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

ABSTRACT Sequencing of a 4.3-kb DNA region from the chromosome ofStreptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomycesantibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of themtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production byStreptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

[1]  S. Donadio,et al.  Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus) , 1989, Journal of bacteriology.

[2]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Rajesh P. N. Rao,et al.  Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens , 1992, Molecular microbiology.

[4]  J. Rohr,et al.  Identification of Two Genes from Streptomyces argillaceus Encoding Glycosyltransferases Involved in Transfer of a Disaccharide during Biosynthesis of the Antitumor Drug Mithramycin , 1998, Journal of bacteriology.

[5]  P. Sharp,et al.  Systematic base composition variation around the genome of Mycoplasma genitalium, but not Mycoplasma pneumoniae , 1997, Molecular microbiology.

[6]  C. Méndez,et al.  Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin , 1997, Journal of bacteriology.

[7]  C. Méndez,et al.  Characterization of Streptomyces argillaceus genes encoding a polyketide synthase involved in the biosynthesis of the antitumor mithramycin. , 1996, Gene.

[8]  F Wright,et al.  Codon usage in the G+C-rich Streptomyces genome. , 1992, Gene.

[9]  G. Besra,et al.  The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[11]  W. Remers.  The chemistry of antitumor antibiotics , 1979 .

[12]  M. Ashburner.  A Laboratory manual , 1989 .

[13]  F. Seela,et al.  Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. , 1986, Nucleic acids research.

[14]  C. Thompson,et al.  Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction. , 1982, Gene.

[15]  J. Distler,et al.  The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites , 1995, Molecular microbiology.

[16]  G. T. van der Horst,et al.  Reconstitution of a group I intron self-splicing reaction with an activator RNA. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. P. Sá.  4-Methyl phthalate catabolism in Surkholderia (Pseudomonas) cepacia Pc701: a gene encoding a phthalate-specific permease forms part of a novel gene , 1996 .

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

[19]  K. Chater,et al.  TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Bibb,et al.  Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. , 1985, Gene.

[21]  C. Khosla,et al.  Deciphering the biosynthetic origin of the aglycone of the aureolic acid group of anti-tumor agents. , 1996, Chemistry & biology.

[22]  J. Vieira,et al.  New pUC-derived cloning vectors with different selectable markers and DNA replication origins. , 1991, Gene.

[23]  Vieira Jeffrey,et al.  New pUC-derived cloning vectors with different selectable markers and DNA replication origins. , 1991 .

[24]  C. Hutchinson,et al.  Purification and characterization of the DNA‐binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius , 1996, Molecular microbiology.

[25]  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.

[26]  B. Steer,et al.  Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP. , 1993, Journal of molecular biology.

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

[28]  A. Ebert,et al.  Gene cluster for streptomycin biosynthesis in Streptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity. , 1987, Nucleic acids research.

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

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

[31]  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.

[32]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

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

[34]  C. Saint,et al.  4-Methylphthalate catabolism in Burkholderia (Pseudomonas) cepacia Pc701: a gene encoding a phthalate-specific permease forms part of a novel gene cluster. , 1996, Microbiology.

[35]  P. Bork,et al.  Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli , 1996, Current Biology.

[36]  H. Baylis,et al.  Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2). , 1987, Genes & development.

[37]  W. Bullock.  XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. , 1987 .

[38]  W. Jacobs,et al.  An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Caballero,et al.  The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of streptomyces , 1991, Cell.