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

Abstract

Actinomycetes are gram-positive bacteria that possess an interesting life cycle involving morphological differentiation (aerial mycelia and spore formation) and specialized secondary metabolism (antibiotic production). To regulate these complex processes, the bacteria appear to have evolved an intricate regulatory hierarchy with several different mechanisms. Some of these processes are unique to the Streptomyces spp., such as the bldA gene (20), and others are well-known in eukaryotes, such as hormone-like molecules (31) and serine-threonine or tyrosine kinases (24). Other mechanisms resemble ones common in prokaryotes. An increasingly important example of the latter is the two-component system (4, 17, 46, 50), which consists of a histidine kinase that undergoes autophosphorylation in response to environmental or developmental changes and then transfers its phosphate group to an aspartyl residue of a response regulator protein (34,35,40).In our study of the biosynthesis of daunorubicin (DNR) and doxorubicin (DXR), commercially important antitumor drugs (Fig. 1), sequence analysis of the dnrN gene (see Fig. 2) showed that its product is very similar to response regulator proteins of the FixJ subfamily (13,32). The N-terminal region of DnrN has a putative phosphorylation site (D-55) and other conserved sites required for activation by phosphorylation and characteristic of response regulators. However, the D-553E (D55E) and D-553N (D55N) substitutions at the putative site of phosphorylation resulted in a reduction rather than a complete loss of DnrN activity (32). Furthermore, analysis of the 60-kb region sequenced around dnrN has not uncovered a putative sensor kinase gene, which often resides near the response regulator gene. Since DnrN lacks two key characteristics of canonical response regulators, we call it a pseudo-response regulator.Our previous results from mutant complementation (32) and transcriptional analysis (23) experiments showed that dnrN is essential for transcription of the dnrI regulatory gene and functions epistatically with respect to dnrI. DnrI, whose sequence is similar to other Streptomyces regulatory proteins such as AfsR (16), RedD (30), and actII-Orf4 (9), in turn activates the DNR and DXR structural and resistance genes (23). Among streptomycetes, this is a clear case in which a putative response regulator gene is known to govern expression of antibiotic biosynthesis and resistance genes in such a direct and essential manner.In spite of considerable information about response regulator proteins in other bacteria (1,11,37,48), so far none of them have been characterized from streptomycetes. Here we report that the DnrN protein binds specifically to the dnrI promoter region and is not likely to require phosphorylation for its binding. We also show that DNR has a positive effect on the DnrN level in Streptomyces peucetius and suggest how DNR could feedback repress dnrI expression.

Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005

“…In addition to these two regulatory genes, there is also a SARP (15) gene cinR1. In some other streptomycete antibiotic gene clusters, for example those for undecylprodigiosin (20) and daunorubicin (21), expression of a SARP is controlled by another regulatory gene. Consequently, we propose that CinK and CinR regulate transcription of cinR1.…”

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

“…Mutants of dnrN containing substitutions (D55E and D55N) at the putative site of phosphorylation were still able to restore antibiotic production to the non-producing dnrN ::aphII mutant strain, although at lower levels than the wild-type dnrN gene (Otten et al, 1995). No phosphorylation of DnrN could be detected in vivo or in vitro, and the DnrN D55N mutant protein that contains a nonphosphorylatable residue at the putative phosphorylation site exhibited the same binding affinity for the dnrI promoter as the wild-type DnrN protein in the presence of phospho donors (Furuya & Hutchinson, 1996). Since the activities of response regulator proteins are typically regulated by phosphorylation, these results raised the interesting question of how the activity of DnrN might be modulated.…”

“…DnrI is related to other Streptomyces regulatory proteins such as actII-Orf4 and redD, all of which belong to the superfamily of SARP proteins (Wietzorrek & Bibb, 1997), and activates the transcription of the structural and resistance genes of the DNR and DXR gene cluster (Madduri & Hutchinson, 1995 ;StutzmanEngwall et al, 1992). DNA-binding studies have shown that DnrI binds near the k35 region of promoters controlling early-and late-acting genes of the DNR biosynthetic pathway (Tang et al, 1996) Daunorubicin biosynthesis gene is in turn regulated by the pseudo response regulator protein DnrN, which activates the transcription of dnrI (Furuya & Hutchinson, 1996 ;Otten et al, 1995). DnrN has been shown to bind specifically to the dnrI promoter region, but not to the promoter regions of three biosynthetic genes nor to its own promoter (Furuya & Hutchinson, 1996).…”

“…DNA-binding studies have shown that DnrI binds near the k35 region of promoters controlling early-and late-acting genes of the DNR biosynthetic pathway (Tang et al, 1996) Daunorubicin biosynthesis gene is in turn regulated by the pseudo response regulator protein DnrN, which activates the transcription of dnrI (Furuya & Hutchinson, 1996 ;Otten et al, 1995). DnrN has been shown to bind specifically to the dnrI promoter region, but not to the promoter regions of three biosynthetic genes nor to its own promoter (Furuya & Hutchinson, 1996).…”

Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

“…Representative proteins are RedZ of the undecylprodigiosin pathway from S. coelicolor A3(2) (Guthrie et al, 1998) and DnrN of the daunorubicin pathway from S. peucetius (Furuya & Hutchinson, 1996). RedZ lacks an Asp residue for phosphorylation, a Tyr residue as a rotameric residue, and a Lys residue for formation of the 'acid pocket' binding site.…”