Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene

• Published in: The Journal of biological chemistry 1993-05, Vol.268 (15), p.11296-11303
• Main Authors: Ivey, D.M., Guffanti, A.A., Zemsky, J., Pinner, E., Karpel, R., Padan, E., Schuldiner, S., Krulwich, T.A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Antiporters; Bacillus - genetics; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Blotting, Southern; Calcium - metabolism; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cation Transport Proteins; Cell Membrane - metabolism; Chromosomes, Bacterial; Cloning, Molecular; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Library; Kinetics; Molecular Sequence Data; Open Reading Frames; Plasmids; Proteins; Sequence Deletion; Sequence Homology, Amino Acid; Sodium - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)82124-X

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.