No abstract

It is difficult to over-state the importance of Zn(II) in biology. It is a ubiquitous essential metal ion and plays a role in catalysis, protein structure and perhaps as a signal molecule, in organisms from all three kingdoms. Of necessity, organisms have evolved to optimise the intracellular availability of Zn(II) despite the extracellular milieu. To this end, prokaryotes contain a range of Zn(II) import, Zn(II) export and/or binding proteins, some of which utilise either ATP or the chemiosmotic potential to drive the movement of Zn(II) across the cytosolic membrane, together with proteins that facilitate the diffusion of this ion across either the outer or inner membranes of prokaryotes. This review seeks to give an overview of the systems currently classified as altering Zn(II) availability in prokaryotes.

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 global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids.

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.

Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn501 was replaced by serine, and the mercury-resistance phenotypes of the mutants were determined in Escherichia coli. Cys-24 and Cys-25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys-76-Ser, Cys-82-Ser or Gly-38-Asp in MerT or Cys-36-Ser in MerP all reduced transport and resistance. Deletion of the merP gene slightly reduced mercuric ion resistance and transport, whereas a Cys-33-Ser mutation in MerP appears to block transport of mercuric ions by MerT. The effects of deleting merP on mutations in merT were tested. The 116-amino-acid MerT protein is sufficient for mercuric ion transport across the cytoplasmic membrane.

Mercury resistance determinants are widespread in Gram-negative bacteria, but vary in the number and identity of genes present. We have shown that the merF gene from plasmid pMER327/419 encodes a 8.7 kDa mercury transport protein, by determining in vivo mercury volatilisation when MerF is expressed in the presence of mercuric reductase. We have confirmed that MerC of Tn21 is also a mercuric ion transporter. We have been able to detect interaction of the periplasmic protein MerP only with the MerT transporter, and not with MerF or MerC. Hydropathy analysis led to the prediction of models for MerT, MerC and MerF having three, four and two transmembrane regions respectively. In all three cases one pair of cysteine residues is predicted to be within the inner membrane with a second pair of cysteine residues on the cytoplasmic face, and the second helix contains a proline and at least one charged residue. The mechanisms of mercuric ion transport may be similar in these transporters even though their structures in the membrane differ.z 2000 Federation of European Biochemical Societies.

Bacterial resistances to metals are heterogeneous in both their genetic and biochemical bases. Metal resistance may be chromosomally-, plasmid- or transposon-encoded, and one or more genes may be involved: at the biochemical level at least six different mechanisms are responsible for resistance. Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species. To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria. How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion. Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms. The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations. It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations. The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals. Potentially, models for selection of resistance to any substance can be derived using this approach.