A-Factor and Phosphate Depletion Signals Are Transmitted to the Grixazone Biosynthesis Genes via the Pathway-Specific Transcriptional Activator GriR

ABSTRACT Grixazone (GX), which is a diffusible yellow pigment containing a phenoxazinone chromophore, is one of the secondary metabolites under the control of A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) in Streptomyces griseus. GX production is also induced by phosphate starvation. The whole biosynthesis gene cluster for GX was cloned and characterized. The gene cluster consisting of 13 genes contained six transcriptional units, griT, griSR, griR, griAB, griCDEFG, and griJIH. During cultivation in a phosphate-depleted medium, the six promoters were activated in the order (i) griR, (ii) griC and griJ, and (iii) griT, griS, and griA. Disruption of griR, which encodes a SARP family transcriptional regulator, abolished the transcriptional activation of all other genes in the cluster. In addition, ectopic expression of griR from a constitutively active promoter resulted in GX overproduction even in the absence of AdpA, a key transcriptional activator in the A-factor regulatory cascade, and in the presence of phosphate at a high concentration. GriR monomers bound direct repeat sequences in the griC and griJ promoters in a cooperative manner. Therefore, the early active genes (griCDEFG and griJIH), all of which, except for griG (which encodes a transporter-like protein), encode the GX biosynthesis enzymes, were directly activated by GriR. The transcription of griR was greatly reduced in the presence of phosphate at a high concentration and was hardly detected in the absence of AdpA. These findings showed that both A-factor and phosphate depletion signals were required for griR transcription and both signals were transmitted to the GX biosynthesis genes solely via the griR promoter.

[1]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[2]  B. Reiss,et al.  Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. , 1982, Gene.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[4]  D. Hopwood.  Genetic manipulation of Streptomyces : a laboratory manual , 1985 .

[5]  K. Narva,et al.  Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2) , 1990, Journal of bacteriology.

[6]  J. Martín,et al.  Phosphate control of pabS gene transcription during candicidin biosynthesis. , 1990, Gene.

[7]  H. Ikeda,et al.  Cloning of large DNA fragments, which hybridize with actinorhodin biosynthesis genes, from kalafungin and nanaomycin A methyl ester producers and identification of genes for kalafungin biosynthesis of the kalafungin producer. , 1991, The Journal of antibiotics.

[8]  E. Schwarz,et al.  The glucose kinase gene of Streptomyces coelicolor A3(2): its nucleotide sequence, transcriptional analysis and role in glucose repression , 1992, Molecular microbiology.

[9]  C. Hutchinson,et al.  Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius , 1992, Journal of bacteriology.

[10]  E. Takano,et al.  Transcriptional regulation of the redD transcriptional activator gene accounts for growth‐phase‐dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2) , 1992, Molecular microbiology.

[11]  M. Bibb,et al.  Stationary‐phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated , 1993, Molecular microbiology.

[12]  H. Shinkawa,et al.  Nucleotide sequence of a principal sigma factor gene (hrdB) of Streptomyces griseus. , 1995, Journal of biochemistry.

[13]  K. Chater,et al.  Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms , 1997, Journal of bacteriology.

[14]  Ann M Stock,et al.  The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor. , 1997, Structure.

[15]  J. Martín,et al.  A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds , 1997, Journal of bacteriology.

[16]  M. Bibb,et al.  A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR‐like DNA‐binding fold , 1997, Molecular microbiology.

[17]  K. Chater,et al.  Developmental Regulation of Transcription ofwhiE, a Locus Specifying the Polyketide Spore Pigment in Streptomyces coelicolor A3(2) , 1998, Journal of bacteriology.

[18]  F. Lombó,et al.  The Mithramycin Gene Cluster of Streptomyces argillaceus Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster , 1999, Journal of bacteriology.

[19]  S. Horinouchi,et al.  The A‐factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus : identification of a target gene of the A‐factor receptor , 1999, Molecular microbiology.

[20]  I. Hunter,et al.  Phosphate Control of Oxytetracycline Production by Streptomyces rimosus Is at the Level of Transcription from Promoters Overlapped by Tandem Repeats Similar to Those of the DNA-Binding Sites of the OmpR Family , 1999, Journal of bacteriology.

[21]  M. Fernández-Moreno,et al.  Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis inStreptomyces coelicolor A3(2) as a DNA-Binding Protein , 1999, Journal of bacteriology.

[22]  T. Kieser.  Practical streptomyces genetics , 2000 .

[23]  T. Beppu,et al.  Characterization of mutants defective in melanogenesis and a gene for tyrosinase of Streptomyces griseus. , 2001, The Journal of antibiotics.

[24]  C. Hutchinson,et al.  Mapping the DNA‐binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI , 2002, Molecular microbiology.

[25]  S. Horinouchi,et al.  A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. , 2002, Frontiers in bioscience : a journal and virtual library.

[26]  P. Bruheim,et al.  High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actII-ORF4 , 2002, Journal of Industrial Microbiology and Biotechnology.

[27]  J. Martín,et al.  CcaR Is an Autoregulatory Protein That Binds to the ccaR and cefD-cmcI Promoters of the Cephamycin C-Clavulanic Acid Cluster in Streptomyces clavuligerus , 2002, Journal of bacteriology.

[28]  J. Martín,et al.  The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Neil Bate,et al.  Positive control of tylosin biosynthesis: pivotal role of TylR , 2004, Molecular microbiology.

[30]  S. Horinouchi,et al.  The A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces , 2004 .

[31]  S. Horinouchi,et al.  Structures of grixazone A and B, A-factor-dependent yellow pigments produced under phosphate depletion by Streptomyces griseus. , 2004, The Journal of antibiotics.

[32]  S. Horinouchi,et al.  DNA‐binding specificity of AdpA, a transcriptional activator in the A‐factor regulatory cascade in Streptomyces griseus , 2004, Molecular microbiology.

[33]  S. Horinouchi,et al.  Transcriptional Control by A-Factor of strR, the Pathway-Specific Transcriptional Activator for Streptomycin Biosynthesis in Streptomyces griseus , 2005, Journal of bacteriology.

[34]  S. Horinouchi,et al.  Autorepression of AdpA of the AraC/XylS family, a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus. , 2005, Journal of molecular biology.

[35]  S. Horinouchi,et al.  AdpA, a Central Transcriptional Regulator in the A-Factor Regulatory Cascade That Leads to Morphological Development and Secondary Metabolism in Streptomyces griseus , 2005, Bioscience, biotechnology, and biochemistry.

[36]  M. Bibb,et al.  Regulation of secondary metabolism in streptomycetes. , 2005, Current opinion in microbiology.

[37]  S. Horinouchi,et al.  Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes*♦ , 2006, Journal of Biological Chemistry.

[38]  S. Horinouchi,et al.  A Novel o-Aminophenol Oxidase Responsible for Formation of the Phenoxazinone Chromophore of Grixazone* , 2006, Journal of Biological Chemistry.

[39]  J. Martín,et al.  The two-component phoR-phoP system of Streptomyces natalensis: Inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis. , 2007, Metabolic engineering.

[40]  S. Horinouchi,et al.  Biosynthesis of γ-butyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces , 2007, Proceedings of the National Academy of Sciences.