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The Czc system ofAlcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2 A cation-proton antiporter. DNA sequencing of the region upstream of theczcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5′ end ofczcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that ofczc . Additional open reading frames upstream ofczc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotideczcN message and a 400-nucleotideczcI message were found, and the presence of 6,200-nucleotideczcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493–503, 1997) was confirmed. Induction ofczcN ,czcI ,czcCBA , andczcDRS 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 fromczcN toczcC and fromczcD toczcS , but not betweenczcA andczcD nor betweenczcS and a 131-amino-acid open reading frame followingczcS . The CzcR putative response regulator was purified and shown to bind in the 5′ region ofczcN . A reporter strain carrying aczcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were ΔczcR and ΔczcS 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 β-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis ofczcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

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