1887

Abstract

Two regulatory genes encoding a antibiotic regulatory protein () and a response regulator () of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in . Disruption of abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of resulted in only a loss of VM biosynthesis, suggesting that is a pathway-specific regulator for both VM and VS biosynthesis, and that is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that controls the biosynthetic genes for VM and VS, and controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of and is under the control of , and also contributes to the expression of VM and VS biosynthetic genes, independent of and . Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

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2009-04-01
2019-11-19
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References

  1. Adamidis, T. & Champness, W. ( 1992; ). Genetic analysis of absB, a Streptomyces coelicolor locus involved in global antibiotic regulation. J Bacteriol 174, 4622–4628.
    [Google Scholar]
  2. 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 Bacteriol 181, 6958–6968.
    [Google Scholar]
  3. Bate, N., Stratigopoulos, G. & Cundliffe, E. ( 2002; ). Differential roles of two SARP-encoding regulatory genes during tylosin biosynthesis. Mol Microbiol 43, 449–458.[CrossRef]
    [Google Scholar]
  4. Bibb, M. ( 1996; ). 1995 Colworth Prize Lecture. The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology 142, 1335–1344.[CrossRef]
    [Google Scholar]
  5. Bierman, M., Logan, R., O'Brien, 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. Gene 116, 43–49.[CrossRef]
    [Google Scholar]
  6. Di Giambattista, M., Chinali, G. & Cocito, C. ( 1989; ). The molecular basis of the inhibitory activities of type A and type B synergimycins and related antibiotics on ribosomes. J Antimicrob Chemother 24, 485–507.[CrossRef]
    [Google Scholar]
  7. Furuya, K. & Hutchinson, C. R. ( 1996; ). The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis. J Bacteriol 178, 6310–6318.
    [Google Scholar]
  8. Guthrie, E. P., Flaxman, C. S., White, J., Hodgson, D. A., Bibb, M. J. & Chater, K. F. ( 1998; ). A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket. Microbiology 144, 727–738.[CrossRef]
    [Google Scholar]
  9. Hanahan, D. ( 1983; ). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[CrossRef]
    [Google Scholar]
  10. Hutchings, M. I., Hoskisson, P. A., Chandra, G. & Buttner, M. J. ( 2004; ). Sensing and responding to diverse extracellular signals? Analysis of the sensor kinases and response regulators of Streptomyces coelicolor A3(2). Microbiology 150, 2795–2806.[CrossRef]
    [Google Scholar]
  11. Kawachi, R., Akashi, T., Kamitani, Y., Sy, A., Wangchaisoonthorn, U., Nihira, T. & Yamada, Y. ( 2000a; ). Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M1 resistance. Mol Microbiol 36, 302–313.[CrossRef]
    [Google Scholar]
  12. Kawachi, R., Wangchaisoonthorn, U., Nihira, T. & Yamada, Y. ( 2000b; ). Identification by gene deletion analysis of a regulator, VmsR, that controls virginiamycin biosynthesis in Streptomyces virginiae. J Bacteriol 182, 6259–6263.[CrossRef]
    [Google Scholar]
  13. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. ( 2000; ). Practical Streptomyces Genetics. Norwich, UK: John Innes Foundation.
  14. Kinoshita, H., Ipposhi, H., Okamoto, S., Nakano, H., Nihira, T. & Yamada, Y. ( 1997; ). Butyrolactone autoregulator receptor protein (BarA) as a transcriptional regulator in Streptomyces virginiae. J Bacteriol 179, 6986–6993.
    [Google Scholar]
  15. Kitani, S., Bibb, M., Nihira, T. & Yamada, Y. ( 2000; ). Conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces lavendulae FRI-5. J Microbiol Biotechnol 10, 535–538.
    [Google Scholar]
  16. Lawlor, E. J., Baylis, H. A. & Chater, K. F. ( 1987; ). Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2). Genes Dev 1, 1305–1310.[CrossRef]
    [Google Scholar]
  17. MacNeil, D. J., Gewain, K. M., Ruby, C. L., Dezeny, G., Gibbons, P. H. & MacNeil, T. ( 1992; ). Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61–68.[CrossRef]
    [Google Scholar]
  18. Namwat, W., Lee, C. K., Kinoshita, H., Yamada, Y. & Nihira, T. ( 2001; ). Identification of the varR gene as a transcriptional regulator of virginiamycin S resistance in Streptomyces virginiae. J Bacteriol 183, 2025–2031.[CrossRef]
    [Google Scholar]
  19. Namwat, W., Kamioka, Y., Kinoshita, H., Yamada, Y. & Nihira, T. ( 2002; ). Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae. Gene 286, 283–290.[CrossRef]
    [Google Scholar]
  20. Nihira, T., Shimizu, Y., Kim, H. S. & Yamada, Y. ( 1988; ). Structure–activity relationships of virginiae butanolide C, an inducer of virginiamycin production in Streptomyces virginiae. J Antibiot (Tokyo) 41, 1828–1837.[CrossRef]
    [Google Scholar]
  21. O'Connor, T. J. & Nodwell, J. R. ( 2005; ). Pivotal roles for the receiver domain in the mechanism of action of the response regulator RamR of Streptomyces coelicolor. J Mol Biol 351, 1030–1047.[CrossRef]
    [Google Scholar]
  22. Paget, M. S., Chamberlin, L., Atrih, A., Foster, S. J. & Buttner, M. J. ( 1999; ). Evidence that the extracytoplasmic function sigma factor σ E is required for normal cell wall structure in Streptomyces coelicolor A3(2). J Bacteriol 181, 204–211.
    [Google Scholar]
  23. Pulsawat, N., Kitani, S. & Nihira, T. ( 2007; ). Characterization of biosynthetic gene cluster for the production of virginiamycin M, a streptogramin type A antibiotic, in Streptomyces virginiae. Gene 393, 31–42.[CrossRef]
    [Google Scholar]
  24. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  25. Sheldon, P. J., Busarow, S. B. & Hutchinson, C. R. ( 2002; ). Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI. Mol Microbiol 44, 449–460.[CrossRef]
    [Google Scholar]
  26. 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 Biol 369, 322–333.[CrossRef]
    [Google Scholar]
  27. Yanagimoto, M. ( 1983; ). Novel actions of inducer in staphylomycin production by Streptomyces virginiae. J Ferment Technol 61, 443–448.
    [Google Scholar]
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Oligonucleotides used in this study [ PDF] (45 kb) Disruption of , and genes in . (a-c) Schematic representation of the strategy for the gene disruption of (a), (b) and (c). (d) Southern blot hybridization analysis of the wild-type strain, and the mutants IC102, IC103 and IC104. Genomic DNAs were digested with HI, I and I for , and mutants, respectively. Plasmid-integrated strains are mutants in which the disruption plasmids were introduced into the genome by single-crossover homologous recombination. [ PDF] (69 kb) Transcriptional analysis of genes in the corresponding mutant. Transcripts from mutants (a) IC102 (Δ ), (b) IC103 (Δ ) and (c) IC104 (Δ ) were analysed. Total RNAs were extracted from mycelia harvested at 14 h cultivation. Each transcript (I) was amplified with the primer sets as used in Fig. 2(a) of the main paper, and each transcript (II) was amplified with primer sets that were designed (Table S1) to detect cDNA corresponding to downstream of the mutated region. The gene was used as a control. -RT indicates PCR product performed without the initial reverse transcription step in RT-PCR. The cycle numbers for PCR were 27 cycles for genes and 25 cycles for . [ PDF] (108 kb)

PDF

Oligonucleotides used in this study [ PDF] (45 kb) Disruption of , and genes in . (a-c) Schematic representation of the strategy for the gene disruption of (a), (b) and (c). (d) Southern blot hybridization analysis of the wild-type strain, and the mutants IC102, IC103 and IC104. Genomic DNAs were digested with HI, I and I for , and mutants, respectively. Plasmid-integrated strains are mutants in which the disruption plasmids were introduced into the genome by single-crossover homologous recombination. [ PDF] (69 kb) Transcriptional analysis of genes in the corresponding mutant. Transcripts from mutants (a) IC102 (Δ ), (b) IC103 (Δ ) and (c) IC104 (Δ ) were analysed. Total RNAs were extracted from mycelia harvested at 14 h cultivation. Each transcript (I) was amplified with the primer sets as used in Fig. 2(a) of the main paper, and each transcript (II) was amplified with primer sets that were designed (Table S1) to detect cDNA corresponding to downstream of the mutated region. The gene was used as a control. -RT indicates PCR product performed without the initial reverse transcription step in RT-PCR. The cycle numbers for PCR were 27 cycles for genes and 25 cycles for . [ PDF] (108 kb)

PDF

Oligonucleotides used in this study [ PDF] (45 kb) Disruption of , and genes in . (a-c) Schematic representation of the strategy for the gene disruption of (a), (b) and (c). (d) Southern blot hybridization analysis of the wild-type strain, and the mutants IC102, IC103 and IC104. Genomic DNAs were digested with HI, I and I for , and mutants, respectively. Plasmid-integrated strains are mutants in which the disruption plasmids were introduced into the genome by single-crossover homologous recombination. [ PDF] (69 kb) Transcriptional analysis of genes in the corresponding mutant. Transcripts from mutants (a) IC102 (Δ ), (b) IC103 (Δ ) and (c) IC104 (Δ ) were analysed. Total RNAs were extracted from mycelia harvested at 14 h cultivation. Each transcript (I) was amplified with the primer sets as used in Fig. 2(a) of the main paper, and each transcript (II) was amplified with primer sets that were designed (Table S1) to detect cDNA corresponding to downstream of the mutated region. The gene was used as a control. -RT indicates PCR product performed without the initial reverse transcription step in RT-PCR. The cycle numbers for PCR were 27 cycles for genes and 25 cycles for . [ PDF] (108 kb)

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