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Journal of Bacteriology, April 1999, p. 2385-2393, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Transcriptional Organization of the czc Heavy-Metal Homeostasis Determinant from Alcaligenes eutrophus

Cornelia Große,1 Gregor Grass,1 Andreas Anton,1 Sylvia Franke,1 Alexander Navarrete Santos,2 Blair Lawley,3 Nigel L. Brown,3 and Dietrich H. Nies1,*

Institut für Mikrobiologie der Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle,1 and Institute of Medical Immunology, Martin-Luther-Universität Halle-Wittenberg, D-06097 Halle,2 Germany, and School of Biological Sciences, The University of Birmingham, Birmingham B15 2TT, United Kingdom3

Received 24 November 1998/Accepted 22 January 1999

The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5' end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotide czcI message were found, and the presence of 6,200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493-503, 1997) was confirmed. Induction of czcN, czcI, czcCBA, and czcDRS followed a similar pattern: transcription was induced best by 300 µM zinc, less by 300 µM cobalt, and only slightly by 300 µM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN to czcC and from czcD to czcS, but not between czcA and czcD nor between czcS and a 131-amino-acid open reading frame following czcS. The CzcR putative response regulator was purified and shown to bind in the 5' region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were Delta czcR and Delta czcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the beta -galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

* Corresponding author. Mailing address: Institut für Mikrobiologie der Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06099 Halle, Germany. Phone: (49)-345-55 26352. Fax: (49)-345-55 27010. E-mail: d.nies{at}mikrobiologie.uni-halle.de.

Journal of Bacteriology, April 1999, p. 2385-2393, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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