chloride, and 5 mM MgC12.Membrane protein was added to 60-70 pgin areaction mixture of 2 ml. The assay medium contained 50 pgof membrane protein in 10 mM Tris-HCI, pH 8.0 or 8.5, plus 140 mM choline chloride and 1p~ quinacrine-HC1 in 2 ml.
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