J. Bacteriol., 11 1996, 6310-6318, Vol 178, No. 21
Copyright © 1996, American Society for Microbiology

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

K Furuya and CR Hutchinson
School of Pharmacy, University of Wisconsin, Madison 53706, USA.

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.

This article has been cited by other articles:

• Tian, Y., Fowler, K., Findlay, K., Tan, H., Chater, K. F. (2007). An Unusual Response Regulator Influences Sporulation at Early and Late Stages in Streptomyces coelicolor. J. Bacteriol. 189: 2873-2885 [Abstract] [Full Text]  
• Gullon, S., Olano, C., Abdelfattah, M. S., Brana, A. F., Rohr, J., Mendez, C., Salas, J. A. (2006). Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin.. Appl. Environ. Microbiol. 72: 4172-4183 [Abstract] [Full Text]  
• Novakova, R., Homerova, D., Feckova, L., Kormanec, J. (2005). Characterization of a regulatory gene essential for the production of the angucycline-like polyketide antibiotic auricin in Streptomyces aureofaciens CCM 3239. Microbiology 151: 2693-2706 [Abstract] [Full Text]  
• Peterson, C. N., Ruiz, N., Silhavy, T. J. (2004). RpoS Proteolysis Is Regulated by a Mechanism That Does Not Require the SprE (RssB) Response Regulator Phosphorylation Site. J. Bacteriol. 186: 7403-7410 [Abstract] [Full Text]  
• 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 [Abstract] [Full Text]  
• Widdick, D. A., Dodd, H. M., Barraille, P., White, J., Stein, T. H., Chater, K. F., Gasson, M. J., Bibb, M. J. (2003). Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005. Proc. Natl. Acad. Sci. USA 100: 4316-4321 [Abstract] [Full Text]  
• Wilson, D. J., Xue, Y., Reynolds, K. A., Sherman, D. H. (2001). Characterization and Analysis of the PikD Regulatory Factor in the Pikromycin Biosynthetic Pathway of Streptomyces venezuelae. J. Bacteriol. 183: 3468-3475 [Abstract] [Full Text]  
• Otten, S. L., Olano, C., Hutchinson, C. R. (2000). The dnrO gene encodes a DNA-binding protein that regulates daunorubicin production in Streptomyces peucetius by controlling expression of the dnrN pseudo response regulator gene. Microbiology 146: 1457-1468 [Abstract] [Full Text]  
• Lomovskaya, N., Otten, S. L., Doi-Katayama, Y., Fonstein, L., Liu, X.-C., Takatsu, T., Inventi-Solari, A., Filippini, S., Torti, F., Colombo, A. L., Hutchinson, C. R. (1999). Doxorubicin Overproduction in Streptomyces peucetius: Cloning and Characterization of the dnrU Ketoreductase and dnrV Genes and the doxA Cytochrome P-450 Hydroxylase Gene. J. Bacteriol. 181: 305-318 [Abstract] [Full Text]  
• Lomovskaya, N., Doi-Katayama, Y., Filippini, S., Nastro, C., Fonstein, L., Gallo, M., Colombo, A. L., Hutchinson, C. R. (1998). The Streptomyces peucetius dpsY and dnrX Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis. J. Bacteriol. 180: 2379-2386 [Abstract] [Full Text]