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.

MerR from Tn501 binds to a single site within the mer operator-promoter, and upon binding mercury positively regulates transcription of the mercury resistance operon (1,2). In the absence of mercury, MerR represses transcription (ϳ2fold). Several lines of evidence support a model in which mercury-MerR activates transcription by realigning abnormally spaced consensus RNA polymerase recognition sequences via underwinding the mer operator-promoter (3,4). Within the fully sequenced genome of the cyanobacterium Synechocystis PCC 6803 (5) is an ORF, 1 sll0794, herein designated coaR, encoding a predicted protein with some sequence similarity to MerR.
The amino-terminal one-third of CoaR, which aligns with MerR, is followed by a polypeptide with sequence similarity to precorrin isomerase (see Fig. 1), a methyl transferase involved in the synthesis of the cobalt-containing corrin ring of vitamin B 12 (6). Unlike Synechocystis PCC 6803, many organisms do not contain the genes for vitamin B 12 biosynthesis, and such organisms have no requirement for cobalt (7). Precorrin isomerase from Pseudomonas denitrificans is known to bind avidly to its product, hydrogenobyrinic acid, which consequently co-purifies with the enzyme (8), suggesting that a domain of CoaR interacts with hydrogenobyrinic acid.
Divergently transcribed from coaR is an ORF, slr0797, designated coaT, encoding a putative P-type ATPase (Fig. 1). CoaT has some sequence features of P 1 - (9) or CPx-type ATPases (10) but lacks an amino-terminal metal binding motif and, most significantly, contains a deduced intramembranous Ser-Pro-Cys motif rather than the characteristic Cys-Pro-Cys/His/Ser (CPx). Known CPx-type ATPases transport larger metal ions and include the cadmium transporter CadA, the yeast copper transporter CCC2, the human copper transporters MNK and WND, the bacterial copper transporters CtaA, PacS, CopA, and CopB (reviewed in Ref. 11), and the zinc transporter ZiaA from Synechocystis PCC 6803 (12) and ZntA from Escherichia coli (13,14). At present, it is not possible to predict which metal ion is transported in which direction, import or export, merely from the sequence of a CPx-type ATPase, but the divergent organization of coaR and coaT encourages the prediction that the product of the former regulates the latter.
Here we describe experiments that confirm that CoaR does bind to and activate expression from the coaT operator-promoter. The activating effector is shown to be cobalt, and CoaT is shown to confer cobalt resistance and exclusion. Following site-directed mutagenesis, it was revealed that a carboxylterminal Cys-His-Cys motif in CoaR is part of the cobalt-sensing site. A partial mutant in the vitamin B 12 biosynthetic pathway at a step preceding precorrin isomerase was generated. Enhanced expression from the coaT operator-promoter in this mutant indicates that this pathway inhibits coaT transcription and that CoaR responds to both activating and inhibitory effectors to attune cobalt export with fluctuations in cellular demand as well as with changing cobalt levels.

EXPERIMENTAL PROCEDURES
Bacterial Strains and DNA Manipulation-Synechocystis PCC 6803 was grown in liquid BG-11 medium (15) or on medium C plates with supplement A 5 (16) using previously described conditions (17). Cells were transformed to antibiotic resistance essentially as described by Hagemann and Zuther (18). E. coli strains JM101 or SURE (Stratagene) were grown in Luria-Bertani medium (19). Standard DNA manipulations were performed as described by Sambrook et al. (19).
A derivative of pLACOA was generated in which codon 10 of coaR was converted from GAA to an ochre stop codon. Primer 5Ј-CCCACT-GCATCTGTGAGTTAACTAATCGTTAAGTGATTAG-3Ј and its reverse complement were used for Quik Change (Stratagene) mutagenesis with pJRJC1.1 as template, creating pJRJC2.1, and the coa sequences were then subcloned into the BamHI/SalI site of pLACPB2 to create pLACOA-OCH.
Two derivatives of pLACOA and pLACOA-OCH were generated with 1 or 2 nucleotides (␦1 or ␦2, see Fig. 7A) removed from the coaT operator-promoter. pJRJC1.1 and pJRJC2.1 were used as templates for Quik Change with primer 5Ј-CCTTCTCAGCCTAACCTTAACATTAGT-GTCAATGTC-3Ј and its reverse complement for the ␦1 deletion, or 5Ј-GACATTGACACTAATGTTAAGGTAGGCTGAGAAGG-3Ј and its reverse complement for ␦2, and the coa sequences were subcloned into the BamHI/SalI site of pLACPB2.
A further derivative of pLACOA was generated in which codons 363 to 365 of coaR, encoding Cys-His-Cys, were converted to encode Ser-Asn-Ser. Primer 5Ј-CATTGATTGCAAAGCCAGATTCGAATTCCTATC-TCACTTGTCTTTAGTGC-3Ј and its reverse complement were used for Quik Change with pJRJC1.1 as template, and the coa sequences were subcloned into the BamHI/SalI site of pLACPB2.
Integration of a coa-lacZ Fusion, or lacZ Alone, into the Synechocystis PCC 6803 Genome-Plasmid pCSCM2 facilitates the integration of translational fusions to lacZ into the Synechocystis PCC 6803 genome (within ORF slr0168) (21). The SacI/PstI fragment from pCSCM2 (containing the truncated 5Ј end of lacZ) was replaced with the SacI/PstI fragment from pLACOA to generate pJRNR1.1, containing the entire lacZ coding region and Shine-Dalgarno motif. The coa sequences from pJRJC1.1 were subcloned into the PstI/SalI site of pJRNR1.1 to create a transcriptional fusion to lacZ. The resulting plasmid, pJRNR1.2, was used to transform Synechocystis PCC 6803 to kanamycin resistance, generating strain JRNR1.2. JRNR1.2 showed no difference in cobalt tolerance to wild type. As a control, lacZ with no associated coa sequences was introduced into Synechocystis PCC 6803 via transformation with pCSCM2 containing full-length lacZ derived from pLACPB2.
␤-Galactosidase Assays-Synechocystis PCC 6803 cultures (final A 595 of 0.18 to 0.35) were exposed (ϳ20 h) to a range of metal ions under standard growth conditions except where stated otherwise. Overnight cultures of E. coli were diluted 100-fold in fresh medium supplemented with a range of metal ions and grown to an A 595 of 0.2 to 0.5. Assays (22) were carried out in triplicate and performed on at least three separate occasions (nine analyses).
Insertional Inactivation of coaT-Synechocystis PCC 6803 genomic DNA was used as a template for PCR with primers 1 and 5Ј-GAAGAAT-TCTAACAGGGCTTAGAGCGTG-3Ј, and the amplification product (3.3 kb), containing coaR and coaT, was ligated to pGEM-T (Promega) to create pJRNR2.1. A 1.3-kb BamHI fragment of pUK4K (Amersham Pharmacia Biotech) containing a kanamycin resistance gene was ligated to the EcoNI site of pJRNR2.1 (within coaT) to create pIN-COAT. pIN-COAT transformants of Synechocystis PCC 6803, designated Synechocystis PCC 6803(coaT), were selected on solid medium containing 20 g ml Ϫ1 kanamycin before growth in liquid medium containing 50 g ml Ϫ1 kanamycin. pJRNR2.1 was used to reintroduce coaT into the chromosome of Synechocystis PCC 6803(coaT), and transformants were selected on medium supplemented with 10 M cobalt (no kanamycin).
Insertional Inactivation of cbiE-Synechocystis PCC 6803 genomic DNA was used as template for PCR with primers 3 (5Ј-GAAGAAT-TCTAGCTTCCGGTGATCC-3Ј) and 5Ј-GAAGAATTCGATCGCCACT-GACC-3Ј, and the amplification product (0.6 kb), containing part of cbiE (ORF sll0099), was ligated to pGEM-T creating pJRNR3.1. pSU19 (23) was used as a template for PCR with primers 5Ј-GAAGATATCGTA-AGTTGGCAGC-3Ј and 5Ј-GAAGATATCGGCACCAATAACTG-3Ј, and the amplification product (0.9 kb) containing the chloramphenicol resistance gene cat was ligated to pGEM-T to create pJRNR3.2. An EcoRV fragment of pJRNR3.2 containing cat was then ligated to the HindIII site of pJRNR3.1 (within cbiE) to create pJRNR3.3, which was used to transform JRNR1.2 to chloramphenicol resistance (7.5 g ml Ϫ1 ). The genotype of transformants was checked by PCR using primers 3 and 5Ј-GATTAACCGTTGACCAGCGCTAG-3Ј, which anneal to cbiE sequences flanking the site of cat insertion. The detected products revealed transformants to be merodiploid, with some chromosomes retaining cbiE and others containing cat within cbiE, with analogy to previous attempts to disrupt an essential cyanobacterial gene (24).
Analyses of Metal Tolerance and Cobalt Compartmentalization-Logarithmically growing cells were subcultured daily (to ϳ1 ϫ 10 6 cells ml Ϫ1 ) for a minimum of 7 days before analyses (to standardize growth rates). Growth of cultures in metal-supplemented BG-11 medium was examined as described (17). To examine cobalt accumulation, cultures (ϳ5 ϫ 10 8 cells) were pelleted, resuspended in 1 ml of fresh BG-11 medium supplemented with 2 M cobalt and 1 kBq of 57 Co, and incubated for 1 h under standard growth conditions. 57 Co-exposed cells were pelleted and washed twice with fresh medium, and the periplasmic contents were extracted into two osmotic shock fractions (25). Assays were carried out in triplicate, and 57 Co compartmentalization was examined on three separate occasions.
Production and Purification of Recombinant CoaR-CoaR and mutants thereof were expressed from the coaR operator-promoter in E. coli cells containing pLACOA (or derivatives described above). In addition, recombinant CoaR was also generated in E. coli as a fusion to glutathione S-transferase.
Synechocystis PCC 6803 DNA was used as template for PCR with primers 1 and 5Ј-GAAGGATCCGGATGAAGACTAATCACTTAACG-3Ј. The amplification product (1.1 kb), containing coaR, was ligated to pGEM-T before subcloning into the BamHI/SmaI site of the glutathione S-transferase gene fusion vector pGEX-3X (Amersham Pharmacia Biotech) to create pGEXCoaR. Recombinant fusion protein was expressed in E. coli (JM101) and purified according to manufacturer's protocols. Extracts from E. coli cells containing pGEXCoaR were fractionated on glutathione Sepharose 4B, and a single protein of ϳ69.6 kDa, corresponding to the predicted size of glutathione S-transferase-CoaR, was detected in fractions containing 5 mM glutathione. Incubation of recombinant protein overnight with factor Xa released a smaller protein corresponding to the predicted size (40.6 kDa) of CoaR plus three residues of glutathione S-transferase. In some preparations a further fragment was also detected, presumed to result from internal cleavage within CoaR; the factor Xa and incubation time were optimized to minimize this.
Gel Retardation Assays-Assays were performed with 0.5 mM spermidine in the binding buffer as described previously (26). Samples were loaded onto 5% polyacrylamide gels and electrophoresed using Trisborate-EDTA (19) buffer. A 77-bp BamHI/EcoRI DNA fragment containing the coa operator-promoter was used as probe. This fragment was released from pSK ϩ containing the PCR product generated using primers 2 and 5Ј-GAAGGATCCCTTTAGTTTACTC-3Ј with pJRJC1.1 as template.

RESULTS
Transcription from the coaT Operator-promoter in Synechocystis PCC 6803 Is Maximally Induced by Cobalt-To identify which, if any, metal ions repress or induce transcription from the coaT operator-promoter, 1.2 kb from upstream of coaT (including the coaT operator-promoter and coaR) were fused to a promoterless lacZ gene to generate plasmid pJRNR1.2. The transcriptional fusion in plasmid pJRNR1.2 is flanked by sequences from Synechocystis PCC 6803, which facilitated integration by homologous recombination into a remote chromosomal site to generate strain JRNR1.2. After exposure to biologically significant concentrations of various metal ions, maximum induction of ␤-galactosidase activity was observed The predicted translational products (larger boxes) of coaT (thin black box) and the divergently transcribed coaR (thin shaded box) are shown above and below the genes, respectively. CoaT contains eight predicted trans-membrane domains (black), an intramembranous Ser-Pro-Cys (SPC) motif within the sixth trans-membrane domain, two larger intracellular loops (larger white blocks) and a shorter aminoterminal intracellular region with no metal binding motif. The first 145 residues of CoaR align (20% identity) with MerR (shaded), residues 174 to 358 align (32% identity) with precorrin isomerase (diagonal lines), and in addition, CoaR contains a carboxyl-terminal Cys-His-Cys (CHC) motif. Hydrogenobyrinic acid (oval) associates with precorrin isomerase (7). with elevated cobalt (Fig. 2). A greater than 10-fold reduction in ␤-galactosidase activity was observed when cells were cultured in modified BG-11 medium devoid of micronutrient (0.15 M) cobalt, and the consequent response to cobalt was enhanced (Fig. 2B). No induction of ␤-galactosidase activity was detected using control cells containing lacZ alone (Fig. 2B). 57 Co in the Cytoplasm-The observation that elevated cobalt enhances transcription from the coaT operatorpromoter suggests that coaT may export, and confer resistance to, cobalt. Mutants, Synechocystis PCC 6803(coaT), with disrupted coaT were generated by integration of plasmid pIN-COAT, which contains coaT interrupted by a kanamycin resistance gene. Growth of Synechocystis PCC 6803(coaT) and wild type was tested in multiple liquid cultures supplemented with a range of levels of cobalt, cadmium, copper, mercury, nickel, silver, and zinc to determine maximum permissive concentrations (data not shown). Only resistance to cobalt appeared to be reduced in Synechocystis PCC 6803(coaT). Subsequently, growth was examined as a function of time in response to selected concentrations of cobalt and three metals, which are known to be transported by CPx-type ATPases (Fig. 3A). Again, only resistance to cobalt was reduced. Restoration of cobalt tolerance was also used as a selectable marker to identify mutants of Synechocystis PCC 6803(coaT) in which coaT had reintegrated into the chromosome by homologous recombination. The genotypes of Synechocystis PCC 6803(coaT) and the mutant with reintegrated coaT were confirmed by Southern analysis; the band of lower M r represents hybridization to coaR on a smaller fragment, due to the disruption of coaT introducing an additional restriction site (Fig. 3B). Fig. 3C shows the phenotypes of Synechocystis PCC 6803(coaT), wild type and cells with coaT reintroduced into the chromosome, on agar plates.

Mutants of Synechocystis PCC 6803 with a Disrupted coaT Gene Have Reduced Tolerance to Cobalt and Increased Accumulation of
Synechocystis PCC 6803(coaT) and wild type cells were exposed for 1 h to 1 kBq of 57 Co in medium containing 2 M cobalt. More 57 Co was located in the cytoplasm of Synechocystis PCC 6803(coaT) compared with wild type cells (Table I), with equivalent observations being made on two further occasions (data not shown). The disruption of coaT impairs the exclusion of cobalt from the cytoplasm.
CoaR Binds to the coa Operator-promoter-A single complex formed between the coa operator-promoter and extracts from Synechocystis PCC 6803 (Fig. 4). Fig. 5B confirms that a single complex is also formed between the coa operator-promoter and total protein from E. coli cells containing pLACOA, whereas, most importantly, this complex is absent when protein is used from cells containing pLACOA-OCH (pLACOA containing a stop codon within the coaR ORF). The complex remains stable in reactions containing 0.1 g l Ϫ1 of poly(dI-dC)⅐poly(dI-dC) competitor DNA (Figs. 4 and 5B). This represents a 1 ϫ 10 5 -fold excess of nonspecific competitor DNA to coa probe DNA and  establishes the specificity of the complex. A similarly migrating complex was also detected with purified recombinant CoaR along with faster migrating complexes, attributed to internal factor Xa cleavage within CoaR (data not shown).
CoaR Is a Cobalt-dependent Activator in E. coli-In all media, other than that supplemented with cobalt, E. coli cells containing pLACOA show reduced (ϳ50%) ␤-galactosidase activity compared with cells containing pLACOA-OCH, with a stop codon introduced within coaR (Fig. 5A). Cells containing pLACOA show highly elevated ␤-galactosidase activity in media supplemented with maximum noninhibitory concentrations of cobalt, and under these conditions, activity in these cells is substantially greater than that observed in cells containing pLACOA-OCH. CoaR activates expression from the coaT operator-promoter in the presence of cobalt, whereas in other conditions, it mediates some repression. E. coli is unable to synthesize the corrin ring of vitamin B 12 (27), the relevant genes being absent. Thus, intermediates in this pathway (e.g. hydrogenobyrinic acid) cannot be required for cobalt-dependent positive regulation by CoaR.
Identification of Cobalt-sensing Residues in CoaR-The carboxyl-terminal 12 residues of CoaR are unlike MerR or precorrin isomerase and include the sequence Cys-His-Cys. Such residues can form metal-thiolate and metal-imidazole bonds and were therefore hypothesized to coordinate cobalt in CoaR. To test whether this motif is required for cobalt-sensing, a variant of pLACOA was generated in which codons 363 and 365, encoding Cys, were converted to encode Ser, whereas codon 364 was converted to encode Asn. Extracts from E. coli cells containing this construct showed equivalent retardation of the coa operator-promoter as extracts containing nonmutant CoaR (Fig. 5B), confirming that the mutant C363S/H364N/ C365S protein is synthesized and can bind to DNA. In the absence of added cobalt, ␤-galactosidase activity in cells containing mutant CoaR was less than in cells containing pLA-COA-OCH (data not shown) and similar to that observed in cells containing pLACOA (Fig. 6), confirming that the mutant C363S/H364N/C365S protein reduces basal expression from the coaT operator-promoter. Most importantly, cobalt-dependent activation was absent in cells containing the mutant CoaR, revealing that the carboxyl-terminal Cys-His-Cys motif is indeed required for cobalt sensing.
Deletions within the coaT Operator-Promoter Enhance Transcription-Known proteins that share sequence similarity to MerR from Tn501 include mercury sensors from other sources (28), the redox sensor SoxR (29), the thiostrepton sensor TipA L (30), BmrR and BltR from Bacillus subtilis (31), and NolA from Bradyrhizobium japonicum (32). These proteins are known, or predicted, to associate with promoters in which consensus Ϫ10 and Ϫ35 elements are separated by 19 or 20 bp rather than 16 to 18 bp. The removal of nucleotides from between such elements revealed that suboptimal spacing is essential for normal regulation of mer transcription, with nucleotide deletions leading to constitutive enhanced expression (33). By analogy, 20 bp separate near consensus Ϫ10 and Ϫ35 sequences in the coaT operator-promoter region (Fig. 7A). A degenerate (1 bp mismatch in 13) hyphenated (6 bp) inverted repeat (13-6-13) (Fig.  7A) in this region contains candidate nucleotides for CoaR binding. To test the importance of suboptimal spacing for regulation of transcription from the coaT operator-promoter, variants of constructs pLACOA and pLACOA-OCH were created in which either 1 (␦1) or 2 (␦2) bp were deleted (Fig. 7A). A single complex with the coaT operator-promoter was detected using extracts from these cells (Fig. 5B). The ␦2 construct conferred highly elevated constitutive ␤-galactosidase activity (Fig. 7B). Elevated expression was also observed with the ␦1 construct, although it was notable that under these conditions the presence of CoaR was inhibitory even in the presence of cobalt. Shortening of the promoter spacing similarly converted the MerR-like redox sensor SoxR from an activator into a repressor regardless of the presence of inducer (34). It is proposed that CoaR functions in an analogous manner to MerR but remodels its target promoter in response to elevated cobalt rather than mercury.
Disruption of cbiE Enhances Transcription from the coaT Operator-Promoter-To test the proposal that interaction be- FIG. 4. In vitro analysis of CoaR binding to the coa operatorpromoter. Gel retardation assays were performed by using ϳ3 fmol of 32 P-labeled 77-bp coa operator-promoter (FP, free probe) incubated with ϳ12 g of protein extract from Synechocystis PCC 6803. Nonspecific competitor poly(dI-dC)⅐poly(dI-dC) was added to binding reactions at the indicated concentrations. tween the precorrin isomerase-like domain of CoaR and intermediates in the vitamin B 12 biosynthetic pathway modulates expression from the coaT operator-promoter, ␤-galactosidase activity was examined in a mutant of strain JRNR1.2 in which the cbiE gene was insertionally inactivated on a proportion of chromosomes. ␤-Galactosidase activity was elevated in the cbiE mutant compared with JRNR1.2 (Fig. 8A), revealing that the vitamin B 12 pathway mediates repression of transcription from the coaT operator-promoter. DISCUSSION Cobalt-transporting CPx-type ATPases have not previously been described. Several lines of evidence indicate that CoaT exports this metal ion. Cobalt is the most potent inducer of transcription from the coaT operator-promoter (Figs. 2 and 5A), insertional inactivation of coaT reduces tolerance to cobalt (Fig.  3A), restoration of cobalt tolerance by coaT can be used as a selectable marker (Fig. 3B), and cells lacking functional coaT have increased accumulation of 57 Co in the cytoplasm (Table I). Eight trans-membrane ␣-helices are predicted for CoaT (Fig.  1), and a Ser-Pro-Cys motif is located in the sixth helix, 42 residues away from a deduced aspartyl kinase site (DKTGT). This is normally the location of the CPx motif, which is thought to associate with larger metal ions during membrane transit, whereas alternative residues flank the conserved proline within this motif in transporters of alkali and alkaline earth metals (10). The SPC-variant motif in CoaT may contribute toward specificity for cobalt. It is known that a metallochaperone can interact with, and donate metal ions to, the aminoterminal metal binding motifs of a CPx-type ATPase (35), and the absence of such sequences in CoaT implies an absence of analogous cobalt transfer.
Association of CoaR with the coaT operator-promoter influences transcriptional activity both negatively, in the absence, and positively, in the presence, of elevated concentrations of cobalt, respectively (Fig. 5). A 20-bp spacing between consensus promoter elements impairs expression from the coaT operatorpromoter and is essential for positive regulation by CoaR (Fig.  7). Deformation of the coaT operator-promoter by cobalt-CoaR, compensating for abnormal spacing, is the inferred mechanism of transcriptional switching. The possibility that activated SoxR/MerR may also interact directly with RNA polymerase has not been eliminated (29), although for cobalt-CoaR, such an interaction would imply a capacity to recognize both Synechocystis PCC 6803 and E. coli RNA polymerase. Sequence similarity to precorrin isomerase initially suggested that CoaR may be solely effected by intermediates in vitamin B 12 biosynthesis, but activation in E. coli (Fig. 5) supports direct interaction with cobalt. Retention of repression, but loss of cobalt-dependent activation (Fig. 6), in a Ser-Asn-Ser mutant of the carboxylterminal Cys-His-Cys motif of CoaR implicates cobalt association with thiol and/or imidazole groups of one or more of these residues in transcriptional switching.
The apparent affinity of mercury-MerR for the mer operatorpromoter is 4-fold less than apo-MerR (4), and this will favor the replacement of mercury-MerR with apo-MerR and, hence, deactivation following removal of cellular mercury. It is notable that a pair of His residues (residues 26 and 28) are located within the deduced helix-turn-helix DNA binding region of CoaR, and it is formally possible that these constitute part of a site that reduces DNA association upon metal binding with some analogy to a model for metal-mediated DNA dissociation by ArsR/SmtB/CadC/ZiaR-like metal-responsive repressors (12, 36 -38). Unlike mercury, cobalt is essential (at least in some organisms), and after deactivation of export, some cellular cobalt may be retained.
The action of cbiE reduces expression from the coaT operator-promoter (Fig. 8A). In a cbiE mutant there will be a reduction in levels of substrate for precorrin isomerase (precorrin-8x) and product (hydrogenobyrinic acid) when enzymes are sub- were grown with no metal supplement or with added Co 2ϩ (1 M) for ϳ20 h immediately before assay. Panels B to E, the proposed mechanism of action of CoaR in Synechocystis PCC 6803. CoaR is shown as two circles, representing the MerR (M) and precorrin isomerase (P)-like domains, with the Cys-His-Cys motif (CHC) at the carboxyl terminus. In low cobalt, CoaR associates with and represses transcription from the coaT operator-promoter (B). In elevated concentrations of cobalt, cobalt binding to the allosteric site (involving the Cys-His-Cys motif) of CoaR effects a conformational change in CoaR (filled symbols) and deformation of the coaT operator-promoter to realign the abnormally spaced promoter elements and activate transcription of coaT triggering cobalt efflux into the periplasm (C). When there is a metabolic requirement for cobalt, hydrogenobyrinic acid (or some other component of the vitamin B 12 biosynthetic pathway) binds to CoaR to inhibit activation of coaT transcription (D). Upon saturation of periplasmic cobalt stores, it is anticipated that a czc/cnr-like operon adjacent to coaR mediates cobalt export across the outer membrane (E). strate limited. In P. denitrificans it is known that hydrogenobyrinic acid precedes the step of cobalt insertion into the corrin ring (6) and is predicted to accumulate when there is insufficient cobalt for vitamin B 12 biosynthesis. An inhibition of CoaT production when hydrogenobyrinic acid accumulates will restrict cobalt export when there is cellular demand (Fig. 8D). Thus, via responses to two effectors, (i) cobalt (positive effector) and (ii) intermediates in the vitamin B 12 pathway (negative effector), CoaR integrates cobalt homeostasis with metabolism. Enzyme recruitment (39) is exemplified by the evolution of the latter response. It is predicted that binding of hydrogenobyrinic acid to the precorrin isomerase domain of CoaR prevents cobalt-mediated conformational change required for activation, possibly occluding the cobalt binding site. Adjacent to the coa divergon in Synechocystis PCC 6803 is a deduced operon, starting with ORF slr0793, with similarity to cnr and czc operons, both of which mediate export of metal ions, including cobalt, across the inner and outer membranes (40). Does transport by CoaT facilitate storage of excess cytoplasmic cobalt in the periplasm while the adjacent genes mediate export across the outer membrane upon saturation of periplasmic stores (Fig.  8E)?
It is now apparent that CoaR senses cobalt (Fig. 5) and some of the residues involved in cobalt sensing have been identified (Fig. 6). During the course of this work, a MerR-like protein from E. coli has been shown to activate transcription from the zntA operator-promoter in response to zinc (41), and a similar activity suggested for a homologue from Proteus mirabilis (42). It will be intriguing to determine how/if responses of ZntR are modified coincident with fluctuating requirements for zinc. Clearly there is a subfamily of MerR-like proteins that switch transcription in response to metal ions, mercury, cobalt, and zinc sensors having now been identified. It is probable that there are further members specific for other metals, which await discovery. It is also now apparent that CoaT is a cobalttransporting variant CPx-type ATPase, adding to the catalogue of resistances (cadmium, copper, zinc, and lead) known to be mediated by these proteins. The next challenge will be to understand metal-specificity.