Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP) | Microbiology Society
Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)
Ram P. Garg1, Ronald J. Parry1
Affiliations: 1 Department of Chemistry, Rice University, Houston, TX 77005, USA
CorrespondenceRonald J. Parry [email protected]
First Published: 01 February 2010 https://doi.org/10.1099/mic.0.033167-0
Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the −35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA–vlmH and vlmI–vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.
Keyword(s): SARP, Streptomyces antibiotic regulatory protein
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Arias P., Fernandez-Moreno M. A., Malpartida F.. 1999; Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J Bacteriol181:6958–6968
Bierman M., Logan R., Obrien K., Seno E. T., Rao R. N., Schoner B. E.. 1992; Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene116:43–49
Chen Y., Wendt-Pienkowski E., Shen B.. 2008; Identification and utility of FdmR1 as a Streptomyces antibiotic regulatory protein activator for fredericamycin production in Streptomyces griseus ATCC 49344 and heterologous hosts. J Bacteriol190:5587–5596
Couch R., Seidle H., Parry R. J.. 2002; Construction of expression vectors to produce affinity-tagged proteins in Pseudomonas. Biotechniques32:1230–1236
Floriano B., Bibb M.. 1996; afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2. Mol Microbiol21:385–396
Garg R. P., Menon A. L., Jacobs K., Robson R. M., Robson R. L.. 1994; The hypE gene completes the gene cluster for H2-oxidation in Azotobacter vinelandii. J Mol Biol236:390–396
Garg R. P., Yindeeyoungyeon W., Gilis A., Denny T. P., Van Der Lelie D., Schell M. A.. 2000; Evidence that Ralstonia eutropha ( Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system. Mol Microbiol38:359–367
Garg R. P., Ma Y., Hoyt J. C., Parry R. J.. 2002; Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin. Mol Microbiol46:505–517
Garg R. P., Gonzalez J. M., Parry R. J.. 2006; Biochemical characterization of VlmL, a seryl-tRNA synthetase encoded by the valanimycin biosynthetic gene cluster. J Biol Chem281:26785–26791
Garg R. P., Qian X. L., Alemany L. B., Moran S., Parry R. J.. 2008; Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA. Proc Natl Acad Sci U S A105:6543–6547
Garg R. P., Alemany L. B., Moran S., Parry R. J.. 2009; Isolation, characterization, and bioconversion of a new intermediate in valanimycin biosynthesis. J Am Chem Soc131:9608–9609
Ma Y., Parry R. J.. 2000; A novel valanimycin-resistance determinant ( vlmF) from Streptomyces viridifaciens MG456-hF10. Microbiology146:345–352
Narva K. E., Feitelson J. S.. 1990; Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2. J Bacteriol172:326–333
Parry R. J., Li W.. 1997a; An NADPH : FAD oxidoreductase from the valanimycin producer, Streptomyces viridifaciens. Cloning, analysis, and overexpression. J Biol Chem272:23303–23311
Parry R. J., Li W.. 1997b; Purification and characterization of isobutylamine N-hydroxylase from the valanimycin producer Streptomyces viridifaciens MG456-hF10. Arch Biochem Biophys339:47–54
Perez-Llarena F. J., Liras P., Rodriguez-Garcia A., Martin J. F.. 1997; A regulatory gene ( ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both β-lactam compounds. J Bacteriol179:2053–2059
Scheu A. K., Martinez E., Soliveri J., Malpartida F.. 1997; abaB, a putative regulator for secondary metabolism in Streptomyces. FEMS Microbiol Lett147:29–36
Stutzman-Engwall K. J., Otten S. L., Hutchinson C. R.. 1992; Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol174:144–154
Tanaka A., Takano Y., Ohnishi Y., Horinouchi S.. 2007; AfsR recruits RNA polymerase to the afsS promoter: a model for transcriptional activation by SARPs. J Mol Biol369:322–333
Tang L., Grimm A., Zhang Y. X., Hutchinson C. R.. 1996; Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius. Mol Microbiol22:801–813
Thomson J. M., Parrott W. A.. 1998; pMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size. Biotechniques24:922–928
Vara J., Lewandowska-Skarbek M., Wang Y. G., Donadio S., Hutchinson C. R.. 1989; Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea ( Streptomyces erythreus. J Bacteriol171:5872–5881
Wasserman H. H., Rodgers G. C., Keith D. D.. 1976; Undecylprodigiosin. Tetrahedron32:1851–1854
Wietzorrek A., Bibb M.. 1997; A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol25:1181–1184
Yamato M., Iinuma H., Naganawa H., Yamagishi Y., Hamada M., Masuda T., Umezawa H.. 1986; Isolation and properties of valanimycin, a new azoxy antibiotic. J Antibiot39:184–191
Yin X., O'Hare T., Gould S. J., Zabriskie T. M.. 2003; Identification and cloning of genes encoding viomycin biosynthesis from Streptomyces vinaceus and evidence for involvement of a rare oxygenase. Gene312:215–224
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.033167-0
Microbiology 156, 472 (2010); https://doi.org/10.1099/mic.0.033167-0
10.1099/mic.0.033167-0
Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP) | Microbiology Society
Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)
Ram P. Garg1, Ronald J. Parry1
Affiliations: 1 Department of Chemistry, Rice University, Houston, TX 77005, USA
CorrespondenceRonald J. Parry [email protected]
First Published: 01 February 2010 https://doi.org/10.1099/mic.0.033167-0
Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the −35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA–vlmH and vlmI–vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.
Keyword(s): SARP, Streptomyces antibiotic regulatory protein
/content/journal/micro/10.1099/mic.0.033167-0
/deliver/fulltext/micro/156/2/472.html?itemId=/content/journal/micro/10.1099/mic.0.033167-0&mimeType=html&fmt=ahah
Arias P., Fernandez-Moreno M. A., Malpartida F.. 1999; Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J Bacteriol181:6958–6968
Bierman M., Logan R., Obrien K., Seno E. T., Rao R. N., Schoner B. E.. 1992; Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene116:43–49
Chen Y., Wendt-Pienkowski E., Shen B.. 2008; Identification and utility of FdmR1 as a Streptomyces antibiotic regulatory protein activator for fredericamycin production in Streptomyces griseus ATCC 49344 and heterologous hosts. J Bacteriol190:5587–5596
Couch R., Seidle H., Parry R. J.. 2002; Construction of expression vectors to produce affinity-tagged proteins in Pseudomonas. Biotechniques32:1230–1236
Floriano B., Bibb M.. 1996; afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2. Mol Microbiol21:385–396
Garg R. P., Menon A. L., Jacobs K., Robson R. M., Robson R. L.. 1994; The hypE gene completes the gene cluster for H2-oxidation in Azotobacter vinelandii. J Mol Biol236:390–396
Garg R. P., Yindeeyoungyeon W., Gilis A., Denny T. P., Van Der Lelie D., Schell M. A.. 2000; Evidence that Ralstonia eutropha ( Alcaligenes eutrophus) contains a functional homologue 
of the Ralstonia solanacearum Phc cell density sensing system. Mol Microbiol38:359–367
Garg R. P., Ma Y., Hoyt J. C., Parry R. J.. 2002; Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin. Mol Microbiol46:505–517
Garg R. P., Gonzalez J. M., Parry R. J.. 2006; Biochemical characterization of VlmL, a seryl-tRNA synthetase encoded by the valanimycin biosynthetic gene cluster. J Biol Chem281:26785–26791
Garg R. P., Qian X. L., Alemany L. B., Moran S., Parry R. J.. 2008; Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA. Proc Natl Acad Sci U S A105:6543–6547
Garg R. P., Alemany L. B., Moran S., Parry R. J.. 2009; Isolation, characterization, and bioconversion of a new intermediate in valanimycin biosynthesis. J Am Chem Soc131:9608–9609
Ma Y., Parry R. J.. 2000; A novel valanimycin-resistance determinant ( vlmF) from Streptomyces viridifaciens MG456-hF10. Microbiology146:345–352
Narva K. E., Feitelson J. S.. 1990; Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2. J Bacteriol172:326–333
Parry R. J., Li W.. 1997a; An NADPH : FAD oxidoreductase from the valanimycin producer, Streptomyces viridifaciens. Cloning, analysis, and overexpression. J Biol Chem272:23303–23311
Parry R. J., Li W.. 1997b; Purification and characterization of isobutylamine N-hydroxylase from the valanimycin producer Streptomyces viridifaciens MG456-hF10. Arch Biochem Biophys339:47–54
Perez-Llarena F. J., Liras P., Rodriguez-Garcia A., Martin J. F.. 1997; A regulatory gene ( ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both β-lactam compounds. J Bacteriol179:2053–2059
Scheu A. K., Martinez E., Soliveri J., Malpartida F.. 1997; abaB, a putative regulator for secondary metabolism in Streptomyces. FEMS Microbiol Lett147:29–36
Stutzman-Engwall K. J., Otten S. L., Hutchinson C. R.. 1992; Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol174:144–154
Tanaka A., Takano Y., Ohnishi Y., Horinouchi S.. 2007; AfsR recruits RNA polymerase to the afsS promoter: a model for transcriptional activation by SARPs. J Mol Biol369:322–333
Tang L., Grimm A., Zhang Y. X., Hutchinson C. R.. 1996; Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius. Mol Microbiol22:801–813
Thomson J. M., Parrott W. A.. 1998; pMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size. Biotechniques24:922–928
Vara J., Lewandowska-Skarbek M., Wang Y. G., Donadio S., Hutchinson C. R.. 1989; Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea ( Streptomyces erythreus. J Bacteriol171:5872–5881
Wasserman H. H., Rodgers G. C., Keith D. D.. 1976; Undecylprodigiosin. Tetrahedron32:1851–1854
Wietzorrek A., Bibb M.. 1997; A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol25:1181–1184
Yamato M., Iinuma H., Naganawa H., Yamagishi Y., Hamada M., Masuda T., Umezawa H.. 1986; Isolation and properties of valanimycin, a new azoxy antibiotic. J Antibiot39:184–191
Yin X., O'Hare T., Gould S. J., Zabriskie T. M.. 2003; Identification and cloning of genes encoding viomycin biosynthesis from Streptomyces vinaceus and evidence for involvement of a rare oxygenase. Gene312:215–224
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.033167-0
Microbiology 156, 472 (2010); https://doi.org/10.1099/mic.0.033167-0
10.1099/mic.0.033167-0