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

Abstract

ABSTRACT The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB 2 A 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 Δ czcR and Δ czcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn 2+ , (ii) induction of the β-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.