Characterisation of the genes involved in the biosynthesis and attachment of the aminodeoxysugar D-forosamine in the auricin gene cluster of Streptomyces aureofaciens CCM3239.

We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The Δsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the Δsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.

A gene determining a new member of the SARP family contributes to transcription of genes for the synthesis of the angucycline polyketide auricin in Streptomyces aureofaciens CCM 3239.

Three regulators, Aur1P, Aur1R and a SARP-family Aur1PR3, have been previously found to control expression of the aur1 cluster for the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Here, we describe an additional regulatory gene, aur1PR4, encoding a homologue from the SARP-family regulators. Its role in auricin regulation was confirmed by its disruption that dramatically affected auricin production. However, transcription from the aur1Ap promoter, directing expression of 22 auricin biosynthetic genes, was not substantially affected in the Δaur1PR4 mutant. A new promoter, sa13p, directing transcription of four putative auricin tailoring genes, was found to be dependent on aur1PR4. Moreover, analysis of the sa13p promoter region revealed the presence of three heptameric repeat sequences corresponding to putative SARP-binding sites. Expression of aur1PR4 is directed by a single promoter, aur1PR4p, which is induced after entry into stationary phase. Transcription from aur1PR4p was absent in a S. aureofaciens Δaur1P mutant strain, and Aur1P was shown to bind specifically to the aur1PR4p promoter. These results indicate a complex network of regulation of the auricin gene cluster. Both Aur1P and Aur1PR3 are involved in regulation of the core aur1A-U biosynthetic genes, and Aur1PR4 in regulation of putative auricin tailoring genes.

The gene cluster aur1 for the angucycline antibiotic auricin is located on a large linear plasmid pSA3239 in Streptomyces aureofaciens CCM 3239.

We previously identified a polyketide synthase gene cluster, aur1, responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. A sequence analysis of the aur1 flanking regions revealed the presence of several genes encoding proteins homologous to those for Streptomyces linear plasmid replication, partitioning and telomere-binding. Pulse-field gel electrophoresis detected the single, 240-kb linear plasmid, pSA3239, in S. aureofaciens CCM3239. The presence of the auricin cluster in pSA3239 was confirmed by several approaches. In addition to aur1, pSA3239 also carries a large number of regulatory genes, and two gene clusters involved in the production of secondary metabolites: the aur2 cluster for an unknown secondary metabolite and the bpsA cluster for the blue pigment indigoidine.

Strict control of auricin production in Streptomyces aureofaciens CCM 3239 involves a feedback mechanism.

The polyketide gene cluster aur1 is responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is regulated in a complex manner involving several regulators, including a key pathway-specific positive regulator Aur1P that belongs to the family of 'atypical' response regulators. Production of auricin is induced after entry into stationary phase. However, auricin was produced in only a short time interval of several hours. We found that the decrease of auricin production was due to a strict regulation of auricin biosynthetic genes at the transcriptional level by a feedback mechanism; auricin and/or its intermediate(s) inhibited binding of Aur1P to its cognate biosynthetic promoter aur1Ap and consequently stopped its activation. In addition, we also determined that synthesised auricin is unstable during growth of S. aureofaciens CCM3239 in the production medium even though purified auricin is stable for days in various organic solvents. The critical parameter affecting its stability was pH. Auricin is stable at acid pH and unstable at neutral and alkaline pH. The drop in auricin concentration was due to an increase of pH shortly after induction of auricin production during cultivation of S. aureofaciens CCM3239.

Genetic manipulation of pathway regulation for overproduction of angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239.

The polyketide gene cluster aur1 is responsible for the production of the antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is low and strictly regulated by two regulators, Aur1P and Aur1R. To improve auricin yield, we genetically manipulated S. aureofaciens CCM 3239 strain to overcome this strict regulation. A regulatory region including aur1R, aur1P, aur1O and the target biosynthetic aur1Ap promoter were replaced by the strong constitutive ermEp* promoter. However, auricin production was decreased in such a genetically manipulated strain. In the second strategy we placed the aur1P gene for auricin pathway-specific activator under the control of the ermEp* promoter. The resulting strain has been shown to produce 2.8-fold higher amount of auricin compared with the WT strain.

The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239.

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

Multidrug resistant P-glycoprotein positive L1210/VCR cells are also cross-resistant to cisplatin via a mechanism distinct from P-glycoprotein-mediated drug efflux activity.

P-glycoprotein (P-gp, a drug transporter found in the plasma membrane)-mediated multidrug resistance of leukemia cells represents a real obstacle in the effective chemotherapeutic treatment of leukemia. While cisplatin (CisPt) is known to be a substance that is untransportable by P-gp, P-gp positive cells were often found to be resistant to CisPt. The aim of the current paper is to study this phenomenon using P-gp positive mouse leukemia cells L1210/VCR in which the overexpression of P-gp was induced by its ability to adapt to growth on vincristine (VCR). L1210/VCR cells are also resistant to CisPt. However, resistance to this substance could not be reversed by addition of the known P-gp inhibitor verapamil. CisPt induced more pronounced entry into apoptosis, as measured using the annexin V/propidium iodide kit, in sensitive L1210 cells than in resistant L1210/VCR cells. In addition, CisPt induced an increase in the proportion of L1210 cells that were in the g2 phase of the cell cycle when compared to L1210/VCR cells, as measured by staining with propidium iodide. Similarly, a higher release of cytochrome c from the mitochondria to the cytosol was induced by CisPt treatment in L1210 than in L1210/VCR cells. While similar levels of Bax and Bcl-2 proteins were observed in sensitive and resistant cells, CisPt induced a more pronounced decrease of the Bcl-2 levels in L1210 cells than in L1210/VCR cells. Consistent with this observation, CisPt induced a larger decrease of the Bcl-2 content in the Bcl-2:Bax heterooligomer in L1210 cells than in L1210/VCR cells. Moreover, CisPt induced a similar apoptotic DNA fragmentation pattern in both resistant and sensitive cells. All of the above observations indicated that L1210/VCR cells are also resistant to CisPt and that this resistance is related to the differences in the regulatory mechanisms responsible for CisPt-induced apoptosis in L1210/VCR cells without any contribution from the drug efflux activity of P-gp.