FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019780345> ?p ?o ?g. }

• W2019780345 endingPage "25832" @default.
• W2019780345 startingPage "25827" @default.
• W2019780345 abstract "CoaR associates with and confers cobalt-dependent activation of the coaT operator-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 coaT operator-promoter but abolished activation by cobalt-CoaR. It is inferred that cobalt effects a transition in CoaR that underwinds the coaT 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 coaT operator-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. CoaR associates with and confers cobalt-dependent activation of the coaT operator-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 coaT operator-promoter but abolished activation by cobalt-CoaR. It is inferred that cobalt effects a transition in CoaR that underwinds the coaT 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 coaT operator-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. open reading frame base pair(s) kilobase pair(s) polymerase chain reaction 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 (1Lund P.A. Ford S.J. Brown N.L. J. Gen. Microbiol. 1986; 132: 465-480PubMed Google Scholar, 2O'Halloran T.V. Frantz B. Shin M.K. Ralston D.M. Wright J.G. Cell. 1989; 56: 119-129Abstract Full Text PDF PubMed Scopus (196) Google Scholar). In the absence of mercury, MerR represses transcription (∼2-fold). 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 (3Ansari A.Z. Chael M.L. O'Halloran T.V. Nature. 1992; 355: 87-89Crossref PubMed Scopus (138) Google Scholar, 4Parkhill J. Ansari A.Z. Wright J.G. Brown N.L. O'Halloran T.V. EMBO J. 1993; 12: 413-421Crossref PubMed Scopus (56) Google Scholar). Within the fully sequenced genome of the cyanobacterium Synechocystis PCC 6803 (5Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Sugiura M. Sasamoto S. Kimura T. et al.DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2133) Google Scholar) 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 B12 (6Roth J.R. Lawrence J.G. Bobik T.A. Rev. Microbiol. 1996; 50: 137-181Crossref Scopus (437) Google Scholar). Unlike Synechocystis PCC 6803, many organisms do not contain the genes for vitamin B12 biosynthesis, and such organisms have no requirement for cobalt (7Fraústo da Silva J.J.R. Williams R.J.P. The Biological Chemistry of the Elements: The Inorganic Chemistry of Life. Clarendon Press, Oxford1993Google Scholar). Precorrin isomerase from Pseudomonas denitrificans is known to bind avidly to its product, hydrogenobyrinic acid, which consequently co-purifies with the enzyme (8Thibaut D. Couder M. Famechon A. Debussche L. Cameron B. Crouzet J. Blanche F. J. Bacteriol. 1992; 174: 1043-1049Crossref PubMed Google Scholar), 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 P1- (9Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar) or CPx-type ATPases (10Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (418) Google Scholar) 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. 11Silver S. Phung L.T. Annu. Rev. Microbiol. 1996; 50: 753-789Crossref PubMed Scopus (1063) Google Scholar), and the zinc transporter ZiaA from Synechocystis PCC 6803 (12Thelwell C. Robinson N.J. Turner-Cavet J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10728-10733Crossref PubMed Scopus (156) Google Scholar) and ZntA from Escherichia coli (13Beard S.J. Hashim R. Membrillo-Hernández J. Hughes M.N. Poole R.K. Mol. Microbiol. 1997; 25: 883-891Crossref PubMed Scopus (160) Google Scholar, 14Rensing C. Sun Y. Mitra B. Rosen B.P. J. Biol. Chem. 1998; 273: 32614-32617Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). 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 carboxyl-terminal Cys-His-Cys motif in CoaR is part of the cobalt-sensing site. A partial mutant in the vitamin B12 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. Synechocystis PCC 6803 was grown in liquid BG-11 medium (15Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) or on medium C plates with supplement A5 (16Kratz W.A. Myers J. Am. J. Bot. 1995; 42: 282-287Crossref Google Scholar) using previously described conditions (17Turner J.S. Morby A.P. Whitton B.A. Gupta A. Robinson N.J. J. Biol. Chem. 1993; 268: 4494-4498Abstract Full Text PDF PubMed Google Scholar). Cells were transformed to antibiotic resistance essentially as described by Hagemann and Zuther (18Hagemann M. Zuther E. Arch. Microbiol. 1992; 158: 429-434Crossref Scopus (41) Google Scholar). E. coli strains JM101 or SURE (Stratagene) were grown in Luria-Bertani medium (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Standard DNA manipulations were performed as described by Sambrook et al. (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Synechocystis PCC 6803 genomic DNA, isolated as described previously (17Turner J.S. Morby A.P. Whitton B.A. Gupta A. Robinson N.J. J. Biol. Chem. 1993; 268: 4494-4498Abstract Full Text PDF PubMed Google Scholar), was used as a template for PCR with primers 1 (5′- GAACCCGGGCACTAAAGACAAGTGAG-3′) and 2 (5′-GAAGAATTCTGGATTTTTACCTTCTCAGCC-3′). The amplification product (1.2 kb) containing coaR and the coaT operator-promoter was ligated into the Hin cII/Eco RI site of pSK+(Stratagene) to create pJRJC1.1, then subcloned into the Bam HI/Sal I site of pLACPB2 (20Scanlan D.J. Bloye S.A. Mann N.H. Hodgson D.A. Carr N.G. Gene. 1990; 90: 43-49Crossref PubMed Scopus (47) Google Scholar) to create pLACOA. A derivative of pLACOA was generated in which codon 10 of coaR was converted from GAA to an ochre stop codon. Primer 5′-CCCACTGCATCTGTGAGTTAACTAATCGTTAAGTGATTAG-3′ 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 Bam HI/Sal I site of pLACPB2 to create pLACOA-OCH. Two derivatives of pLACOA and pLACOA-OCH were generated with 1 or 2 nucleotides (δ1 or δ2, see Fig. 7 A) removed from the coaT operator-promoter. pJRJC1.1 and pJRJC2.1 were used as templates for Quik Change with primer 5′-CCTTCTCAGCCTAACCTTAACATTAGTGTCAATGTC-3′ and its reverse complement for the δ1 deletion, or 5′-GACATTGACACTAATGTTAAGGTAGGCTGAGAAGG-3′ and its reverse complement for δ2, and the coa sequences were subcloned into the Bam HI/Sal I 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 5′-CATTGATTGCAAAGCCAGATTCGAATTCCTATCTCACTTGTCTTTAGTGC-3′ and its reverse complement were used for Quik Change with pJRJC1.1 as template, and the coa sequences were subcloned into the Bam HI/Sal I site of pLACPB2. Plasmid pCSCM2 facilitates the integration of translational fusions to lac Z into the Synechocystis PCC 6803 genome (within ORF slr0168) (21Milkowski C. Quinones A. Hagemann M. Curr. Microbiol. 1998; 37: 108-116Crossref PubMed Scopus (8) Google Scholar). The Sac I/Pst I fragment from pCSCM2 (containing the truncated 5′ end of lacZ) was replaced with the Sac I/Pst I 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 Pst I/Sal I 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. Synechocystis PCC 6803 cultures (final A595 of 0.18 to 0.35) were exposed (∼20 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 A595of 0.2 to 0.5. Assays (22Morby A.P. Turner J.S. Huckle J.W. Robinson N.J. Nucleic Acids Res. 1993; 21: 921-925Crossref PubMed Scopus (137) Google Scholar) were carried out in triplicate and performed on at least three separate occasions (nine analyses). Synechocystis PCC 6803 genomic DNA was used as a template for PCR with primers 1 and 5′-GAAGAATTCTAACAGGGCTTAGAGCGTG-3′, 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 Bam HI fragment of pUK4K (Amersham Pharmacia Biotech) containing a kanamycin resistance gene was ligated to the Eco NI 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 μg ml−1 kanamycin before growth in liquid medium containing 50 μg ml−1 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 μm cobalt (no kanamycin). Synechocystis PCC 6803 genomic DNA was used as template for PCR with primers 3 (5′-GAAGAATTCTAGCTTCCGGTGATCC-3′) and 5′-GAAGAATTCGATCGCCACTGACC-3′, and the amplification product (0.6 kb), containing part of cbiE (ORF sll0099), was ligated to pGEM-T creating pJRNR3.1. pSU19 (23Martinez E. Bartolome B. de la Cruz F. Gene. 1988; 68: 159-162Crossref PubMed Scopus (253) Google Scholar) was used as a template for PCR with primers 5′-GAAGATATCGTAAGTTGGCAGC-3′ and 5′-GAAGATATCGGCACCAATAACTG-3′, and the amplification product (0.9 kb) containing the chloramphenicol resistance gene cat was ligated to pGEM-T to create pJRNR3.2. An Eco RV fragment of pJRNR3.2 containing cat was then ligated to the Hin dIII site of pJRNR3.1 (within cbiE) to create pJRNR3.3, which was used to transform JRNR1.2 to chloramphenicol resistance (7.5 μg ml−1). The genotype of transformants was checked by PCR using primers 3 and 5′-GATTAACCGTTGACCAGCGCTAG-3′, 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 (24Bird A.J. Turner-Cavet J.S. Lakey J.H. Robinson N.J. J. Biol. Chem. 1998; 273: 21246-21252Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Logarithmically growing cells were subcultured daily (to ∼1 × 106 cells ml−1) 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 (17Turner J.S. Morby A.P. Whitton B.A. Gupta A. Robinson N.J. J. Biol. Chem. 1993; 268: 4494-4498Abstract Full Text PDF PubMed Google Scholar). To examine cobalt accumulation, cultures (∼5 × 108 cells) were pelleted, resuspended in 1 ml of fresh BG-11 medium supplemented with 2 μm cobalt and 1 kBq of 57Co, and incubated for 1 h under standard growth conditions. 57Co-exposed cells were pelleted and washed twice with fresh medium, and the periplasmic contents were extracted into two osmotic shock fractions (25Ames G.F.-L. Methods Enzymol. 1994; 235: 234-241Crossref PubMed Scopus (12) Google Scholar). Assays were carried out in triplicate, and 57Co compartmentalization was examined on three separate occasions. 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 5′-GAAGGATCCGGATGAAGACTAATCACTTAACG-3′. The amplification product (1.1 kb), containing coaR, was ligated to pGEM-T before subcloning into the Bam HI/Sma I 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 ∼69.6 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. Assays were performed with 0.5 mm spermidine in the binding buffer as described previously (26Turner J.S. Glands P.D. Samson A.C.R. Robinson N.J. Nucleic Acids Res. 1996; 24: 3714-3721Crossref PubMed Scopus (86) Google Scholar). Samples were loaded onto 5% polyacrylamide gels and electrophoresed using Tris-borate-EDTA (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) buffer. A 77-bp Bam HI/Eco RI DNA fragment containing the coa operator-promoter was used as probe. This fragment was released from pSK+ containing the PCR product generated using primers 2 and 5′-GAAGGATCCCTTTAGTTTACTC-3′ with pJRJC1.1 as template. 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 β-galactosidase activity was observed with elevated cobalt (Fig.2). A greater than 10-fold reduction in β-galactosidase activity was observed when cells were cultured in modified BG-11 medium devoid of micronutrient (0.15 μm) cobalt, and the consequent response to cobalt was enhanced (Fig.2 B). No induction of β-galactosidase activity was detected using control cells containing lacZ alone (Fig.2 B). The observation that elevated cobalt enhances transcription from the coaT operator-promoter 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. 3 A). 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 Mr represents hybridization to coaR on a smaller fragment, due to the disruption of coaT introducing an additional restriction site (Fig. 3 B). Fig.3 C shows the phenotypes of Synechocystis PCC 6803(coaT), wild type and cells with coaT reintroduced into the chromosome, on agar plates. Synechocystis PCC 6803(coaT) and wild type cells were exposed for 1 h to 1 kBq of 57Co in medium containing 2 μm cobalt. More 57Co 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.Table ICompartmentalization of cobalt in wild type and Synechocystis PCC 6803(coaT)FractionAccumulated cobaltWild typeSynechocystis PCC 6803(coaT)pmol cobalt mg protein−1Cytoplasm442 ± 17656 ± 26Periplasm817 ± 31658 ± 31EDTA wash5290 ± 2904944 ± 85The means and S.D. of triplicate estimations of the amount of cobalt in the cytoplasm, periplasm (osmotic shock fraction), and EDTA wash fraction (before osmotic shock) of cells exposed to 2 μmCo2+ for 1 h. Open table in a new tab The means and S.D. of triplicate estimations of the amount of cobalt in the cytoplasm, periplasm (osmotic shock fraction), and EDTA wash fraction (before osmotic shock) of cells exposed to 2 μmCo2+ for 1 h. A single complex formed between the coa operator-promoter and extracts from Synechocystis PCC 6803 (Fig.4). Fig.5 B 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 μg μl−1 of poly(dI-dC)·poly(dI-dC) competitor DNA (Figs. 4 and 5 B). This represents a 1 × 105-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).Figure 5Metal-induced expression from the coaT operator-promoter in E. coli. Panel A, E. coli (JM101) containing pLACOA (filled bars) or pLACOA-OCH (open bars) were grown with no metal supplement or in the presence of maximum noninhibitory concentrations of Ag+ (50 μm), Cd2+ (0.1 mm), Co2+ (0.1 mm), Cu2+ (0.8 mm), Hg2+ (4 μm), Ni2+ (0.8 mm), or Zn2+ (0.1 mm). Panel B, gel retardation assays were performed using ∼2 fmol of32P-labeled 77-bp coa operator-promoter as probe with 0.1 μg μl−1 poly(dI-dC)·poly(dI-dC) and ∼6 μg of protein extract from E. coli containing pLACOA-OCH (lane 1), pLACOA (lane 2), a variant of pLACOA with mutated (C363S/H364N/C365S) coaR (lane 3), or variants of pLACOA with the δ1 (lane 4) or δ2 (lane 5) deletions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In all media, other than that supplemented with cobalt, E. coli cells containing pLACOA show reduced (∼50%) β-galactosidase activity compared with cells containing pLACOA-OCH, with a stop codon introduced within coaR (Fig.5 A). Cells containing pLACOA show highly elevated β-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 B12 (27Raux E. Lanois A. Levillayer F. Warren M. Brody E. Rambach A. Thermes C. J. Bacteriol. 1996; 178: 753-767Crossref PubMed Scopus (96) Google Scholar), the relevant genes being absent. Thus, intermediates in this pathway (e.g. hydrogenobyrinic acid) cannot be required for cobalt-dependent positive regulation by 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. 5 B), confirming that the mutant C363S/H364N/C365S protein is synthesized and can bind to DNA. In the absence of added cobalt, β-galactosidase activity in cells containing mutant CoaR was less than in cells containing pLACOA-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. Known proteins that share sequence similarity to MerR from Tn501 include mercury sensors from other sources (28O'Halloran T.V. Science. 1993; 261: 715-725Crossref PubMed Scopus (558) Google Scholar), the redox sensor SoxR (29Hidalgo E. Leautaud V. Demple B. EMBO J. 1998; 17: 2629-2636Crossref PubMed Scopus (60) Google Scholar), the thiostrepton sensor TipAL (30Chiu M.L. Folcher M. Griffin P. Holt T. Klatt T. Thompson C.J. Biochemistry. 1996; 35: 2332-2341Crossref PubMed Scopus (44) Google Scholar), BmrR and BltR from Bacillus subtilis (31Ahmed M. Lyass L. Markham P.N. Taylor S.S. Vázquez-Laslop N. Neyfakh A.A. J. Bacteriol. 1995; 177: 3904-3910Crossref PubMed Scopus (145) Google Scholar), and NolA from Bradyrhizobium japonicum (32Sadowsky M.J. Cregan P.B. Gottfert M. Sharma A. Gerhold D. Rodriguez-Quinones F. Keyser H.H. Hennecke H. Stacey G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 637-641Crossref PubMed Scopus (86) Google Scholar). These proteins are known, or predicted, to associate with promoters in which consensus −10 and −35 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 (33Parkhill J. Brown N.L. Nucleic Acids Res. 1990; 18: 5157-5162Crossref PubMed Scopus (56) Google Scholar). By analogy, 20 bp separate near consensus −10 and −35 sequences in the coaT operator-promoter region (Fig. 7 A). A degenerate (1 bp mismatch in 13) hyphenated (6 bp) inverted repeat (13-6-13) (Fig.7 A) 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 (δ1) or 2 (δ2) bp were deleted (Fig. 7 A). A single complex with the coaT operator-promoter was detected using extracts from these cells (Fig. 5 B). The δ2 construct conferred highly elevated constitutive β-galactosidase activity (Fig. 7 B). Elevated expression was also observed with the δ1 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 (34Hildago E. Demple B. EMBO J. 1997; 16: 1056-1065Crossref PubMed Scopus (90) Google Scholar). 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. To test the proposal that interaction between the precorrin isomerase-like domain of CoaR and intermediates in the vitamin B12 biosynthetic pathway modulates expression from the coaT operator-promoter, β-galactosidase activity was examined in a mutant of strain JRNR1.2 in which the cbiE gene was insertionally inactivated on a proportion of chromosomes. β-Galactosidase activity was elevated in the cbiE mutant compared with JRNR1.2 (Fig.8 A), revealing that the vitamin B12 pathway mediates repression of transcription from the coaT operator-promoter. 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 5 A), insertional inactivation of coaT reduces tolerance to cobalt (Fig. 3 A), restoration of cobalt tolerance by coaT can be used as a selectable marker (Fig.3 B), and cells lacking functional coaT have increased accumulation of 57Co in the cytoplasm (Table I). Eight trans-membrane α-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 (10Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (418) Google Scholar). 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 amino-terminal metal binding motifs of a CPx-type ATPase (35Pufahl R.A. Singer C.P. Peariso K.L. Lin S.-J. Schmidt P.J. Fahrni C.J. Cizewski-Culotta V. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (595) Google Scholar), 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 operator-promoter 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 (29Hidalgo E. Leautaud V. Demple B. EMBO J. 1998; 17: 2629-2636Crossref PubMed Scopus (60) Google Scholar), 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 B12 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 carboxyl-terminal 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 operator-promoter is 4-fold less than apo-MerR (4Parkhill J. Ansari A.Z. Wright J.G. Brown N.L. O'Halloran T.V. EMBO J. 1993; 12: 413-421Crossref PubMed Scopus (56) Google Scholar), 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 (12Thelwell C. Robinson N.J. Turner-Cavet J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10728-10733Crossref PubMed Scopus (156) Google Scholar, 36Shi W. Wu J. Rosen B.P. J. Biol. Chem. 1994; 269: 19826-19829Abstract Full Text PDF PubMed Google Scholar, 37Cook W.J. Kar S.R. Taylor K.B. Hall L.M. J. Mol. Biol. 1998; 275: 337-346Crossref PubMed Scopus (125) Google Scholar, 38Endo G. Silver S. J. Bacteriol. 1995; 177: 4437-4441Crossref PubMed Google Scholar). 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. 8 A). 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 substrate limited. In P. denitrificans it is known that hydrogenobyrinic acid precedes the step of cobalt insertion into the corrin ring (6Roth J.R. Lawrence J.G. Bobik T.A. Rev. Microbiol. 1996; 50: 137-181Crossref Scopus (437) Google Scholar) and is predicted to accumulate when there is insufficient cobalt for vitamin B12 biosynthesis. An inhibition of CoaT production when hydrogenobyrinic acid accumulates will restrict cobalt export when there is cellular demand (Fig. 8 D). Thus, via responses to two effectors, (i) cobalt (positive effector) and (ii) intermediates in the vitamin B12 pathway (negative effector), CoaR integrates cobalt homeostasis with metabolism. Enzyme recruitment (39Hawkins A.R. Lamb H.K. Moore J.D. Roberts C.F. Gene (Amst.). 1993; 136: 49-54Crossref PubMed Scopus (17) Google Scholar) 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 (40Nies D.H. Brown N.L. Silver S. Walden W. Metal Ions in Gene Regulation. Chapman & Hall, New York1998: 77-103Crossref Google Scholar). 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.8 E)? 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 (41Brocklehurst K.R. Hobman J.L. Lawley B. Blank L. Marshall S.J. Brown N.L. Morby A.P. Mol. Microbiol. 1999; 31: 893-903Crossref PubMed Scopus (210) Google Scholar), and a similar activity suggested for a homologue from Proteus mirabilis (42Noll M. Petrukhin K. Lutsenko S. J. Biol. Chem. 1998; 273: 21393-21401Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). 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 cobalt-transporting 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. We thank Martin Hagemann for an essential strain, plasmid, and advice." @default.
• W2019780345 created "2016-06-24" @default.
• W2019780345 creator A5052719640 @default.
• W2019780345 creator A5058467039 @default.
• W2019780345 creator A5085587138 @default.
• W2019780345 date "1999-09-01" @default.
• W2019780345 modified "2023-10-16" @default.
• W2019780345 title "Cobalt-dependent Transcriptional Switching by a Dual-effector MerR-like Protein Regulates a Cobalt-exporting Variant CPx-type ATPase" @default.
• W2019780345 cites W1504738268 @default.
• W2019780345 cites W1512113747 @default.
• W2019780345 cites W1771723362 @default.
• W2019780345 cites W1963506606 @default.
• W2019780345 cites W1963921610 @default.
• W2019780345 cites W1967859158 @default.
• W2019780345 cites W1974552460 @default.
• W2019780345 cites W1978491890 @default.
• W2019780345 cites W1979542194 @default.
• W2019780345 cites W1979680370 @default.
• W2019780345 cites W1982872831 @default.
• W2019780345 cites W1995293508 @default.
• W2019780345 cites W2011557428 @default.
• W2019780345 cites W2012323300 @default.
• W2019780345 cites W2018029111 @default.
• W2019780345 cites W2022571490 @default.
• W2019780345 cites W2045300018 @default.
• W2019780345 cites W2049339567 @default.
• W2019780345 cites W2057489118 @default.
• W2019780345 cites W2061040884 @default.
• W2019780345 cites W2066006292 @default.
• W2019780345 cites W2067057319 @default.
• W2019780345 cites W2074715696 @default.
• W2019780345 cites W2079357395 @default.
• W2019780345 cites W2080131435 @default.
• W2019780345 cites W2101039716 @default.
• W2019780345 cites W2118881590 @default.
• W2019780345 cites W2119373372 @default.
• W2019780345 cites W2130253484 @default.
• W2019780345 cites W2146766740 @default.
• W2019780345 cites W2155260714 @default.
• W2019780345 cites W2158282653 @default.
• W2019780345 cites W2165645937 @default.
• W2019780345 cites W2166999322 @default.
• W2019780345 cites W2211402137 @default.
• W2019780345 cites W2217853554 @default.
• W2019780345 cites W4295127969 @default.
• W2019780345 doi "https://doi.org/10.1074/jbc.274.36.25827" @default.
• W2019780345 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10464323" @default.
• W2019780345 hasPublicationYear "1999" @default.
• W2019780345 type Work @default.
• W2019780345 sameAs 2019780345 @default.
• W2019780345 citedByCount "121" @default.
• W2019780345 countsByYear W20197803452012 @default.
• W2019780345 countsByYear W20197803452013 @default.
• W2019780345 countsByYear W20197803452014 @default.
• W2019780345 countsByYear W20197803452016 @default.
• W2019780345 countsByYear W20197803452017 @default.
• W2019780345 countsByYear W20197803452018 @default.
• W2019780345 countsByYear W20197803452019 @default.
• W2019780345 countsByYear W20197803452020 @default.
• W2019780345 countsByYear W20197803452021 @default.
• W2019780345 countsByYear W20197803452022 @default.
• W2019780345 countsByYear W20197803452023 @default.
• W2019780345 crossrefType "journal-article" @default.
• W2019780345 hasAuthorship W2019780345A5052719640 @default.
• W2019780345 hasAuthorship W2019780345A5058467039 @default.
• W2019780345 hasAuthorship W2019780345A5085587138 @default.
• W2019780345 hasBestOaLocation W20197803451 @default.
• W2019780345 hasConcept C124952713 @default.
• W2019780345 hasConcept C142362112 @default.
• W2019780345 hasConcept C179104552 @default.
• W2019780345 hasConcept C181199279 @default.
• W2019780345 hasConcept C185592680 @default.
• W2019780345 hasConcept C23265538 @default.
• W2019780345 hasConcept C2780980858 @default.
• W2019780345 hasConcept C515602321 @default.
• W2019780345 hasConcept C51785407 @default.
• W2019780345 hasConcept C55493867 @default.
• W2019780345 hasConcept C86803240 @default.
• W2019780345 hasConcept C95444343 @default.
• W2019780345 hasConceptScore W2019780345C124952713 @default.
• W2019780345 hasConceptScore W2019780345C142362112 @default.
• W2019780345 hasConceptScore W2019780345C179104552 @default.
• W2019780345 hasConceptScore W2019780345C181199279 @default.
• W2019780345 hasConceptScore W2019780345C185592680 @default.
• W2019780345 hasConceptScore W2019780345C23265538 @default.
• W2019780345 hasConceptScore W2019780345C2780980858 @default.
• W2019780345 hasConceptScore W2019780345C515602321 @default.
• W2019780345 hasConceptScore W2019780345C51785407 @default.
• W2019780345 hasConceptScore W2019780345C55493867 @default.
• W2019780345 hasConceptScore W2019780345C86803240 @default.
• W2019780345 hasConceptScore W2019780345C95444343 @default.
• W2019780345 hasIssue "36" @default.
• W2019780345 hasLocation W20197803451 @default.
• W2019780345 hasOpenAccess W2019780345 @default.
• W2019780345 hasPrimaryLocation W20197803451 @default.
• W2019780345 hasRelatedWork W1750541350 @default.
• W2019780345 hasRelatedWork W1757607567 @default.
• W2019780345 hasRelatedWork W1985830493 @default.

• next