The role of the TetR-family transcriptional regulator Aur1R in negative regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Abstract

Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.

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

“…The standard DNA manipulations in E. coli were done as described by Ausubel et al (1995), and those in Streptomyces according to Kieser et al (2000). Hybridization of a S. aureofaciens CCM 3239 cosmid library (Sau3AI partially digested DNA fragments in the BamHI site of SuperCos-1) with a probe comprising the aur1M-aur1N genes revealed 28 positive recombinant cosmids, pCosSA1-28 (Novakova et al, 2010a). One cosmid, pCosSA25, containing the largest region upstream of the aur1 cluster has been used in further studies for aur1PR2 and aur1PR3 disruption.…”

“…The Streptomyces integrative plasmid pPM927 (Smokvina et al, 1990) was used for complementation. Growth of S. aureofaciens CCM 3239 strains was carried out as described by Novakova et al (2010a). The phenotype of the S. aureofaciens Daur1PR2 and Daur1PR3 mutants was analysed after growth on solid minimal MM medium (Kieser et al, 2000) and rich Bennet medium (Horinouchi et al, 1983) and after growth in liquid minimal NMP medium (Kieser et al, 2000) and rich Bennet medium.…”

“…per pmol of probe). The S1 probes were prepared as follows: probe 1 (for the aur1PR2p promoter) was prepared by amplification of the cosmid pCosSA25 by PCR using the 59 end-labelled primer SRP4REV (59-GGGT-AGAGCGCGAGGAGCGCC-39) from the aur1PR2 coding region and the unlabelled primer SRP4DIR (59-CGGAATCCCGAAGACG-GCCCG-39) from aur1PR2 upstream region; probe 2 (for the aur1PR3p promoter) was prepared similarly by PCR amplification using 59 end-labelled primer SARP1 (59-CGAGCAGGGCCA-GCACCTGACG-39) from the aur1PR3 coding region and the unlabelled primer SARP2 (59-CCTTCTGGTCAGGAAACTCCC-39) from aur1PR3 upstream region, the probes for the aur1Ap and aur1Pp promoters were described previously (Novakova et al, 2010a), and the control hrdBp2 promoter probe was described by Kormanec & Farkasovský (1993 together with G+A and T+C sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980). Analysis of auricin production.…”

“…S. aureofaciens CCM 3239 wild-type was from the Czechoslovak Collection of Microorganisms, Brno, Czech Republic. The S. aureofaciens Daur1R mutant strain was described by Novakova et al (2010a). Escherichia coli SURE (Stratagene) was used as a host, and plasmid pBluescript II SK 2 (Stratagene) was used for E. coli cloning experiments.…”

“…A 266 bp DNA fragment (probe 2) comprising the aur1PR3p promoter region [nucleotide positions 2124 to +142 with respect to the transcription start point (TSP) for the aur1PR3p promoter] was prepared by PCR amplification from pCosSA25 using the 59 end-labelled primer SARP1 and unlabelled primer SARP2. EMSA with purified Aur1R protein or cell-free extracts was done as described recently (Novakova et al, 2010a). The 32 P-labelled DNA fragments (0.04 ng, approx.…”