copA Gene Research Articles

Biochemical Characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. CopA formed a phosphorylated intermediate with [gamma-(32)P]ATP. Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm. CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.

Structure and expression of Strabismus 1 gene on human chromosome 1q21-q23

Xenopus Strabismus (Stbm) is a negative regulator of the WNT - beta-catenin signaling pathway. Strabismus 1 (STB1/VangL2) and Strabismus 2 (STB2/Vangl1) are human homologues of Xenopus Stbm and Drosophila Stbm/ Van Gogh (Vang) STB1 and STB2 are four-transmembrane-type proteins with Dishevelled-binding motif. STB2 and CASQ2 genes are located on human chromosome 1p13.3-p11 with an interval less than 5 kb. Here, STB1 gene and CASQ1 gene were found to be located on human chromosome 1q21-q23 with an interval of about 210 kb including Nicastrin, COPA, PXF, H326 and PEA15 genes. Exon-intron structure was well conserved between STB1 and STB2 genes. STB1-CASQ1 gene cluster and STB2-CASQ2 gene cluster might be generated due to duplication of ancestral gene cluster, and several genes might be inserted into the STB1-CASQ1 intergenic region during or after gene-cluster duplication. STB1 mRNA was relatively highly expressed in prostate, trachea, thymus, lymph node, placenta, fetal kidney, fetal brain, and fetal lung. In adult brain, STB1 mRNA was more highly expressed in cerebellum, corpus callosum, amygdala, and medulla oblongata. STB1 mRNA was moderately expressed in K-562 (chronic myelogenous leukemia), G-361 (melanoma), and MKN7 (gastric cancer). On the other hand, STB1 mRNA was almost undetectable in several human cancer cell lines, and was down-regulated in 4 out of 14 cases of primary kidney tumors, and in 2 out of 3 cases of primary lung cancer. Loss-of-function mutation of STB1 gene might lead to carcinogenesis through activation of the WNT - beta-catenin signaling pathway.

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively.

Copper metalloregulation of gene expression

Bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae . Cu metalloregulation of transcription occurs for ATPase genes in bacteria and certain fungi. The En hirae CopY is a transcriptional repressor that limits the expression of the CopA and CopB P-type ATPases in cells cultured in medium containing minimal Cu(lI) levels. An increase in medium Cu(II) levels results in Cu ion uptake, routing of the Cu(l) ions to CopY by the CopZ metallochaperone, and the subsequent dissociation of CuCopY from the copA and copB genes. In contrast to the En. hirae CopY metalloregulation, Sa. Cerevisiae copper metalloregulation involves two transcriptional activators, Acel and Macl. Both the factors reside within the yeast nucleus and are regulated in their function in a Cu-dependent manner. Addition of Cu salts to the growth medium (at greater than micromolar concentrations of Cu) induces Ace1-dependent transcription within 10 min. Cu binding to Ace1 stabilizes a distinct conformation that enables CuAce1 to bind to DNA in a specific manner. Cu(I) ions activate Acel by formation of a tetracopperthiolate cluster within the DNA-binding domain.

Escherichia coli CopA N-Terminal Cys(X)2Cys Motifs Are Not Required for Copper Resistance or Transport

Escherichia coli CopA is a Cu(I)-translocating P-type ATPase that is involved in copper export and resistance. It is an orthologue of the human Menkes and Wilson disease-related proteins. Each of those two human copper pumps has six N-terminal Cys(X)2Cys sequences, but their function in transport is unclear. CopA has two N-terminal Cys(X)2Cys sequences, GLSC14GHC17 and GMSC110ASC113. The requirement of these cysteine motifs was investigated by mutagenesis of the codons for all four cysteine residues, singly and in combination. Cells of a copA deletion strain expressing genes for the mutant genes were nearly as resistant to copper as the wild type. In addition, everted membrane vesicles from cells expressing the mutant copA genes exhibited ATP-coupled accumulation of copper similar to that of the wild type. The results indicate that neither of two N-terminal Cys(X)2Cys motifs is required for either resistance or transport.

The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions.

Transcription of the ybcZ--ylcA ylcBCD--ybdE region of the Escherichia coli K38 chromosome was analysed by Northern RNA--DNA hybridization, RT-PCR and primer extension. Transcription of a dicistronic ybcZ--ylcA mRNA and a tetracistronic ylcBCD--ybdE mRNA was induced by silver and was initiated from the sigma-70 promoters ylcAp and ylcBp. Expression of beta-galactosidase activity from a Phi(ylcBp--lacZ) operon fusion was also induced by Ag(+) and Cu(2+), but not by Zn(2+). In-frame deletion of ybdE from the chromosome yielded a silver-sensitive E. coli mutant strain which did not differ in its copper resistance from its wild-type strain. On the other hand, deletion of the copA gene for the copper-exporting P-type ATPase CopA resulted in copper sensitivity, but not in silver sensitivity. A Delta ybdE Delta copA double mutant strain behaved towards copper as the Delta copA strain and towards silver as the Delta ybdE strain. Thus, in E. coli, the YlcBCD--YbdE system may be involved in silver- but not in copper resistance, and CopA may be involved in copper- but not in silver resistance.

CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA.

We have shown that the open reading frame ybbI in the genomic sequence of Escherichia coli K-12 encodes the regulator of expression of the copper-exporting ATPase, CopA. In vivo studies showed that ybbI (designated cueR for copper export regulator gene) was required for copper tolerance during growth, that disruption of cueR caused loss of copA expression and that copA gene expression was regulated by cueR and by copper or silver ions. Expression of a lacZ reporter gene under the control of the copA promoter was approximately proportional to the concentration of cupric ions in the medium, but increased more rapidly in response to silver ion concentrations. The start of the copA transcript was located by primer extension mapping, and DNase I protection assays showed that the CueR protein binds in vitro to a dyad symmetrical sequence within a 19 bp spacer sequence in the copA promoter. CueR binding occurs in vitro in both the presence and the absence of RNA polymerase with or without copper ions present but, in the presence of CueR, RNA polymerase and copper ions, permanganate-sensitive transcription complexes were formed. CueR is predicted to have an N-terminal helix-turn-helix sequence and shows similarity to MerR family regulators.

Control of copper homeostasis in Escherichia coli by a P-type ATPase, CopA, and a MerR-like transcriptional activator, CopR

We have isolated and characterized a copper sensitive Escherichia coli mutant that is deficient in the copper transporting P-type ATPase encoded by the copA gene (previously ybaR). Measurements of uptake and efflux of 64Cu by wild-type and mutant cells implicated the CopA protein in copper efflux from the cytoplasm, and further demonstrated that cell-associated copper in intact E. coli cells is distributed between two kinetically distinguishable pools, the ratio of which was dramatically disturbed by the copA mutation. Using a copA-lacZ gene fusion the copA promoter was found to be specifically induced by copper, and this induction was shown to be dependent on a MerR-like transcriptional activator encoded by a previously uncharacterized gene, copR (previously ybbI). In the copA deficient background the copA-lacZ fusion was super induced to very high levels even in the absence of copper addition to the medium, and this induction was dependent on CopR. These results indicated that the cytoplasmic copper concentration was dramatically increased in the copA mutant, in agreement with the 64Cu uptake experiments. Moreover, they implied, that the copper concentration in wild type cells is determined primarily by the CopA efflux pump, while copper is taken up by an essentially constitutive mechanism.

Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria

Tn phoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of Tn phoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons.

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

Identification of the mouse β`-COP golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse Chromosome 9

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated β′-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the β′-COP gene Copb2 to mouse Chromosome 9, region E3–F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.

Alpha coat protein COPA (HEP-COP): presence of an Alu repeat in cDNA and identity of the amino terminus to xenin.

We previously sequenced the 4333-nucleotide cDNA of the COPA (HEP-COP) gene which encodes the human homologue of the alpha-subunit of the coatomer protein complex, involved in intracellular protein transport. Within the 3' untranslated region at nucleotides 4049-4333 was observed an Alu repeat containing conserved A and B block elements, and showing high homology to the human Alu-Sx subfamily consensus sequence. Upstream of the Alu repeat were noted a TATA box, a CAAT motif and two activating transcription factor (ATF)-like binding sites, which represent putative regulatory elements directing Alu transcription. In addition, the 25 and 35 N-terminal amino acid residues of COPA and its bovine homologue were identical to xenin-25 and proxenin, respectively. Xenin-25 is a gastrointestinal hormone that stimulates exocrine pancreatic secretion. This peptide is related to xenopsin, neurotensin and neuromedin N which are bioactive peptides derived from larger precursors via proteolytic cleavage by cathepsin E at processing sites determined by conserved C-terminal sequences, i.e. proline/valine-X-X-hydrophobic amino acid. Given the conformity of the C-terminal residues of xenin-25 (PWIL) and of its progenitor molecule, proxenin (VIQL), it is proposed that these peptides are generated by a similar mechanism of post-translational modification involving a parent precursor represented by the alpha-subunit of coatomer.

Occurrence of cop-like copper resistance genes among bacteria isolated from a water distribution system

The occurrence of cop-like copper resistance determinants homologous to the cop genes of Pseudomonas syringae among bacteria isolated from a water distribution system experiencing copper corrosion was investigated in this study. It was found that at least 49% of the copper-resistant bacteria and less than 15% of the copper-sensitive isolates possessed a cop homolog. The occurrence of this determinant in the copper-resistant population correlated with the degree of copper tolerance exhibited by the bacteria. The effect of organic substances present in the culture media on the empirical degree of bacterial copper tolerance is also discussed.Key words: copper resistance genes, water distribution system, cop.

Illuminating plant development

AbstractThroughout 1994 remarkable progress was made with molecular and genetic studies on signal transduction pathways of photomorphogenesis, the lightdependent development of plants. Analysis of Arabidopsis DET and COP genes suggests that they are involved in suppression of photomorphogenic development in the dark and that this is then reversed by light. Studies with COP1 indicate that this is achieved by redistribution of COP1 from the nucleus, in the dark, to the cytosol in the light(1). Overexpression of COP1 in the light, however, was able to partially suppress photomorphogenic development, confirming its role as a light‐inactivatable repressor(2). Evidence also suggests that some of the COP gene products may interact directly to form a large complex(3).

Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae

Copper-resistant and copper-sensitive strains of Pseudomonas syringae, as well as many other pseudomonads, contain chromosomal DNA homologous to the plasmid-borne copper resistance operon (copABCD). cop homologs were cloned from the chromosome of P. syringae pv. tomato PT12.2, which had an elevated level of resistance to copper compared with typical copper-sensitive strains of other P. syringae pathovars and showed an unusually high frequency of spontaneous mutation to high levels of copper resistance. Two chromosomal cop homolog regions were cloned. Homolog 1 hybridized with copA and copB, and homolog 2 hybridized with copA, copB, copC, and the copper-responsive regulatory genes copRS. Homolog 1 had no detectable function when transferred to a copper-sensitive strain of P. syringae. However, homolog 2 conferred the low level of copper resistance observed with PT12.2 and produced proteins related to CopA and CopC. In addition, homolog 2 conferred a high frequency of mutation to full copper resistance. In a spontaneously mutated derivative of the cloned homolog 2 (pCOPH2R) that conferred copper resistance, an increased level of CopA was observed. pCOPH2R also supported a higher level of transcriptional activity of the cop promoter that was fused to lacZ and provided in trans (pCOP38), suggesting that the spontaneous mutation was regulatory, probably involving the copRS homologs. Homolog 2 was similar but not identical to the plasmid-borne cop operon, and it did not complement site-specific mutations in cop genes.

Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae

We cloned an operon, copAB, from Enterococcus hirae encoding two P-type ATPases of 727 and 745 amino acids, respectively. Both enzymes display heavy metal ion binding motifs in their polar N-terminal region. With an antibody against CopB, we showed on Western blots that expression of the operon is induced by either low or high ambient copper concentrations. Disruption of the copA gene renders the cells dependent, whereas copper disruption of copB results in a copper-sensitive phenotype. CopA exhibits 35% sequence similarity to CopB and 43% similarity to the ATPase encoded by the recently cloned human Mc1 gene, a gene responsible for the Menkes inborn error of copper metabolism. Our results imply that CopA and CopB are heavy metal ion ATPases that regulate the cytoplasmic copper activity, with CopA serving in the uptake and CopB in the extrusion of copper.

Control of Mini-R1 Plasmid Replication: A Computer Simulation

A molecular model for the control of plasmid R1 replication has been proposed by Nordström. Molin and Light ( Plasmid 12, 71-90, 1984), involving three genes: repA, copA, copB. RepA codes for a polypeptide whose synthesis is required for initiation; replication is controlled by regulating this synthesis. CopA encodes a small, unstable, untranslated RNA molecule that inhibits translation of the repA message whereas copB produces a protein that inhibits transcription from the repA promoter. We have recast this model into precise mathematical terms and tested it by computer simulation of a synchronous culture in steady-state balanced growth, composed of individual Escherichia coli cells harboring the small, unstable derivative, mini-R1. All single-cell steady-state distributions obtained are independent of initial conditions, and the average values of various plasmid-related variables are similar to those measured experimentally. The relationship between the number of replication events per cell and the copy number at birth, as predicted by the model, mitigates against a sensitive correction mechanism for cells born with other than average copy number and is much closer quantitatively to a nonresponse system, although there is a weak dependence on copy number. The effect of the convergent transcription initiated at the repA and copB promoters on the expression of the copA gene is found to contribute little to the stability of mini-R1 replicons under steady-state growth conditions or to their potential for survival following infection. In fact, the role of the entire CopB control loop is shown to be quite minor, both in steady state and after infection. It is pointed out that genetic manipulations are far more easily performed in silico than in vivo but that results of the kind presented here are very often possible only when simulating individual cells.

Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation.

The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis.

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.