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.
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.
SmtB is a member of a family of repressors which dissociate from DNA in the presence of metals; Zn2+ being the most potent inducer of metallothionein gene (smtA) transcription in vivo. In Synechococcus PCC 7942 cells devoid of chromosomal smtB, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed lacZ expression driven by the smtA operator-promoter. Gel retardation assays with extracts from the complemented cells detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB. Elevated [Zn2+] alleviated repression in vivo by all of the mutants except H105R/H106R. These His residues (one or both) are therefore essential for Zn2+-sensing while, contrary to expectations, Cys residues are not. Hence different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the spectra of metals sensed. Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm that additional unknown factors form specific associations with the smt operator-promoter in elevated [Zn2+].
Genomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal-tolerant eukaryotes. Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus, smt, in cells of Synechococcus PCC 6301 selected for Cd tolerance. Following the characterization of this locus, the altered smt region has now been isolated from a Cd-tolerant cell line, C3.2, and its nucleotide sequence determined. This has identified a deletion within smtB, which encodes a trans-acting repressor of smt transcription. Two identical palindromic octanucleotides (5'-GCGATC-GC-3') traverse both borders of the excised element. This palindromic sequence is highly represented in the smt locus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL/DDBJ DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences from Synechococcus strains (estimated to occur at an average frequency of once every c. 664 bp). HIP1 is also abundant in the genomes of other cyanobacteria. The functional significance of smtB deletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteria are discussed.
Metallothioneins have been extensively studied in many different eukaryotes where they sequester, and hence detoxify, excess amounts of certain metal ions. However, the precise functions of many of these molecules are not fully understood. This article reviews literature concerning their namesakes in prokaryotes.
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