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Juan F Martin - One of the best experts on this subject based on the ideXlab platform.
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characterization of dna binding sequences for ccar in the Cephamycin clavulanic acid supercluster of streptomyces clavuligerus
Molecular Microbiology, 2011Co-Authors: Irene Santamarta, M T Lopezgarcia, A Kurt, N Nardiz, Ruben Alvarezalvarez, Rosario Perezredondo, Juan F Martin, Paloma LirasAbstract:RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for Cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the Cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.
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streptomyces clavuligerus rela null mutants overproduce clavulanic acid and Cephamycin c negative regulation of secondary metabolism by p ppgpp
Microbiology, 2008Co-Authors: Juan Pablo Gomezescribano, Juan F Martin, Andrew Hesketh, Maureen J Bibb, Paloma LirasAbstract:The (p)ppGpp synthetase gene, relA, of Streptomyces clavuligerus was cloned, sequenced and shown to be located in a genomic region that is highly conserved in other Streptomyces species. relA-disrupted and relA-deleted mutants of S. clavuligerus were constructed, and both were unable to form aerial mycelium or to sporulate, but regained these abilities when complemented with wild-type relA. Neither ppGpp nor pppGpp was detected in the S. clavuligerus relA-deletion mutant. In contrast to another study, clavulanic acid and Cephamycin C production increased markedly in the mutants compared to the wild-type strain; clavulanic acid production increased three- to fourfold, while that of Cephamycin C increased about 2.5-fold. Complementation of the relA-null mutants with wild-type relA decreased antibiotic yields to approximately wild-type levels. Consistent with these observations, transcription of genes involved in clavulanic acid (ceaS2) or Cephamycin C (cefD) production increased dramatically in the relA-deleted mutant when compared to the wild-type strain. These results are entirely consistent with the growth-associated production of both Cephamycin C and clavulanic acid, and demonstrate, apparently for the first time, negative regulation of secondary metabolite biosynthesis by (p)ppGpp in a Streptomyces species of industrial interest.
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ccar is an autoregulatory protein that binds to the ccar and cefd cmci promoters of the Cephamycin c clavulanic acid cluster in streptomyces clavuligerus
Journal of Bacteriology, 2002Co-Authors: Irene Santamarta, Rosario Perezredondo, Juan F Martin, Antonio Rodriguezgarcia, Paloma LirasAbstract:The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the Cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of Cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.
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The Nine Genes of the Nocardia lactamdurans Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region
Journal of bacteriology, 1998Co-Authors: Francisco J. Enguita, Paloma Liras, J J Coque, Juan F MartinAbstract:The nine biosynthesis genes of the Nocardia lactamdurans Cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.
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the clar gene of streptomyces clavuligerus encoding a lysr type regulatory protein controlling clavulanic acid biosynthesis is linked to the clavulanate 9 aldehyde reductase car gene
Gene, 1998Co-Authors: Rosario Perezredondo, Juan F Martin, Antonio Rodriguezgarcia, Paloma LirasAbstract:Abstract Two genes, cla R and car , encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb Bgl II– Eco RI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the Cephamycin and clavulanic acid biosynthesis gene cluster. cla R encoded a protein of 431 amino acids (deduced M r , 47 080), that showed a significant degree of homology with several transcriptional activators of the LysR family. The ClaR protein contained two helix–turn–helix (HTH) motifs in the amino and carboxyl terminal regions. The second gene, car , encoded a protein of 247 amino acids ( M r 26 629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus . Amplification of the cla R gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas Cephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or Cephamycin production. Both cla R and car are expressed as monocistronic transcripts; the level of transcript declined rapidly at 24 and 48 h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96 h. cla R and car were not significantly expressed in mutants disrupted in the cca R gene, a regulatory gene that controls positively clavulanic acid and Cephamycin biosynthesis. These results indicate that clavulanic acid and Cephamycin biosynthesis in S. clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.
Paloma Liras - One of the best experts on this subject based on the ideXlab platform.
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characterization of dna binding sequences for ccar in the Cephamycin clavulanic acid supercluster of streptomyces clavuligerus
Molecular Microbiology, 2011Co-Authors: Irene Santamarta, M T Lopezgarcia, A Kurt, N Nardiz, Ruben Alvarezalvarez, Rosario Perezredondo, Juan F Martin, Paloma LirasAbstract:RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for Cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the Cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.
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homologous expression of aspartokinase ask gene in streptomyces clavuligerus and its hom deleted mutant effects on Cephamycin c production
Bioengineered bugs, 2010Co-Authors: Gulay Ozcengiz, Paloma Liras, Sezer Okay, Eser Unsaldi, Bilgin Taskin, Jacqueline PiretAbstract:In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on Cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the wild strain resulted in a maximum of 3.1- and 3.3-fold increase in specific, 1.7- and 1.9-fold increase in volumetric Cephamycin C production when grown in trypticase soy broth (TSB) and a modified chemically defined medium (mCDM), respectively. However, expression of multicopy ask gene in a hom-deleted background significantly decreased Cephamycin C yields when the cells were grown in either TSB or mCDM, most probably due to physiological disturbance resulting from enzyme overexpression and high copy number plasmid burden in an auxotrophic host, respectively.
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streptomyces clavuligerus rela null mutants overproduce clavulanic acid and Cephamycin c negative regulation of secondary metabolism by p ppgpp
Microbiology, 2008Co-Authors: Juan Pablo Gomezescribano, Juan F Martin, Andrew Hesketh, Maureen J Bibb, Paloma LirasAbstract:The (p)ppGpp synthetase gene, relA, of Streptomyces clavuligerus was cloned, sequenced and shown to be located in a genomic region that is highly conserved in other Streptomyces species. relA-disrupted and relA-deleted mutants of S. clavuligerus were constructed, and both were unable to form aerial mycelium or to sporulate, but regained these abilities when complemented with wild-type relA. Neither ppGpp nor pppGpp was detected in the S. clavuligerus relA-deletion mutant. In contrast to another study, clavulanic acid and Cephamycin C production increased markedly in the mutants compared to the wild-type strain; clavulanic acid production increased three- to fourfold, while that of Cephamycin C increased about 2.5-fold. Complementation of the relA-null mutants with wild-type relA decreased antibiotic yields to approximately wild-type levels. Consistent with these observations, transcription of genes involved in clavulanic acid (ceaS2) or Cephamycin C (cefD) production increased dramatically in the relA-deleted mutant when compared to the wild-type strain. These results are entirely consistent with the growth-associated production of both Cephamycin C and clavulanic acid, and demonstrate, apparently for the first time, negative regulation of secondary metabolite biosynthesis by (p)ppGpp in a Streptomyces species of industrial interest.
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ccar is an autoregulatory protein that binds to the ccar and cefd cmci promoters of the Cephamycin c clavulanic acid cluster in streptomyces clavuligerus
Journal of Bacteriology, 2002Co-Authors: Irene Santamarta, Rosario Perezredondo, Juan F Martin, Antonio Rodriguezgarcia, Paloma LirasAbstract:The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the Cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of Cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.
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The Nine Genes of the Nocardia lactamdurans Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region
Journal of bacteriology, 1998Co-Authors: Francisco J. Enguita, Paloma Liras, J J Coque, Juan F MartinAbstract:The nine biosynthesis genes of the Nocardia lactamdurans Cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.
Irene Santamarta - One of the best experts on this subject based on the ideXlab platform.
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e-mail: degplp!unileon.es Area de Microbiologı!a,
2015Co-Authors: Victor Herna Ndo-rico, Irene Santamarta, Juan F. Martı!n, Facultad De CienciasAbstract:of the ask-asd operon and formation of aspartokinase subunits in the Cephamycin producer ‘Amycolatopsis lactamdurans
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characterization of dna binding sequences for ccar in the Cephamycin clavulanic acid supercluster of streptomyces clavuligerus
Molecular Microbiology, 2011Co-Authors: Irene Santamarta, M T Lopezgarcia, A Kurt, N Nardiz, Ruben Alvarezalvarez, Rosario Perezredondo, Juan F Martin, Paloma LirasAbstract:RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for Cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the Cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.
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ccar is an autoregulatory protein that binds to the ccar and cefd cmci promoters of the Cephamycin c clavulanic acid cluster in streptomyces clavuligerus
Journal of Bacteriology, 2002Co-Authors: Irene Santamarta, Rosario Perezredondo, Juan F Martin, Antonio Rodriguezgarcia, Paloma LirasAbstract:The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the Cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of Cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.
Antonio Rodriguezgarcia - One of the best experts on this subject based on the ideXlab platform.
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ccar is an autoregulatory protein that binds to the ccar and cefd cmci promoters of the Cephamycin c clavulanic acid cluster in streptomyces clavuligerus
Journal of Bacteriology, 2002Co-Authors: Irene Santamarta, Rosario Perezredondo, Juan F Martin, Antonio Rodriguezgarcia, Paloma LirasAbstract:The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the Cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of Cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.
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the clar gene of streptomyces clavuligerus encoding a lysr type regulatory protein controlling clavulanic acid biosynthesis is linked to the clavulanate 9 aldehyde reductase car gene
Gene, 1998Co-Authors: Rosario Perezredondo, Juan F Martin, Antonio Rodriguezgarcia, Paloma LirasAbstract:Abstract Two genes, cla R and car , encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb Bgl II– Eco RI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the Cephamycin and clavulanic acid biosynthesis gene cluster. cla R encoded a protein of 431 amino acids (deduced M r , 47 080), that showed a significant degree of homology with several transcriptional activators of the LysR family. The ClaR protein contained two helix–turn–helix (HTH) motifs in the amino and carboxyl terminal regions. The second gene, car , encoded a protein of 247 amino acids ( M r 26 629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus . Amplification of the cla R gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas Cephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or Cephamycin production. Both cla R and car are expressed as monocistronic transcripts; the level of transcript declined rapidly at 24 and 48 h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96 h. cla R and car were not significantly expressed in mutants disrupted in the cca R gene, a regulatory gene that controls positively clavulanic acid and Cephamycin biosynthesis. These results indicate that clavulanic acid and Cephamycin biosynthesis in S. clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.
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a regulatory gene ccar required for Cephamycin and clavulanic acid production in streptomyces clavuligerus amplification results in overproduction of both beta lactam compounds
Journal of Bacteriology, 1997Co-Authors: Francisco J Perezllarena, Antonio Rodriguezgarcia, Paloma Liras, Juan F MartinAbstract:A regulatory gene (ccaR), located within the Cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for Cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize Cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of Cephamycin and clavulanic acid.
Brenda K. Leskiw - One of the best experts on this subject based on the ideXlab platform.
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Printed in Great Britain The positive activator of Cephamycin C and
2013Co-Authors: Nicole K. Trepanier, Susan E Jensen, Dylan C. Alex, Brenda K. LeskiwAbstract:clavulanic acid production in Streptomyces clavuligerus is mistranslated in a bldA mutan
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Expression of ccaR, encoding the positive activator of Cephamycin C and clavulanic acid production in Streptomyces clavuligerus, is dependent on bldG
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Dawn R. D. Bignell, Kapil Tahlan, Susan E Jensen, Kimberley R. Colvin, Brenda K. LeskiwAbstract:In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as Cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce Cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.