Characterization of a copper-transport operon, copYAZ, from Streptococcus mutans

Department of Microbiology and Immunology, Faculty of Medicine 1 , and Department of Applied Oral Sciences, Faculty of Dentistry 2 , Dalhousie University, Halifax, Nova Scotia, CanadaB3H 3J5 Author for correspondence: Song F. Lee (Applied Oral Sciences). Tel: +1 902 494 8799. Fax: +1 902 494 6621. e-mail: Song.Lee{at}Dal.Ca A copper-transport ( copYAZ ) operon was cloned from the oral bacterium Streptococcus mutans JH1005. DNA sequencing showed that the operon contained three genes ( copY , copA and copZ ), which were flanked by a single promoter and a factor-independent terminator. copY encoded a small protein of 147 aa with a heavy-metal-binding motif (CXCX 4 CXC) at the C-terminus. CopY shared extensive homology with other bacterial negative transcriptional regulators. copA encoded a 742 aa protein that shared extensive homology with P-type ATPases. copZ encoded a 67 aa protein that also contained a heavy-metal-binding motif (CXXC) at the N-terminus. Northern blotting showed that a 3·2 kb transcript was produced by Cu 2+ -induced Strep. mutans cells, suggesting that the genes were synthesized as a polycistronic message. The transcriptional start site of the cop operon was mapped and shown to lie within the inverted repeats of the promoter–operator region. Strep. mutans wild-type cells were resistant to 800 µM Cu 2+ , whereas cells of a cop knock-out mutant were killed by 200 µM Cu 2+ . Complementation of the cop knock-out mutant with the cop operon restored Cu 2+ resistance to wild-type level. The wild-type and the mutant did not show any differences in susceptibility to other heavy metals, suggesting that the operon was specific for copper. By using a chloramphenicol acetyltransferase reporter gene fusion, the cop operon was shown to be negatively regulated by CopY and could be derepressed by Cu 2+ . Keywords: copper transport, copper resistance, P-type ATPase, heavy metals Abbreviations: CAT, chloramphenicol acetyltransferase; THA, Todd–Hewitt agar; THB, Todd–Hewitt broth; TYG, tryptone/yeast extract glucose broth The GenBank accession number for the cop operon sequence reported in this paper is AF296446 .