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. 1996 Nov;178(21):6310–6318. doi: 10.1128/jb.178.21.6310-6318.1996

The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis.

K Furuya 1, C R Hutchinson 1
PMCID: PMC178506  PMID: 8892835

Abstract

DnrN, a protein essential for the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius, was overproduced in Escherichia coli and S. peucetius. The cell-free extract from E. coli was used to conduct DNA-binding assays. The results of gel mobility shift analysis showed that DnrN binds specifically to the dnrI promoter region with a high affinity (Kd = 50 nM). Neither acetyl phosphate nor ATP affected the binding ability, and there was no difference in binding between wild-type DnrN and a mutant form (D-55-->N) lacking the putative phosphorylation site (aspartate 55) of a response regulator protein. Therefore, phosphorylation of DnrN apparently is not necessary for DNA binding. DNase I footprinting analysis indicated binding regions at 37 to 55 bp and 62 to 100 bp upstream of the transcriptional start point of dnrI. Interestingly, the sequence of these regions includes consecutive overlapping triplets [5'-(A/T)GC, 5'-(A/T)CG, 5'-(A/T)C(A/T)] that have been shown to be the preferential binding site of daunorubicin (J. B. Chaires and J. E. Herrera, Biochemistry 29:6145-6153, 1990). This may explain why daunorubicin appeared to inhibit the binding of DnrN to the dnrI promoter, which could result in feedback repression of daunorubicin production. The results of Western blotting (immunoblotting) analysis with His-tagged DnrN antiserum showed that dnrN expression is coincident with daunorubicin production and that the maximum level of DnrN is 0.01% of total protein in the wild-type S. peucetius strain. Since the level of DnrN was lowered in mutant strains that do not produce daunorubicin, we speculate that dnrN and dnrI expression are regulated by daunorubicin.

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Selected References

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  1. Boucher P. E., Stibitz S. Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. J Bacteriol. 1995 Nov;177(22):6486–6491. doi: 10.1128/jb.177.22.6486-6491.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  4. Brian P., Riggle P. J., Santos R. A., Champness W. C. Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system. J Bacteriol. 1996 Jun;178(11):3221–3231. doi: 10.1128/jb.178.11.3221-3231.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carey J. Gel retardation. Methods Enzymol. 1991;208:103–117. doi: 10.1016/0076-6879(91)08010-f. [DOI] [PubMed] [Google Scholar]
  6. Chaires J. B. Allosteric conversion of Z DNA to an intercalated right-handed conformation by daunomycin. J Biol Chem. 1986 Jul 5;261(19):8899–8907. [PubMed] [Google Scholar]
  7. Chaires J. B., Herrera J. E., Waring M. J. Preferential binding of daunomycin to 5'ATCG and 5'ATGC sequences revealed by footprinting titration experiments. Biochemistry. 1990 Jul 3;29(26):6145–6153. doi: 10.1021/bi00478a006. [DOI] [PubMed] [Google Scholar]
  8. Fernández-Moreno M. A., Caballero J. L., Hopwood D. A., Malpartida F. The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. Cell. 1991 Aug 23;66(4):769–780. doi: 10.1016/0092-8674(91)90120-n. [DOI] [PubMed] [Google Scholar]
  9. Galinier A., Garnerone A. M., Reyrat J. M., Kahn D., Batut J., Boistard P. Phosphorylation of the Rhizobium meliloti FixJ protein induces its binding to a compound regulatory region at the fixK promoter. J Biol Chem. 1994 Sep 23;269(38):23784–23789. [PubMed] [Google Scholar]
  10. Grimm A., Madduri K., Ali A., Hutchinson C. R. Characterization of the Streptomyces peucetius ATCC 29050 genes encoding doxorubicin polyketide synthase. Gene. 1994 Dec 30;151(1-2):1–10. doi: 10.1016/0378-1119(94)90625-4. [DOI] [PubMed] [Google Scholar]
  11. Gross R., Aricò B., Rappuoli R. Families of bacterial signal-transducing proteins. Mol Microbiol. 1989 Nov;3(11):1661–1667. doi: 10.1111/j.1365-2958.1989.tb00152.x. [DOI] [PubMed] [Google Scholar]
  12. Guilfoile P. G., Hutchinson C. R. A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8553–8557. doi: 10.1073/pnas.88.19.8553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horinouchi S., Kito M., Nishiyama M., Furuya K., Hong S. K., Miyake K., Beppu T. Primary structure of AfsR, a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2). Gene. 1990 Oct 30;95(1):49–56. doi: 10.1016/0378-1119(90)90412-k. [DOI] [PubMed] [Google Scholar]
  14. Ishizuka H., Horinouchi S., Kieser H. M., Hopwood D. A., Beppu T. A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp. J Bacteriol. 1992 Dec;174(23):7585–7594. doi: 10.1128/jb.174.23.7585-7594.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iuchi S., Lin E. C. Purification and phosphorylation of the Arc regulatory components of Escherichia coli. J Bacteriol. 1992 Sep;174(17):5617–5623. doi: 10.1128/jb.174.17.5617-5623.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lawlor E. J., Baylis H. A., Chater K. F. Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2). Genes Dev. 1987 Dec;1(10):1305–1310. doi: 10.1101/gad.1.10.1305. [DOI] [PubMed] [Google Scholar]
  18. Lewis M., Chang G., Horton N. C., Kercher M. A., Pace H. C., Schumacher M. A., Brennan R. G., Lu P. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science. 1996 Mar 1;271(5253):1247–1254. doi: 10.1126/science.271.5253.1247. [DOI] [PubMed] [Google Scholar]
  19. Lomovskaya N., Hong S. K., Kim S. U., Fonstein L., Furuya K., Hutchinson R. C. The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production. J Bacteriol. 1996 Jun;178(11):3238–3245. doi: 10.1128/jb.178.11.3238-3245.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Madduri K., Hutchinson C. R. Functional characterization and transcriptional analysis of the dnrR1 locus, which controls daunorubicin biosynthesis in Streptomyces peucetius. J Bacteriol. 1995 Mar;177(5):1208–1215. doi: 10.1128/jb.177.5.1208-1215.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matsumoto A., Hong S. K., Ishizuka H., Horinouchi S., Beppu T. Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase. Gene. 1994 Aug 19;146(1):47–56. doi: 10.1016/0378-1119(94)90832-x. [DOI] [PubMed] [Google Scholar]
  22. McCleary W. R., Stock J. B. Acetyl phosphate and the activation of two-component response regulators. J Biol Chem. 1994 Dec 16;269(50):31567–31572. [PubMed] [Google Scholar]
  23. McCleary W. R., Stock J. B., Ninfa A. J. Is acetyl phosphate a global signal in Escherichia coli? J Bacteriol. 1993 May;175(10):2793–2798. doi: 10.1128/jb.175.10.2793-2798.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCleary W. R., Zusman D. R. Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity. J Bacteriol. 1990 Dec;172(12):6661–6668. doi: 10.1128/jb.172.12.6661-6668.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Meurer G., Hutchinson C. R. Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens. J Bacteriol. 1995 Jan;177(2):477–481. doi: 10.1128/jb.177.2.477-481.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moore M. H., Hunter W. N., d'Estaintot B. L., Kennard O. DNA-drug interactions. The crystal structure of d(CGATCG) complexed with daunomycin. J Mol Biol. 1989 Apr 20;206(4):693–705. doi: 10.1016/0022-2836(89)90577-9. [DOI] [PubMed] [Google Scholar]
  27. Narva K. E., Feitelson J. S. Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2). J Bacteriol. 1990 Jan;172(1):326–333. doi: 10.1128/jb.172.1.326-333.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Onaka H., Ando N., Nihira T., Yamada Y., Beppu T., Horinouchi S. Cloning and characterization of the A-factor receptor gene from Streptomyces griseus. J Bacteriol. 1995 Nov;177(21):6083–6092. doi: 10.1128/jb.177.21.6083-6092.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Otten S. L., Ferguson J., Hutchinson C. R. Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus. J Bacteriol. 1995 Mar;177(5):1216–1224. doi: 10.1128/jb.177.5.1216-1224.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Otten S. L., Stutzman-Engwall K. J., Hutchinson C. R. Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius. J Bacteriol. 1990 Jun;172(6):3427–3434. doi: 10.1128/jb.172.6.3427-3434.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pao G. M., Saier M. H., Jr Response regulators of bacterial signal transduction systems: selective domain shuffling during evolution. J Mol Evol. 1995 Feb;40(2):136–154. doi: 10.1007/BF00167109. [DOI] [PubMed] [Google Scholar]
  32. Parkinson J. S. Signal transduction schemes of bacteria. Cell. 1993 Jun 4;73(5):857–871. doi: 10.1016/0092-8674(93)90267-t. [DOI] [PubMed] [Google Scholar]
  33. Quon K. C., Marczynski G. T., Shapiro L. Cell cycle control by an essential bacterial two-component signal transduction protein. Cell. 1996 Jan 12;84(1):83–93. doi: 10.1016/s0092-8674(00)80995-2. [DOI] [PubMed] [Google Scholar]
  34. Roggiani M., Dubnau D. ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J Bacteriol. 1993 May;175(10):3182–3187. doi: 10.1128/jb.175.10.3182-3187.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stutzman-Engwall K. J., Hutchinson C. R. Multigene families for anthracycline antibiotic production in Streptomyces peucetius. Proc Natl Acad Sci U S A. 1989 May;86(9):3135–3139. doi: 10.1073/pnas.86.9.3135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stutzman-Engwall K. J., Otten S. L., Hutchinson C. R. Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol. 1992 Jan;174(1):144–154. doi: 10.1128/jb.174.1.144-154.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Summers R. G., Wendt-Pienkowski E., Motamedi H., Hutchinson C. R. Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase. J Bacteriol. 1992 Mar;174(6):1810–1820. doi: 10.1128/jb.174.6.1810-1820.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ueda K., Miyake K., Horinouchi S., Beppu T. A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators. J Bacteriol. 1993 Apr;175(7):2006–2016. doi: 10.1128/jb.175.7.2006-2016.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vara J., Lewandowska-Skarbek M., Wang Y. G., Donadio S., Hutchinson C. R. Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J Bacteriol. 1989 Nov;171(11):5872–5881. doi: 10.1128/jb.171.11.5872-5881.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Walker M. S., DeMoss J. A. NarL-phosphate must bind to multiple upstream sites to activate transcription from the narG promoter of Escherichia coli. Mol Microbiol. 1994 Nov;14(4):633–641. doi: 10.1111/j.1365-2958.1994.tb01302.x. [DOI] [PubMed] [Google Scholar]
  42. Wang A. H., Ughetto G., Quigley G. J., Rich A. Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution. Biochemistry. 1987 Feb 24;26(4):1152–1163. doi: 10.1021/bi00378a025. [DOI] [PubMed] [Google Scholar]
  43. Wray L. V., Jr, Fisher S. H. The Streptomyces coelicolor glnR gene encodes a protein similar to other bacterial response regulators. Gene. 1993 Aug 16;130(1):145–150. doi: 10.1016/0378-1119(93)90359-b. [DOI] [PubMed] [Google Scholar]
  44. Zhang R. G., Joachimiak A., Lawson C. L., Schevitz R. W., Otwinowski Z., Sigler P. B. The crystal structure of trp aporepressor at 1.8 A shows how binding tryptophan enhances DNA affinity. Nature. 1987 Jun 18;327(6123):591–597. doi: 10.1038/327591a0. [DOI] [PubMed] [Google Scholar]

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