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.
Streptomyces bacteria are major producers of bioactive natural products, including many antibiotics. We identified a gene cluster, aur1, in a large linear plasmid of Streptomyces aureofaciens CCM3239. The cluster is responsible for the production of a new angucycline polyketide antibiotic auricin. Several tailoring biosynthetic genes were scatted in rather distant aur1 flanking regions. Auricin was produced in a very narrow growth phase interval of several hours after entry into stationary phase, after which it was degraded to non-active metabolites because of its instability at the high pH values reached after the production stage. Strict transcriptional regulation of the auricin biosynthetic gene cluster has been demonstrated, including feed-forward and feedback control by auricin intermediates via several of the huge number of regulatory genes present in the aur1 cluster. The complex mechanism may ensure strict confinement of auricin production to a specific growth stage.
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.
Streptomyces lavendulae subsp. lavendulae CCM 3239 produces the angucycline antibiotic auricin and was thought to be the type strain of Streptomyces aureofaciens. We report the complete genome sequence of this strain, which consists of a linear chromosome and the linear plasmid pSA3239, and demonstrate it to be S. lavendulae subsp. lavendulae.
The γ-butyrolactone (GBL) autoregulator-receptor systems play a role in controlling secondary metabolism and/or morphological differentiation in many Streptomyces species. We previously identified the aur1 gene cluster, located on the Streptomyces aureofaciens CCM 3239 large linear plasmid pSA3239, which is responsible for the production of the angucycline antibiotic auricin. Here, we describe the characterisation of two genes, sagA and sagR, encoding GBL autoregulatory signalling homologues, which lie in the upstream part of the aur1 cluster. SagA was similar to GBL synthases and SagR to GBL receptors. The expression of each gene is directed by its own promoter, sagAp for sagA and sagRp for sagR. Both genes were active mainly during the exponential phase, and their transcription was interdependent. The disruption of sagA abolished auricin production, while the disruption of sagR resulted in precocious but dramatically reduced auricin production. Transcription from the aur1Pp and aur1Rp promoters, which direct the expression of auricin-specific cluster-situated regulators (CSRs), was also precocious and increased in the sagR mutant strain. In addition, SagR was also shown to specifically bind both promoters in vitro. These results indicated that the SagA-SagR GBL system regulates auricin production. Unlike many other GBL receptors, SagR does not bind its own promoter, but Aur1R, an auricin-specific repressor from the family of pseudo GBL receptors, does bind both sagAp and sagRp promoters. Moreover, the expression of both promoters was deregulated in an aur1R mutant, indicating that the SagA-SagR GBL system is regulated by a feedback mechanism involving the auricin-specific CSR Aur1R, which regulates downstream.
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.
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