Jennifer S. Cavet

Zn, Cu and Co in cyanobacteria: selective control of metal availability

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.

A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

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 cyanobacterium Synechococcus PCC 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 Zn4Cys9His2 cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys4 and two ZnCys3His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn4Cys11 cluster 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.

Cobalt-dependent Transcriptional Switching by a Dual-effector MerR-like Protein Regulates a Cobalt-exporting Variant CPx-type ATPase

CoaR associates with and confers cobalt-dependent activation of the coaToperator-promoter. A CoaR mutant (Ser-Asn-Ser) in a carboxyl-terminal Cys-His-Cys motif bound the coaT operator-promoter but did not activate expression in response to cobalt, implicating thiolate and/or imidazole ligands at these residues in an allosteric cobalt binding site. Deletion of 1 or 2 nucleotides from between near consensus, but with aberrant (20 base pairs) spacing, −10 and −35 elements enhanced expression from the coaToperator-promoter but abolished activation by cobalt-CoaR. It is inferred that cobalt effects a transition in CoaR that underwinds thecoaT operator-promoter to realign promoter elements. In the absence of cobalt, CoaR represses expression (∼50%). CoaR is a fusion of ancestral MerR (mercury-responsive transcriptional activator)- and precorrin isomerase (enzyme of vitamin B12biosynthesis)-related sequences. Expression from the coaToperator-promoter was enhanced in a partial mutant of cbiE(encoding an enzyme preceding precorrin isomerase in B12biosynthesis), revealing that this pathway “inhibits”coaT expression. Disruption of coaT reduced cobalt tolerance and increased cytoplasmic 57Co accumulation. coaT-mediated restoration of cobalt tolerance has been used as a selectable marker.

Copper Homeostasis in Salmonella Is Atypical and Copper-CueP Is a Major Periplasmic Metal Complex

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.

Copper homeostasis in bacteria.

Els ts Kin A . T he properties of copper 217 B . C opper requiring proteins 218 C . P rinciples of copper homeostasis 219 II. M echanisms of Copper Trafficking and Resistance 220 A . P 1B-type ATPases 222 B . C opper acquisition 223 C . C opper detoxification 225 D . S ensors of elevated copper levels 231 E . C opper-chaperones 233 III. C opper and Bacterial Pathogenicity 234 IV. C opper as a Biocide 237 V. C oncluding Remarks 238 Refere nces 239

Bacterial metal-sensing proteins exemplified by ArsR–SmtB family repressors

Detecting deficiency and excess of different metal ions is fundamental for every organism. Our understanding of how metals are detected by bacteria is exceptionally well advanced, and multiple families of cytoplasmic DNA-binding, metal-sensing transcriptional regulators have been characterised(ArsR-SmtB, MerR, CsoR-RcnR, CopY, DtxR, Fur, NikR). Some of the sensors regulate a single gene while others act globally controlling transcription of regulons. They not only modulate the expression of genes directly associated with metal homeostasis, but can also alter metabolism to reduce the cellular demand for metals in short supply. Different representatives of each of the sensor families can regulate gene expression in response to different metals, and the residues that form the sensory metal-binding sites have been defined in a number of these proteins. Indeed, in the case of theArsR-SmtB family, multiple distinct metal-sensing motifs (and one non-metal-sensing motif) have been identified which correlate with the detection of different metals. This review summarises the different families of bacterial metal-sensing transcriptional regulators and discusses current knowledge regarding the mechanisms of metal-regulated gene expression and the structural features of sensory metal-binding sites focusing on the ArsR-SmtB family. In addition, recent progress in understanding the principles governing the ability of the sensors to detect specific metals within a cell and the coordination of the different sensors to control cellular metal levels is discussed.

Mycobacterial cells have dual nickel-cobalt sensors: sequence relationships and metal sites of metal-responsive repressors are not congruent.

A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterized from mycobacteria. Mutants of Mycobacterium tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays and fluorescence anisotropy show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. Incubation of KmtR with cobalt inhibits DNA complex assembly and metal-protein binding was confirmed. KmtR is the second, to NmtR, characterized ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, whereas NmtR retains a response to nickel and cobalt under these conditions. KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB-related proteins are listed in databases. KmtR has none of the previously defined metal-sensing sites. Substitution of His88, Glu101, His102, His110, or His111 with Gln generated KmtR variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory motifs revealing that only the latter should be used to predict metal sensing.

CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA

The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown. DeltacopA was hypersensitive to copper and contained more copper atoms cell(-1) than wild-type. Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export. A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance. Activity of copper-requiring cytochrome caa(3) oxidase was retained in deltacopZ and deltacopA. DeltacopZ was only slightly copper-hypersensitive but deltacopZ/deltacopA was more sensitive than deltacopA, implying some action of CopZ that is independent of CopA. Significantly, deltacopZ contained fewer copper atoms cell(-1) than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.

A Cadmium-Lead-sensing ArsR-SmtB Repressor with Novel Sensory Sites: COMPLEMENTARY METAL DISCRIMINATION BY NMTR AND CMTR IN A COMMON CYTOSOL.

We report a cadmium- and lead-detecting transcriptional repressor from Mycobacterium tuberculosis designated CmtR. Two genes were co-transcribed with cmtR, one encoding a deduced P1 type ATPase. Purified CmtR bound to the cmt operator-promoter, and repression of transcription was lost after introduction of a stop codon into cmtR. Assays of metal-dependent expression from cmt and nmt operator-promoters established that the metal specificity of CmtR in vivo was perfectly inverted relative to the nickel-cobalt sensor NmtR from the same organism, with CmtR totally insensitive to Co(II) or Ni(II) and NmtR totally insensitive to Cd(II) or Pb(II). Absorption spectroscopy of Cd(II)-, Co(II)-, and Ni(II)-substituted CmtR revealed S– to metal-charge-transfer which was absent in NmtR, providing diagnostic metal-difference spectra that discriminated between metal-binding to these two proteins. Ni(II)-binding isothermal titrations of CmtR are complex, with Kapp = 1.8 × 104 m–1 for site1, three orders of magnitude weaker than KNi for NmtR. Mixing equimolar apo-NmtR and apo-CmtR with 0.9 equivalents of Cd(II) gave Cd(II)-dependent difference spectra almost identical to Cd(II)0.9-CmtR. Thus, Cd(II) bound to CmtR in preference to NmtR, whereas the converse was true for Ni(II); this correlates faithfully with and provides a simplistic basis for metal-sensing preferences. In contrast, CmtR and NmtR had similar affinities for Co(II), and alternative explanations for Co(II) sensitivities are invoked. ArsR-SmtB repressors detect metals through derivatives of one or both of two possible allosteric sites at either carboxyl-terminal α5 helices or helix α3 proximal to the DNA-binding site. Unexpectedly, neither site was required for inducer recognition by CmtR. The mutants in potential metal ligands in, or near, these regions, Cys4, Cys35, Asp79, His81, Asp97, Asp99, Glu105, Glu111, and Glu114, retained both repression and inducer recognition. Crucially, substitution of Cys57, Cys61, and Cys102 with Ser revealed that each of these three residues is obligatory for Cd(II) detection, and this defines completely new sensory sites.

The copper supply pathway to a Salmonella Cu,Zn‐superoxide dismutase (SodCII) involves P1B‐type ATPase copper efflux and periplasmic CueP

Periplasmic Cu,Zn‐superoxide dismutases (Cu,Zn‐SODs) are implicated in bacterial virulence. It has been proposed that some bacterial P1B‐type ATPases supply copper to periplasmic cupro‐proteins and such transporters have also been implicated in virulence. Here we show that either of two P1B‐type ATPases, CopA or GolT, is needed to activate a periplasmic Cu,Zn‐SOD (SodCII) in Salmonella enterica serovar Typhimurium. A ΔcopA/ΔgolT mutant accumulates inactive Zn‐SodCII which can be activated by copper‐supplementation in vitro. In contrast, either single ATPase mutant accumulates fully active Cu,Zn‐SodCII. A contribution of GolT to copper handling is consistent with its copper‐responsive transcription mediated by DNA‐binding metal‐responsive activator GolS. The requirement for duplicate transcriptional activators CueR and GolS remains unclear since both have similar tight KCu. Mutants lacking periplasmic cupro‐protein CueP also accumulate inactive Zn‐SodCII and while CopA and GolT show functional redundancy, both require CueP to activate SodCII in vivo. Zn‐SodCII is also activated in vitro by incubation with Cu‐CueP and this coincides with copper transfer as monitored by electron paramagnetic resonance spectroscopy. These experiments establish a role for CueP within the copper supply pathway for Salmonella Cu,Zn‐SodCII. Copper binding by CueP in this pathogen may confer protection of the periplasm from copper‐mediated damage while sustaining vital cupro‐enzyme activity.