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Journal of Bacteriology, November 2001, p. 6645-6653, Vol. 183, No. 22
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.22.6645-6653.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na+(Li+)/H+ Antiporter Activity on Escherichia coli: Characterization of the Recovered Genes and the Corresponding Gene Products

Alan Majerník,1 Gerhard Gottschalk,2,3 and Rolf Daniel2,*

Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, 90028 Ivanka pri Dunaji, Slovak Republic,1 and Abteilung Allgemeine Mikrobiologie2 and Göttingen Genomics Laboratory,3 Institut für Mikrobiologie und Genetik der Georg-August-Universität, 37077 Göttingen, Germany

Received 8 June 2001/Accepted 30 August 2001

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na+(Li+)/H+ antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li+-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na+/H+ antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na+/H+ antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The Km values of both antiporters for Na+ were approximately 10-fold higher than the values for Li+.

* Corresponding author. Mailing address: Institut für Mikrobiologie und Genetik der Georg-August-Universität, Grisebachstr. 8, 37077 Göttingen, Germany. Phone: 49-551-393827. Fax: 49-551-393808. E-mail: rdaniel{at}gwdg.de.

Journal of Bacteriology, November 2001, p. 6645-6653, Vol. 183, No. 22
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.22.6645-6653.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.