Abstract
A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no homology between the alkaliphile gene, which we propose to call nhaC, and the nhaA gene of Escherichia coli, nor with other genes in digests of DNA from E. coli, Bacillus subtilis, or Bacillus alcalophilus. Although there was also no significant similarity between the deduced protein products of the alkaliphile gene and the nhaA gene of E. coli, there was a small region of significant similarity between the deduced alkaliphile gene product and the protein encoded by a human Na+/H+ antiporter gene (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280).
Highlights
RESULTSE. coli strain NM81 was transformed with the DNA library 1 from B. firmus OF4 in pSPT18
A gene has been cloned from a DNA library from species have offered some of the most compelling suggestions alkaliphilic Bacillus firrnus OF4 that functioncoamlly- of theparticular role of asecondaryelectrogenic Na+/H+
The insert was used to isolate clones containing antiporter have been found deficient in mutants of two difthe complete openreading frame, whichwould bepre- ferent alkaliphiles that were no longer able to grow at very dicted to encode a protein with a molecular weight of alkaline pH[5].Interest in the alkaliphile antiporteisr further enhanced by the finding thatgenetic variants of at least one
Summary
E. coli strain NM81 was transformed with the DNA library 1 from B. firmus OF4 in pSPT18. Thetransformants from three plates with numerous colonies were resuspended ligated with thechromosomal DNA fragments. DNA from the ligation mix was used to transform E. coli DH5nMCR(GIBCO/BRL Life ' Theabbreviations used are: PCR, polymerase chainreaction; Technologies), and approximately 10"transformants were pooled in EIPA, ethylisopropylamiloride; IPTG, isopropyl-1-thio-(-t-galacto-. The culture was plated onto solid LB medium, pH 7.5, containing 0.7 M NaCl. Colonies that arose on these plates were purified by restreaking on the same medium and were shown to be Growth on m - E.coli NM81 p H 7.5+.6M NaCl Li*+rnelibiose. 2 dozen such isolates were screened from each library, by analysis of minipreps, for the Transform with. Subsequent studies revealed that the phenotype was not conferred by the plasmid, designatedpEM271, but was a property of the pEM271-bearing host that hadbeen isolated
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