Homeostatic systems for essential and non-essential metals create the cellular environments in which the correct metals are acquired by metalloproteins while the incorrect ones are somehow avoided. Cyanobacteria have metal requirements often absent from other bacteria; copper in thylakoidal plastocyanin, zinc in carboxysomal carbonic anhydrase, cobalt in cobalamin but magnesium in chlorophyll, molybdenum in heterocystous nitrogenase, manganese in thylakoidal water-splitting oxygen-evolving complex. This article reviews: an intracellular trafficking pathway for inward copper supply, the sequestration of surplus zinc by metallothionein (also present in other bacteria) and the detection and export of excess cobalt. We consider the influence of homeostatic proteins on selective metal availability.

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene,smtA, is required for normal zinc-tolerance in the cyanobacteriumSynechococcusPCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn₄Cys₉His₂cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys₄and two ZnCys₃His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn₄Cys₁₁cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys₄-coordinated zinc ions in SmtA readily exchanges with exogenous metal (¹¹¹Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of β-strand and α-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

ORF slr0798, now designated ziaA , from Synechocystis PCC 6803 encodes a polypeptide with sequence features of heavy metal transporting P-type ATPases. Increased Zn ²⁺ tolerance and reduced ⁶⁵ Zn accumulation was observed in Synechococcus PCC 7942, strain R2-PIM8( smt ), containing ziaA and upstream regulatory sequences, compared with control cells. Conversely, reduced Zn ²⁺ tolerance was observed following disruption of ziaA in Synechocystis PCC 6803, and ziaA -mediated restoration of Zn ²⁺ tolerance has subsequently been used as a selectable marker for transformation. Nucleotide sequences upstream of ziaA , fused to a promoterless lacZ gene, conferred Zn ²⁺ -dependent β-galactosidase activity when introduced into R2-PIM8( smt ). The product of ORF sll0792, designated ZiaR, is a Zn ²⁺ -responsive repressor of ziaA transcription. Reporter gene constructs lacking ziaR conferred elevated Zn ²⁺ -independent expression from the ziaA operator–promoter in R2-PIM8( smt ). Gel retardation assays detected ZiaR-dependent complexes forming with the zia operator–promoter and ZiaR–DNA binding was enhanced by treatment with a metal-chelator in vitro . Two mutants of ZiaR (C71S/C73S and H116R) bound to, and repressed expression from, the ziaA operator–promoter but were unable to sense Zn ²⁺ . Metal coordination to His-imidazole and Cys-thiolate ligands at these residues of ZiaR is thus implicated in Zn ²⁺ -perception by Synechocystis PCC 6803.

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P₁-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P₁-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.

NmtR from Mycobacterium tuberculosis is a new member of the ArsR-SmtB family of metal sensor transcriptional repressors. NmtR binds to the operator-promoter of a gene encoding a P(1) type ATPase (NmtA), repressing transcription in vivo except in medium supplemented with nickel or, to some extent, cobalt. In a cyanobacterial host, Synechococcus PCC 7942 strain R2-PIM8(smt), NmtR-mediated repression is alleviated by cobalt but not nickel or zinc addition, while the related sensor SmtB responds exclusively to zinc. Quantification of the number of atoms of nickel per cell shows that NmtR nickel sensitivity correlates with cytosolic nickel contents. Differential metal discrimination in a common cytosol by SmtB (zinc) and NmtR (cobalt) is not simply explained by affinities at equilibrium; although NmtR does bind nickel substantially more tightly than SmtB, it has a higher affinity for zinc than for cobalt and binds cobalt more weakly than SmtB. SmtB is known to bind and sense zinc at interhelical four-coordinate, tetrahedral sites across the C-terminal alpha 5 helices, while absorption spectroscopy of Co(II)- and Ni(II)-substituted NmtR reveals five- and six-coordinate metal complexes. Site-directed mutagenesis identifies six potential cobalt/nickel ligands that are obligatory for inducer recognition but not repression by NmtR, four of which (Asp(91), His(93), His(104), His(107)) align with alpha 5 ligands of SmtB with two additional His provided by a carboxyl-terminal "extension" (designated alpha 5C). Gel retardation assays reveal that zinc does not allosterically regulate NmtR-DNA binding at concentrations where lower affinity cobalt does. These data suggest that two additional ligands form hexacoordinate metal complexes and are crucial for driving allosteric regulation of DNA binding by NmtR, thereby allowing NmtR to preferentially sense metals that favor higher coordination numbers relative to SmtB.