In eukaryotes, metallothioneins (MTs) are involved in cellular responses to elevated concentrations of certain metal ions. We report the isolation and analysis of a prokaryotic MT locus from Synechococcus PCC 7942. The MT locus (smt) includes smtA, which encodes a class II MT, and a divergently transcribed gene, smtB. The sites of transcription initiation of both genes have been mapped and features within the smt operator-promoter region identified. Elevated concentrations of the ionic species of Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn elicited an increase in the abundance of smtA transcripts. There was no detectable effect of elevated metal (Cd) on smtA transcript stability. Sequences upstream of smtA, fused to a promoterless lacZ gene, conferred metal-dependent beta-galactosidase activity in Synechococcus PCC 7942 (strain R2-PIM8). At maximum permissive concentrations, Zn was the most potent elicitor in vivo, followed by Cu and Cd with slight induction by Co and Ni. The deduced SmtB polypeptide has similarity to the ArsR and CadC proteins involved in resistance to arsenate/arsenite/antimonite and to Cd, contains a predicted helix-turn-helix DNA-binding motif and is shown to be a repressor of transcription from the smtA operator-promoter.

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The smt locus of Synechococcus PCC 7942 contains a metal-regulated gene (smtA), which encodes a class II metallothionein, and a divergently transcribed gene, smtB, which encodes a repressor of smtA transcription. Regions containing cis-acting elements required for efficient induction, and required for smtB-dependent repression, of the smtA operator-promoter were identified. Specific interactions between proteins extracted from Synechococcus PCC 7942 and defined regions surrounding the smtA operator-promoter were detected by electrophoretic mobility shift assays. Three metallothionein operator-promoter associated complexes were identified, one of which (MAC1) showed Zn-dependent dissociation and involved a region of DNA immediately upstream of smtA. Treatment with Zn-chelators facilitated re-association of MAC1 in vitro. MAC1 was not observed in extracts from smt deficient mutants but was restored in extracts from mutants complemented with a plasmid borne smtB. SmtB is thus required for the formation of a Zn-responsive complex with the smt operator-promoter and based upon the predicted structure of SmtB we propose direct SmtB-DNA interaction exerting metal-ion inducible negative control.

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