DnrI of Streptomyces peucetius binds to the resistance genes, drrAB and drrC but is activated by daunorubicin.

The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

Daunorubicin forms a specific complex with a secreted serine protease of Streptomyces peucetius.

Daunorubicin forms specific complex with an extracellular protease in the Streptomyces peucetius culture. The drug-protein complex co-migrates in non-denaturing PAGE as a red band. De novo peptide sequencing by nano-LC-ESI-MS/MS and MASCOT analysis identified the daunorubicin binding protein as serine protease precursor. The same protease precursor was purified sans the daunorubicin, from the mutant named ΔDPSAmut, which is deficient in daunorubicin production. Daunorubicin was added to ΔDPSAmut culture and the protease readily formed the daunorubicin-protease complex. Ability of serine protease precursor to form a selective complex with daunorubicin was confirmed by this study. Selective binding of protease to daunorubicin was seen as self-resistance determinant for the organism to survive toxic levels of the drug outside the cell. Daunorubicin-protease complex placed on S. peucetius lawn did not produce clearing zone around it, whereas daunorubicin purified from the complex did produce the clearing zone. Thereby it is concluded that the protease sequesters daunorubicin to prevent its entry into cells. Sequestration of daunorubicin by extracellular protease helps the organism to maintain a steady state sub-inhibitory level of drug around the cells. A new self-resistance determinant is reported here.

Regulation of daunorubicin biosynthesis in Streptomyces peucetius - feed forward and feedback transcriptional control.

Streptomyces are a major group of soil bacteria that produce wide range of bioactive compounds including antibiotics. Daunorubicin is a chemotherapeutic agent for treatment of certain types of cancer, which is produced as a secondary metabolite by S. peucetius. Owing to the significance of this drug in treating cancer, understanding the molecular mechanism of its biosynthesis will assist in the genetic manipulation of this strain for better drug yields. Additionally, the knowledge can also be applied to design hybrid antibiotics that can be made in vivo by transferring genes from one Streptomyces species to another. Biosynthesis of daunorubicin in S. peucetius is accomplished by the function of 30 enzyme-coding genes in a sequential and coordinated fashion. In addition to these enzymes, three transcriptional regulators DnrO, DnrN and DnrI regulate this multi-step process by forming a coherent feed forward loop regulatory circuit, consequently controlling the entire enzyme coding genes. Since daunorubicin is a DNA intercalating drug, maintaining an optimal intracellular drug concentration is pivotal to prevent self-toxicity. Commencement of daunorubicin biosynthesis also activates the feedback mechanisms mediated by the metabolite. At exceeding intracellular concentrations, daunorubicin intercalates into DNA sequences and impedes the binding of these transcription factors. This feedback repression is relieved by a group of self-resistance genes, which concurrently efflux the excess intracellular daunorubicin. This review will discuss the mechanistic role of each transcription factor and their interplay in initiating and maintaining the biosynthesis of daunorubicin in S. peucetius.

A novel protein that binds to dnrN-dnrO intergenic region of Streptomyces peucetius purified by DNA affinity capture has dihydrolipoamide dehydrogenase activity.

An antitumour chemotherapeutic, daunorubicin (DNR), produced by Streptomyces peucetius exhibits cytotoxic activity through topoisomerase-mediated interaction with DNA, thereby inhibiting DNA replication and repair and RNA and protein synthesis. It is synthesized by the type II polyketide pathway. Understanding molecular mechanisms that drive expression of antibiotic biosynthetic genes in response to diverse signals and chemical inducers is of considerable interest. Intergenic DNA between regulatory genes dnrN and dnrO of DNR biosynthesis pathway in S. peucetius has a promoter for transcription of dnrN in one strand and three promoters in the opposite strand for dnrO. Studies have shown that DnrO binds to a specific sequence in this region to activate transcription of dnrN. In the present study, using biotinylated intergenic DNA in combination with streptavidin magnetic beads, we have purified a protein that binds to this target sequence. The protein has been characterized by nano LC ESI MS/MS mass spectrometry. Sequence similarity searches for effective identification of protein by genome databases comparisons led to identification of a sequence-specific DNA binding protein that exhibits dihydrolipoamide dehydrogenase (DLDH) activity suggesting that this protein may be involved in regulation of DNR biosynthesis.

DrrC protein of Streptomyces peucetius removes daunorubicin from intercalated dnrI promoter.

DrrC is a DNA-binding protein of Streptomyces peucetius that provides self-resistance against daunorubicin, the antibiotic produced by the organism. DrrC was expressed in E.coli and purified by using N-terminal MBP-tag which retained DNA-binding property in spite of the tag. Mobility shift assay confirmed the interaction of 313bp DNA that has the dnrI promoter, daunorubicin and MBP-DrrC in the presence of ATP. Biotinylated and immobilized 313bp DNA was intercalated with daunorubicin to observe the release of the drug when MBP-DrrC is allowed to act on the DNA. The release of daunorubicin was recorded by absorption and fluorescence spectroscopy. The experiments proved that daunorubicin was released from DNA in the presence of MBP-DrrC. Fluorescence emission of daunorubicin had a maximum peak at 591nm. However, emission spectrum of released daunorubicin showed hypochromism with a maximum peak at 584nm that is possibly because it is in complex with MBP-DrrC. We propose that DrrC naturally binds at intercalated sites to eject daunorubicin; in the process both drug and protein are dislodged from DNA. Like UvrA, DrrC possibly scans the DNA for intercalated daunorubicin. When it encounters daunorubicin, DrrC dislodges it, thereby allowing DNA replication and transcription to go on unhindered. Thus a novel self resistance mechanism by DNA repair is mediated by DrrC.

Biophysical characterization of in vitro bound Streptomyces peucetius daunorubicin-serine protease complex.

A serine protease of Streptomyces peucetius is found in association with daunorubicin in the culture filtrate and co-purifies as a complex as reported earlier by us (Dubey et al., 2013). The same protease was purified without drug attachment from dpsA(-) mutant of S. peucetius, which does not produce daunorubicin. Drug-protein complex was made in vitro by mixing daunorubicin and the protease. Spectral analysis and circular dichroism (CD) analysis were employed to determine the interaction between daunorubicin and the protease. Our study showed that interaction of daunorubicin with the protease affects the spectral characteristics of the drug and changes the secondary structure of the protein. Thin layer chromatography (TLC) analysis showed that the drug-protein interaction results in partial conversion of the drug to aglyconic form. The complex formation implies sequestration of the drug when it attains potentially lethal level in the extracellular milieu of S. peucetius culture.

The activator/repressor protein DnrO of Streptomyces peucetius binds to DNA without changing its topology.

Regulatory proteins that bind to upstream un-translated region often control transcription of prokaryotic genes. Many of these proteins bend or distort their DNA binding sites, and the induced DNA curvature facilitates protein-protein or protein-DNA contacts essential for transcriptional regulation. DnrO is an essential transcription regulator of Streptomyces peucetius that activates daunorubicin biosynthetic pathway. It binds to a specific sequence adjacent to dnrN promoter to activate transcription. The same binding event represses its own transcription. DNA binding domain of DnrO is within 60 aa from N-terminal end of the 340 aa protein. Helix-turn-helix motif in DnrO is similar to BirA repressor of E. coli. In this study, we show that this dual functional protein does not cause any localized bending of DNA as observed by circular permutation gel shift assay. This observation complements the functional role of DnrO as an activator/repressor, since the change in DNA topology might impede the activation or repression function if this protein. This is in variance with DNA bending property of BirA repressor and many other transcription factors. The possibility of G+C rich sequences in the target DNA not favoring distortion of major groove upon protein binding is discussed.

Modulation of dnrN expression by intracellular levels of DnrO and daunorubicin in Streptomyces peucetius.

DnrO is a transcription factor that regulates biosynthesis of secondary metabolite daunorubicin (DNR) in Streptomyces peucetius. DNR is a DNA-intercalating drug widely used in cancer chemotherapy. Binding of DnrO close to its promoter fulfils dual functions, namely activation of dnrN and repression of dnrO. DnrN protein binds to a sequence close to the dnrI promoter to activate it, which is essential for turning on biosynthetic genes. In this study, we analyzed the inhibition of DNA-DnrO complex formation by DNR and its effect on dnrO and dnrN expression. The intracellular concentration of drug required to alter the expression of these two genes was determined in vitro. Based on the results, a model is proposed which describes the modulation of dnrN and dnrO expression by intracellular stoichiometric concentration of the drug DNR and protein DnrO. This regulatory mechanism would maintain optimal intracellular drug concentrations in S. peucetius. This would imply that the organism has an adaptive mechanism to escape the cytotoxicity of DNR in addition to its self-resistance.

Daunorubicin efflux in Streptomyces peucetius modulates biosynthesis by feedback regulation.

Streptomyces peucetius self-resistance genes drrA and drrB encode membrane-associated proteins that function like an ABC transporter for the efflux of daunorubicin and to maintain a constant subinhibitory physiological concentration of the drug within the cell. In this study, the drrA and drrB operons were disrupted for investigating drug production, self-resistance and regulation. The drrA-drrB null mutant was highly sensitive to daunorubicin. A 10-fold decrease in drug production was observed in the null mutant compared with the wild-type strain. We propose that the absence of a drug-specific efflux pump increases the intracellular concentration of daunorubicin, which is sensed by the organism to turn down drug production. Quantitative real-time PCR analysis of the mutant showed a drastic reduction in the expression of the key regulator dnrI and polyketide synthase gene dpsA. However, the expression of regulatory genes dnrO and dnrN was increased. Feedback regulation based on the intracellular daunorubicin concentration is discussed.

A novel extracellular protein of Streptomyces peucetius binds to daunorubicin but does not inhibit the bioactivity of the antibiotic.

Extracellular proteins of Streptomyces peucetius that bind to a red pigment were identified during the course of isolation of mutants defective in daunorubicin production. Two pigment-protein complexes were partially purified and this complex inhibited the growth of Bacillus subtilis. Routine solvent extraction could not separate the pigment from purified pigment-protein complex. Treatment with 2% SDS at 100 degrees C followed by solvent extraction also failed to separate the protein from the pigment. However, the pigment could be separated from the purified pigment-protein complex by heating it in 0.1M HCl at 100 degrees C followed by solvent extraction. The pigment extracted from the complex was analyzed by HPLC and found to be daunorubicin.