Characterization of a regulatory gene essential for the production of the angucycline-like polyketide antibiotic auricin in Streptomyces aureofaciens CCM 3239

Renata Novakova, Dagmar Homerova, Lubomira Feckova and Jan Kormanec

Institute of Molecular Biology, Centre of Excellence for Molecular Medicine, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovak Republic

Correspondence
Jan Kormanec
jan.kormanec{at}savba.sk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A gene, aur1P, encoding a protein similar to the response regulators of bacterial two-component signal transduction systems, was identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. Expression of the gene was directed by a single promoter, aur1Pp, which was transcribed at low levels during the exponential phase and induced just before the stationary phase. A divergently transcribed gene, aur1R, has been identified upstream of aur1P, encoding a protein homologous to transcriptional repressors of the TetR family. The aur1P gene was disrupted in the S. aureofaciens CCM 3239 chromosome by homologous recombination. The mutation in the aur1P gene had no effect on growth and differentiation. However, biochromatographic analysis of culture extracts from the S. aureofaciens aur1P-disrupted strain revealed that auricin was not produced in the mutant. This indicated that aur1P is essential for auricin production. Transcription from the previously characterized aur1Ap promoter, directing expression of the first gene, aur1A, in the auricin gene cluster, was dramatically decreased in the S. aureofaciens CCM 3239 aur1P mutant strain. Moreover, the Aur1P protein, overproduced in Escherichia coli, was shown to bind specifically upstream of the aur1Ap promoter region. The results indicated that the Aur1P regulator activates expression of the auricin biosynthesis genes.


Abbreviations: SARP, Streptomyces antibiotic regulatory protein; TSP, transcription start point

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY956334.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Gram-positive soil bacteria of the genus Streptomyces undergo a complex process of morphological differentiation, accompanied by the production of biologically active secondary metabolites, including the majority of known antibiotics (Chater, 1998). Most of the antibiotics are produced by complex biosynthetic pathways encoded by physically clustered genes. The gene clusters are usually regulated by pathway-specific transcriptional activators that are located in these clusters. In addition, various global regulatory genes have been identified, which affect antibiotic production indirectly and have pleiotropic roles in stress response and morphological differentiation. Most of these pleiotropic regulatory genes have been shown to influence the activity of the pathway-specific regulatory genes. Expression of both types of regulatory gene is influenced by a variety of physiological and environmental factors, including growth rate, signalling molecules, imbalances in metabolism and various physiological stresses (Chater & Bibb, 1997). Investigation of the regulatory mechanisms of antibiotic production is of great interest, as these studies provide a potential platform for manipulating industrially important strains to increase production of their secondary metabolites. There are several examples of studies describing elevated levels of antibiotic production in streptomycetes as a result of overexpression of positive regulatory genes (Gramajo et al., 1993; Takano et al., 1992; Stratigopoulos et al., 2004).

In streptomycetes, several antibiotic regulatory genes have been identified that belong to the family of response regulators known as bacterial two-component signal transduction systems. These widespread regulatory systems transduce signals of external or internal conditions into the expression of particular genes. They consist of a sensor histidine kinase and a response regulator. The histidine kinase is autophosphorylated at a histidine residue in response to a specific signal, and this phoshoryl group is transferred to an aspartate residue of the response regulator. The resulting phosphorylated response regulator activates the transcription of target genes (Stock et al., 2000). The analysis of the Streptomyces coelicolor A3(2) genome sequence has revealed the presence of a high number (67) of gene pairs encoding two-component signal transduction systems (Hutchings et al., 2004). Some of the two-component systems (e.g. AbsA1/AbsA2 and CutR/CurS) belong to global regulators affecting antibiotic production in streptomycetes (Brian et al., 1996; Chang et al., 1996). However, some of the pathway-specific streptomycete antibiotic response-regulator genes (e.g. redZ, jadR1, dnrN and brpA) do not appear to be associated with a histidine kinase sensor gene and lack a conserved phosphorylated aspartate residue (Guthrie et al., 1998; Yang et al., 2001; Furuya & Hutchinson, 1996; Raibaud et al., 1991).

In Streptomyces aureofaciens CCM 3239, we have previously identified a type II polyketide synthase gene cluster, aur1, which is responsible for production of the angucycline-like polyketide antibiotic auricin (Novakova et al., 2002). Although auricin has been readily detected in the strain grown on solid Bennet medium (Novakova et al., 2002), its purification by HPLC for structural analysis has been hampered by very low yields (J. Kormanec, unpublished results). Similar problems were recently described for other homologous angucycline clusters (Lombo et al., 2004; Metsa-Ketela et al., 2004; Pang et al., 2004). It therefore seems that these polyketide synthase clusters are very tightly regulated. Investigation of this regulation may help to overcome the problem of low yield, as this knowledge could facilitate efforts to engineer strains that overproduce these secondary metabolites.

In a search for S. aureofaciens CCM 3239 promoters dependent on the homologue of the principal sigma factor HrdA (Kormanec et al., 1993), we identified a promoter directing expression of a gene, aur1P, encoding an unusual homologue of the family of response regulators of bacterial two-component systems, which proved to belong to the auricin cluster. Streptomycetes contain several close homologues of principal sigma factors. In S. aureofaciens CCM 3239, four genes, hrdA, hrdB, hrdD and hrdE, encoding homologues of principal sigma factors were identified (Kormanec et al., 1992). However, only the hrdB gene is essential for viability and has been suggested to encode a functional principal sigma factor. The function of other hrd-encoded homologues is unclear, although they are expressed at various stages during differentiation (Buttner & Lewis, 1992; Kormanec et al., 1993; Kormanec & Farkasovsky, 1993). In this paper, we provide evidence that aur1P is essential for auricin production in S. aureofaciens CCM 3239. We further describe its transcriptional regulation and show that Aur1P is the transcriptional activator that binds to the promoter that directs expression of the aur1 cluster.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
S. aureofaciens CCM 3239 wild-type was from the Czechoslovak Collection of Microorganisms, Brno, Czech Republic. S. aureofaciens CCM 3239-A4, containing the hrdA-disrupted allele (Kormanec et al., 1993), was previously prepared in our laboratory. Escherichia coli XL1Blue, which was used as a host, and plasmid pBluescript II SK+ were from Stratagene. Plasmid pSB40 (Park et al., 1989) was kindly provided by Dr M. K. Winson, University of Nottingham, UK. pPM927 (Smokvina et al., 1990) was provided by Mark J. Buttner, John Innes Institute, Norwich, UK. The plasmids pAPHII1, containing the kanamycin resistance gene aphII, and pTSR1, containing the thiostrepton resistance gene tsr, had been prepared previously (Kormanec et al., 1998). The E. coli expression plasmid pAC5mut2, containing a p15A origin of replication and a strong IPTG-inducible Ptrc promoter, is described in Novakova et al. (1998). E. coli BL21(DE3) pLysS and the vector pET28a, used for aur1P overexpression, were obtained from Novagen. Growth and transformation of S. aureofaciens strains were carried out as described in Kormanec et al. (1993). The phenotype of the S. aureofaciens CCM 3239, aur1P : : tsr mutant was analysed after growth on solid minimal medium (MM) (Kieser et al., 2000) and rich Bennet medium (Horinouchi et al., 1983). For RNA isolation from E. coli cultures, strains were inoculated into 20 ml liquid LB medium (Ausubel et al., 1995) supplemented with ampicillin (50 µg ml–1), chloramphenicol (40 µg ml–1) and 1 mM IPTG, and grown at 37 °C for 16 h. For RNA isolation from liquid-grown cultures, 109 c.f.u. of the particular S. aureofaciens strain was inoculated into 50 ml NMP liquid medium (Kieser et al., 2000) containing mannitol (0·5 %, w/v) as the carbon source, and grown at 30 °C to different growth phases. For RNA isolation from surface cultures, 108 c.f.u. of S. aureofaciens was spread on sterile cellophane membranes placed on Bennet medium (Horinouchi et al., 1983), and grown to an appropriate phase of development at 30 °C. Conditions for E. coli growth and transformation are described by Ausubel et al. (1995).

DNA manipulations.
DNA manipulations in E. coli were done as described in Ausubel et al. (1995), and those in Streptomyces according to Kieser et al. (2000). Chromosomal DNA from S. aureofaciens strains was isolated according to Kieser et al. (2000). Colony blot hybridization was performed as described in Ausubel et al. (1995). DNA fragments for S1-nuclease mapping and binding studies were isolated from agarose gels as described in Kormanec (2001). The DNA fragments and oligonucleotides were labelled at their 5' ends with [{gamma}-32P]ATP (ICN, 1·665x1014 Bq mmol–1) and T4 polynucleotide kinase (Biolabs), as described in Ausubel et al. (1995). Nucleotide sequencing was performed with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and analysed on an Applied Biosystems model 373 DNA sequencer. DNA sequence ladders G+A and T+C for S1-nuclease mapping were performed by the chemical method (Maxam & Gilbert, 1980). Site-directed mutagenesis was done with the Chameleon mutagenesis kit from Promega.

Detection of E. coli clones containing the S. aureofaciens hrdA-dependent promoter fragment.
The S. aureofaciens CCM 3239 hrdA gene (Kormanec et al., 1993) was mutagenized to introduce a single NdeI site in the start codon using a mutagenic primer MUT29 (5'-GGAGGTCGCCCATATGCAGACCCAGAC-3'). The gene was then cloned as a 1750 bp NdeI–HindIII fragment in pAC5mut2, resulting in plasmid pAC-hrdA1. An S. aureofaciens CCM 3239 genomic library was prepared by cloning partially Sau3AI-digested chromosomal DNA fragments (0·5–1·2 kb) into the BamHI site of pSB40. The library (about 80 000 clones) was transformed into E. coli XL1Blue containing the compatible plasmid pAC-hrdA1. The positive fragments containing potential hrdA-dependent promoters were screened on LBACIX plates by the procedure described by Novakova et al. (1998).

RNA isolation and S1-nuclease mapping.
Total RNA from E. coli and from S. aureofaciens CCM 3239 was prepared as described by Kormanec (2001). The integrity of the RNA was indicated by sharp rRNA bands after electrophoresis in agarose containing 2·2 M formaldehyde. High-resolution S1-nuclease mapping was done as in Kormanec (2001). Samples (40 µg) of RNA (estimated spectrophotometrically) were hybridized to approximately 0·02 pmol of suitable DNA probe labelled at the 5' end with [{gamma}-32P]ATP [~106 d.p.m. (pmol probe)–1]. The S1 probes used (Fig. 1a) were prepared as follows: probe 1 was prepared by PCR amplification from plasmid pHRDA5 using the 5' end-labelled universal oligonucleotide primer –47 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') from the lacZ{alpha} coding region and the primer mut80 (5'-GGGTTCCGCGCACATTTCCCCG-3') from the 5' region flanking the polylinker of pSB40; probe 2 was a 550 bp BamHII–NotI fragment uniquely labelled on the 5' end at the BamHI site; probe 3 was a 970 bp BsiWI–BamHI fragment uniquely labelled on the 5' end at the BsiWI site. The control hrdBp2 promoter probe has been described in Kormanec & Farkasovsky (1993). The protected DNA fragments were analysed on DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980).



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Fig. 1. (a) Genetic organization of the S. aureofaciens CCM 3239 aur1 cluster (Novakova et al., 2002). Genes are indicated by arrows, and the new regulatory genes are shown by darker arrows. The expansion shows the 3344 bp TaqI fragment cloned in the ClaI site of pBluescript II SK+, resulting in pJUP3. The hatched box marks the position of the tsr gene cloned between the NcoI and SacI sites of plasmid pJUP3, resulting in plasmid pJUP3H, which was used for the aur1P gene replacement experiments. The open box indicates the corresponding position of a 1046 bp Sau3AI positive fragment (comprising the aur1Pp promoter) of plasmid pHRDA5. The thin lines below the map represent DNA fragments (5' labelled at the end marked with an asterisk) that were used as probes in S1-nuclease mapping experiments. Bent arrows indicate the positions and direction of transcription from the aur1Ap and aur1Pp promoters. Relevant restriction sites are indicated. (b) High-resolution S1-nuclease mapping of the transcription start point (TSP) of the aur1Pp promoter in the E. coli two-plasmid system. Total RNA isolation and high-resolution S1-nuclease mapping were performed as described in Methods. The 5'-labelled DNA fragment corresponding to S1 probe 1 (Fig. 1a) was hybridized with 40 µg RNA isolated from stationary-phase-grown E. coli containing pHRDA5 and pAC5mut2 (lane 1), and pHRDA5 and pAC5-hrdA1 (lane 2). E. coli tRNA was used as control (lane C). The RNA-protected DNA fragments were analysed on DNA sequencing gels together with G+A (lane A) and T+C (lane T) sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980). The thin horizontal arrow indicates the position of the RNA-protected fragment and the thick bent vertical arrow indicates the nucleotide corresponding to the TSP. Before assigning the TSP, 1·5 nt was subtracted from the length of the protected fragment to account for the difference in the 3' ends resulting from S1-nuclease digestion and the chemical sequencing reactions. All S1-nuclease mapping experiments were performed twice with independent sets of RNA with similar results.

 
Disruption of the S. aureofaciens CCM 3239 aur1P gene cluster.
The plasmid pJUP3 contained a 3344 bp TaqI fragment in the ClaI site of pBluescript II SK+ (Fig. 1a). A 1050 bp HindIII (blunt-ended) –SacI DNA fragment from pTSR1, containing the tsr gene of Streptomyces azureus, was inserted between the NcoI (blunt-ended) and SacI sites of pJUP3. A 1300 bp KpnI–SmaI fragment of pAPHII1, containing the kanamycin-resistance gene of Tn5, was inserted between the KpnI and XhoI (blunt-ended) sites of the resulting plasmid pJUP3G, to create pJUP3H (Fig. 1a), which was finally used to transform S. aureofaciens CCM 3239 protoplasts to thiostrepton resistance, as described in Kormanec et al. (1993). Since pJUP3H was unable to replicate in S. aureofaciens CCM 3239, thiostrepton-resistant transformants were expected to arise from homologous recombination between the S. aureofaciens insert in the plasmid and the corresponding region in the chromosome. The thiostrepton-resistant clones were further examined for kanamycin sensitivity that would indicate a double crossover event. Three kanamycin-sensitive clones were identified. All the clones had similar phenotypes. One clone, S. aureofaciens CCM 3239, aur1P : : tsr, was chosen for further study.

The plasmid pPMaur1P, used for complementation of the aur1P mutation, was prepared by inserting a 1·2 kb BclI–MluI (blunt-ended) fragment of pJUP3 (containing the aur1P gene with its promoter) in pPM927 cut with BamHI and KpnI (blunt-ended).

Analysis of secondary metabolites.
S. aureofaciens CCM 3239 strains were grown for 7 days on Bennet agar medium (Horinouchi et al., 1983). The agar from three plates was extracted three times with 0·5 vol. ethylacetate. Residual water was removed by sodium sulphate, and extracts were evaporated under vacuum. The pellets were dissolved in a small amount of methanol and subjected to TLC analysis on silica gel 60 F254 plates (Merck) with n-butanol saturated with water. Dried TLC plates were placed on LB agar plates and overlaid with soft nutrient agar (Kieser et al., 2000) containing 0·1 ml of an overnight culture of Bacillus subtilis, and the agar was allowed to solidify. The agar plates were incubated for 16 h at 37 °C and screened visually for growth-inhibition zones.

Overexpression of aur1P in E. coli and protein purification.
The S. aureofaciens CCM 3239 aur1P gene was mutagenized to introduce a single NdeI site in the start codon using a mutagenic primer JAD1 (5'-GGGGGGCATATGAACCAGCGG-3') in the plasmid pJUP3 (Figs 1a and 3b). The mutagenized aur1P with the introduced NdeI site was cloned as a 2000 bp NdeI–EcoRI fragment (Fig. 1a) in the E. coli expression plasmid pET28a, and cut with NdeI/EcoRI, resulting in plasmid pET-aur1P. The DNA sequence of the fusion region was verified by sequencing. The host strain for pET series expression plasmids, E. coli BL21(DE3) pLysS, transformed with the plasmid pET-aur1P, was grown in LB medium containing 30 µg chloramphenicol ml–1 and 40 µg kanamycin ml–1 at 30 °C to OD600 0·5. Expression was induced with 1 mM IPTG. After 3 h, the cells were harvested by centrifugation at 12 000 g for 10 min, and washed by ice-cold 0·9 % (w/v) NaCl. The lysis of cells and native purification of His-tagged Aur1P protein on His-Tag Bind resin (Novagen) were carried out as directed by the manufacturer. The eluted proteins were dialysed overnight at 4 °C against the storage buffer (12·5 mM Tris/HCl, pH 7·9, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 50 %, v/v, glycerol), cleared by centrifugation at 30 000 g for 10 min, and the supernatant was stored in aliquots at –20 °C.



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Fig. 3. (a) High-resolution S1-nuclease mapping of the transcription start point (TSP) of the aur1Pp promoter directing expression of the S. aureofaciens CCM 3239 aur1P gene. Total RNA isolation and high-resolution S1-nuclease mapping were performed as described in Methods. The 5'-labelled DNA fragment corresponding to S1 probe 2 (Fig. 1a) was hybridized with 40 µg RNA isolated from S. aureofaciens CCM 3239 wild-type and hrdA mutant CCM 3239-A4 (Kormanec et al., 1993) strains grown in liquid NMP medium to the time points indicated, which corresponded to the different growth phases: exponential phase (12 h), end of exponential phase (20 h) and stationary phase (30 h). E. coli tRNA was used as a control (lane C). The RNA-protected DNA fragments were analysed on DNA sequencing gels together with G+A (lane A) and T+C (lane T) sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980). The thin horizontal arrow indicates the position of the RNA-protected fragment and the thick bent vertical arrow indicates the nucleotide corresponding to the TSP. Before assigning the TSP, 1·5 nt was subtracted from the length of the protected fragment to account for the difference in the 3' ends resulting from S1-nuclease digestion and the chemical sequencing reactions. All S1-nuclease mapping experiments were performed twice with independent sets of RNA with similar results. (b) The nucleotide sequence of the S. aureofaciens CCM 3239 aur1Pp promoter region. The deduced protein products are given in the single-letter amino-acid code in the second position of each codon. The TSP of the aur1Pp promoter is indicated by a bent arrow. The –10 and –35 boxes of the promoter are in bold type and underlined. An arrow is used to show the position and direction of the oligonucleotide primer JAD1 used for site directed mutagenesis to incorporate an NdeI site into the aur1P start codon. The sequence numbers refer to the deposited nucleotide sequence in the GenBank/EMBL/DDBJ databases under accession number AY956334.

 
Gel mobility-shift assays.
A 200 bp SmaI–EcoRI DNA fragment uniquely labelled on the 5' end at the EcoRI site was used for the binding studies. The fragment contained the aur1Ap promoter region from –167 to +32 with respect to the transcription start point (TSP) (Figs 1a and 6d). The assays were performed essentially as described by Ausubel et al. (1995). The 32P-labelled DNA fragment (0·8 ng, 1000 d.p.m.) was incubated with increasing amounts of purified Aur1P protein for 15 min at 30 °C in a 15 µl total volume of the binding buffer [12·5 mM Tris, pH 7·9, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 12 % (v/v) glycerol], 2 µg sonicated salmon sperm DNA and 4·5 µg BSA. After incubation, protein-bound and free DNA were resolved on a non-denaturing polyacrylamide gel containing 4 % (w/v) acrylamide, 0·05 % (w/v) bisacrylamide and 2·5 % (w/v) glycerol, running at 4 °C (after a 1 h prerun at 30 mA) in a high-ionic-strength buffer containing 50 mM Tris, 380 mM glycine, 2 mM EDTA, pH 8·5, at the same current. The gels were dried and exposed to X-ray film.



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Fig. 6. (a) Overproduction of Aur1P in E. coli. Samples were analysed by SDS-PAGE (12·5 % acrylamide). Cell-free extracts were prepared from E. coli BL21(DE3) pLysS carrying the corresponding plasmid grown at 30 °C before induction (lane 2) and after 3 h induction with IPTG (lanes 1 and 3). Lane 1, E. coli containing pET28a; lanes 2 and 3, E. coli containing pET-aur1P; lane 4, purified His-tagged Aur1P protein after Ni2+-affinity chromatography. Lane S, molecular mass markers. (b) Gel-mobility shift-assays of 0·8 ng of a 32P-labelled 200 bp SmaI–EcoRI fragment containing the aur1Ap promoter region (Fig. 6c) with increasing amounts of purified His-tagged Aur1P. Lane 1, labelled fragment in the absence of protein; lanes 2, 3 and 4, 180 ng, 1·8 µg and 3·6 µg, respectively, of the purified His-tagged Aur1P protein. Addition of 100 ng of the unlabelled 200 bp aur1Ap-promoter DNA fragment was used to demonstrate Aur1P binding specificity (lane 5). The arrows indicate the free DNA fragment and shifted fragments corresponding to the proposed complexes. (c) DNase I footprints of Aur1P binding to the 5' end-labelled 200 bp aur1Ap promoter DNA fragment (2 ng). The vertical bar indicates the position of the Aur1P binding site. The numbering is relative to the TSP of the aur1Ap promoter (Fig. 6c). Lane 1 is without the His-tagged Aur1P protein sample. Lanes 2 and 3 contain 5·6 and 16·8 µg, respectively, of the purified His-tagged Aur1P protein. Lanes A and T represent G+A and C+T sequencing ladders, respectively (Maxam & Gilbert, 1980). All binding experiments were performed twice with independent sets of protein samples, giving similar results. (d) The nucleotide sequence of the S. aureofaciens CCM 3239 aur1Ap promoter region. The deduced protein products are given in the single-letter amino-acid code in the second position of each codon. The TSP of the aur1Ap promoter is indicated by a bent arrow. The –10 and –35 boxes of the promoter are in bold type and underlined. The nucleotides that were protected from DNase I by Aur1P binding are shaded. Arrows denote the positions of tandem repeat sequences in the protected region. Relevant restriction sites are indicated. The sequence numbers refer to the deposited nucleotide sequence in the GenBank/EMBL/DDBJ databases under accession number AY956334.

 
DNase I footprinting.
Binding reactions were performed in 30 µl of binding buffer, essentially under the same conditions as for the gel mobility-shift assays with 2 ng of the 32P-labelled DNA fragment (8000 d.p.m.) and increasing amounts of purified Aur1P. After incubation for 15 min at 30 °C, 3 µl DNase I solution [5 U DNase I ml–1 (Boehringer Mannheim) in 100 mM MgCl2, 100 mM DTT] was added to the binding reaction. The reaction was incubated for 40 s at 37 °C, stopped by adding 7·5 µl DNase I stop buffer (3 M ammonium acetate, 0·25 M EDTA, 0·1 mg t-RNA ml–1), and extracted with 30 µl of alkaline phenol/chloroform. The aqueous phase was precipitated by adding three volumes of ethanol. The resulting pellet, after washing with 70 % (v/v) ethanol and Speed Vac drying, was suspended in 5 µl Maxam loading buffer [80 % (v/v) formamide, 1 mM EDTA, 10 mM NaOH, 0·05 % (w/v) bromophenol blue, 0·05 % (w/v) xylene cyanol FF]. The DNA fragments were analysed on 6 % DNA sequencing gels together with G+A and T+C sequencing ladders derived from the end-labelled fragment (Maxam & Gilbert, 1980). After electrophoresis, the gels were dried and exposed to an X-ray film.

Protein analysis.
Protein concentrations were determined according to Bradford (1976) with BSA as standard. Denaturing SDS-PAGE of proteins was done as described by Laemmli (1970), and gels were stained with Coomassie blue R250.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of the S. aureofaciens CCM 3239 chromosomal fragment containing the putative hrdA-dependent promoter
In order to identify S. aureofaciens CCM 3239 promoters recognized by the principal sigma factor homologue HrdA (Kormanec et al., 1993), an S. aureofaciens CCM 3239 genomic library (Methods) was screened by the procedure previously established (Novakova et al., 1998) in E. coli containing the compatible plasmid pAC-hrdA1, having the hrdA gene under the control of the IPTG-inducible trc promoter. Sequence analysis of one of the identified positive clones (the plasmid pHRDA5 carrying a 1046 bp Sau3AI DNA fragment; Fig. 1a) that was positive only in the background of the hrdA-encoded sigma factor (E. coli containing pAC-hrdA1) revealed a 3'-truncated gene encoding a protein with a high sequence similarity to response-regulator proteins of bacterial two-component signal transduction systems.

In order to identify a potential TSP for the putative hrdA-dependent promoter in E. coli, high-resolution S1-nuclease mapping was performed using 5'-labelled probe 1 and RNA isolated from E. coli containing the plasmids pHRDA5 and pAC-hrdA1 (Fig. 1a). A single RNA-protected fragment with a TSP at A, 35 bp upstream of the most likely translation initiation codon ATG of the putative response regulator gene, was identified (Fig. 1b, lane 2). This TSP was 4 bp downstream of the sequence TTGACA–N18–TATCTT, which is similar to the consensus sequence (TTGACN–N16–18–TAGAPuT) of the promoters recognized by the principal sigma factor HrdB of S. coelicolor (Brown et al., 1992). A much weaker RNA-protected fragment was identified with a control RNA from E. coli containing the plasmids pHRDA5 and pAC5mut2 (Fig. 1b, lane 1), which indicates partial recognition of the promoter by the E. coli RNA polymerase containing the principal sigma factor {sigma}70.

To clone the region downstream of the putative hrdA-dependent promoter, we used the chromosome-walking procedure. An S. aureofaciens CCM 3239 genomic library [TaqI partially digested chromosomal fragments (2–4 kb) cloned into the ClaI site of pBluescript II SK+] was hybridized with the 1050 bp EcoRI positive DNA fragment from pHRDA5. One positive clone, the plasmid pJUP3 (Fig. 1a) containing an overlapping 3·3 kb TaqI DNA fragment, was identified, and the fragment was sequenced on both strands. Interestingly, analysis of the sequence identified a complete gene encoding a putative response regulator which was immediately followed by the aur1O gene, previously identified at the beginning of the aur1 polyketide synthase gene cluster that is involved in the biosynthesis of an angucycline-like polyketide antibiotic, auricin (Novakova et al., 2002). Based on its role in auricin biosynthesis (see below), we named the response-regulator-like gene aur1P, and the putative hrdA-dependent promoter directing its expression as aur1Pp. Another divergently transcribed gene has been located upstream of the aur1P gene. This gene was named aur1R (Fig. 1a).

Characterization of the deduced protein products of aur1P and aur1R
Comparison of the deduced protein product of the aur1P gene with databases revealed significant end-to-end sequence similarity to response-regulator proteins of bacterial two-component signal transduction systems. Response regulators are characterized by an N-terminal response regulatory domain of the CheY family containing four highly conserved residues that are believed to compose the active site, with phosphorylation occurring at the second aspartate residue (amino acid 89 in Aur1P) (Stock et al., 2000). Almost all of these residues are conserved in Aur1P, except for the aspartic acid residue (amino acid 47) closest to the N-terminal end. This residue is conservatively replaced by a glutamate residue in Aur1P. However, the conservation of this aspartic residue does not seem to be so critical for the function of response regulators in streptomycetes (Hutchings et al., 2004). Moreover, all the conserved residues which make up the hydrophobic core in the response regulatory domain are also highly conserved in Aur1P (Fig. 2a).



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Fig. 2. (a) Alignment of Aur1P with the most similar response-regulator-like proteins from the Streptomyces antibiotic gene clusters and with two representatives of the OmpR family of response regulators, PhoB (P08402) and OmpR (P03025) from E. coli. Other protein sequences and accession numbers are: JadR1 (AAB36584, LndI (AAU04840, LanI (AAO32359, Med-ORF30 (BAC79018, SimReg1 (AAK06808. Conserved amino acid residues predicted to be important in ‘acid pocket’ formation of the N-terminal CheY-like regulatory domain (Stock et al., 2000) are indicated by asterisks above the sequence. Conserved residues that make up the hydrophobic core of response regulators are marked by filled squares above the sequences. Highly conserved residues of the OmpR family DNA-binding effector domain and conserved hydrophobic core residues (Martinez-Hackert & Stock, 1997) are marked by filled triangles and circles, respectively, above the sequence. The locations of the secondary structure elements of OmpR determined from its tertiary structure (Martinez-Hackert & Stock, 1997) are shown below the sequence. (b) Sequence comparison of Aur1R with the TetR-family repressor protein JadR2 from the S. venezuelae ISP5230 jadomycin B gene cluster (Yang et al., 1995) and TylQ from the Streptomyces fradiae tylosin gene cluster (Bate et al., 1999). The region with the predicted helix–turn–helix motif is indicated above the sequence. Identical residues are highlighted in black. Similar residues are shaded. The numbers refer to the deposited nucleotide sequence in databases. Sequences were retrieved from GenBank and aligned using CLUSTALX.

 
Response regulators have been categorized into several subfamilies (OmpR, NarL and NtrC) on the basis of conservation of the C-terminal effector DNA-binding domain that generally controls the adaptive response (Stock et al., 2000). Aur1P is clearly a member of the OmpR subfamily, and shows significant sequence similarity in the whole region of the OmpR effector domain (Fig. 2a). The OmpR subfamily is characterized by a specific fold that contains several {alpha}-helices and antiparallel {beta}-sheets in the C-terminal region, leading to specific winged-helical structures. The two helices, {alpha}2 and {alpha}3, and a 10-residue loop between them (Fig. 2a), function in an analogous fashion to the canonical helix–turn–helix motif. Helix {alpha}3 and the loop connecting two C-terminal {beta}-strands, {beta}6 and {beta}7, are DNA recognition sites, while the loop connecting helices {alpha}2 and {alpha}3 may interact with the {alpha}-subunit of RNA polymerase in promoting the initiation of transcription. Amino acids that form part of {alpha}1 and {alpha}2, as well as hydrophobic core residues, are highly conserved. The most conserved residues form part of the recognition helix {alpha}3 and the recognition wing W1 (Martinez-Hackert & Stock, 1997). Aup1P contains all the conserved residues of the OmpR family signature motifs. Moreover, 18 of the 22 residues that constitute the hydrophobic core of the OmpR DNA-binding fold (Martinez-Hackert & Stock, 1997) are also highly conserved in Aur1P (Fig. 2a).

Interestingly, the proteins to which Aur1P exhibited the highest overall similarity were several recently characterized Streptomyces response-regulator-like proteins that act as positive regulators for angucycline-like biosynthesis gene clusters, including JadR1 from the Streptomyces venezuelae ISP5230 jadomycin B biosynthesis gene cluster (74 % identity) (Yang et al., 2001), and LndI and LanI from the Streptomyces globisporus 1912 and Streptomyces cyanogenus S136 landomycin E and landomycin A gene clusters, (66 and 62 % identity, respectively) (Rebets et al., 2003). Two further proposed Streptomyces antibiotic regulatory proteins have also been identified as being similar to Aur1P, and they both likely belong to the same family of proteins (Fig. 2a). One of them, SimReg1 (43 % identity), is a putative regulator from the simocyclinone cluster of Streptomyces antibioticus Tu 6040, which produces an angucycline class antibiotic (Trefzer et al., 2002), but interestingly, another homologue of this family, Med-ORF30 (56 % identity), has been found in the medermycin gene cluster of Streptomyces sp. AM-7161, which produces a benzoisochromanequinone class polyketide, not an angucycline (Ichinose et al., 2003).

A comparison of the amino acid sequence of Aur1R encoded by the divergently transcribed gene with sequences in databases showed that the closest resemblance is to the potential repressor JadR2 of the S. venezuelae ISP5230 jadomycin B biosynthesis gene cluster (55 % identity) (Yang et al., 1995) and to a group of repressor proteins of the TetR family, including {gamma}-butyrolactone-binding repressor proteins from different Streptomyces species (Fig. 2b).

Transcriptional analysis of the aur1Pp promoter
In order to investigate the activity of the aur1Pp promoter in S. aureofaciens CCM 3239 and its proposed dependence upon hrdA, high-resolution S1-nuclease mapping was performed using probe 2 (Fig. 1a) and RNA isolated from S. aureofaciens CCM 3239 and its isogenic hrdA mutant, S. aureofaciens CCM 3239-A4 (Kormanec et al., 1993), during growth in liquid NMP medium with mannitol as the carbon source. As shown in Fig. 3a, a single RNA-protected fragment was identified that corresponded to the aur1Pp promoter, with a TSP at A, which is at an identical position to that of the aur1Pp promoter in the E. coli two-plasmid system. This position is 35 nt upstream of the most likely translation initiation codon, ATG (Fig. 3b). No RNA-protected fragment was identified with tRNA as a control (Fig. 3a, lane C). The promoter was induced at the late exponential phase. However, when RNA from the same time points was prepared from the S. aureofaciens CCM 3239 hrdA mutant, RNA-protected fragments with similar time-course intensities were identified that corresponded to the aur1Pp promoter (Fig. 3a). These results indicated that this promoter is likely not dependent in vivo upon hrdA in S. aureofaciens CCM 3239. A possible explanation for this discrepancy may be that this putative hrdA-dependent promoter is recognized by the principal sigma factor HrdB or some of its homologues, such as HrdD and HrdE.

Disruption of the S. aureofaciens CCM 3239 aur1P gene
In order to investigate the function of the aur1P gene, a chromosomal copy of S. aureofaciens aur1P was inactivated by a double crossover using a method for disruption of S. aureofaciens CCM 3239 genes (Kormanec et al., 1993). The thiostrepton-resistance gene, tsr, was used to replace a 347 bp NcoI–SacI fragment, removing the 5'-coding region of the aur1P gene (Fig. 1a), and this construct was inserted into the chromosome of S. aureofaciens CCM 3239 by homologous recombination, resulting in the aur1P-disrupted strain S. aureofaciens CCM 3239, aur1P : : tsr (Methods). The correct integration through a double crossover was confirmed by Southern blot hybridization (data not shown). The disruption did not affect growth and differentiation of the bacterium. S. aureofaciens CCM 3239, aur1P : : tsr was investigated for production of auricin using the Gram-positive bacterium Bacillus subtilis. Ethyl acetate extracts from solid-grown wild-type and aur1P-disrupted strains were analysed by TLC followed by a bioassay against B. subtilis (Methods). The inhibition zones, including the spot for auricin, could be identified in the case of the wild-type S. aureofaciens CCM 3239 strain, as reported previously (Novakova et al., 2002). However, the extract from the aur1P-disrupted strain lacked the inhibition zone corresponding to auricin (Fig. 4). To verify that this phenotype was solely due to the deletion of aur1P, S. aureofaciens CCM 3239, aur1P : : tsr was complemented in trans by transformation with the plasmid pPMaur1P, which contained the aur1P gene, including its promoter, cloned in the integrative plasmid pPM927 (Methods). As shown in Fig. 4, production of auricin was fully restored to the complemented strain, showing that the lack of auricin is indeed due to aur1P disruption. Thus, it indicated that the aur1P gene was essential for auricin production.



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Fig. 4. Biochromatography assay for auricin production. Extracts from S. aureofaciens CCM 3239 wild-type (lane 1), S. aureofaciens CCM 3239, aur1P : : tsr (lane 2) and S. aureofaciens CCM 3239, aur1P : : tsr transformed with pPMaur1P (lane 3) were resolved by TLC and overlaid with B. subtilis as described in Methods. The inhibition zone corresponding to auricin (Novakova et al., 2002) is indicated by an arrow.

 
Transcriptional analysis of the aur1Ap promoter in the S. aureofaciens CCM 3239 aur1P mutant
We had previously characterized the aur1Ap promoter, which directs the first characterized gene of the auricin cluster, aur1A, which in turn encodes a putative oxygenase. Expression of the promoter is induced at the time of aerial mycelium formation (Novakova et al., 2002). To investigate whether the aur1P disruption has an effect on aur1Ap transcription, S1-nuclease mapping was performed using RNA isolated from S. aureofaciens CCM 3239 wild-type and aur1P-mutant strains during differentiation on solid Bennet medium. A single RNA-protected fragment corresponding to the aur1Ap promoter was identified using probe 3 with the RNA isolated from the wild-type strain; the time-course of expression was similar to that previously published (Fig. 5a). However, the level of aur1Ap mRNA from all developmental stages was dramatically decreased in the aur1P mutant (Fig. 5a). The results indicated that the aur1P mutation dramatically affected transcription from the aur1Ap promoter. Thus, the promoter is directly or indirectly dependent upon aur1P. As an internal control, S1-nuclease mapping was performed with the same RNA samples using a probe fragment specific for the S. aureofaciens hrdBp2 promoter, which is expressed fairly constantly during differentiation. RNA-protected fragments corresponding to the hrdBp2 promoter were identified with all RNA samples (Fig. 5b).



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Fig. 5. High-resolution S1-nuclease mapping of the TSP for the aur1Ap promoter (Novakova et al., 2002) directing expression of the S. aureofaciens CCM 3239 aur1A gene. (a) The 5'-labelled DNA fragment corresponding to S1 probe 3 (Fig. 1a) was hybridized with 40 µg RNA isolated from surface cultures of S. aureofaciens CCM 3239 wild-type and the aur1P mutant, S. aureofaciens CCM 3239, aur1P : : tsr, on solid Bennet medium to the time points indicated, which corresponded to the different phases of morphological differentiation: vegetative substrate mycelium (13 h), the beginning of aerial mycelium formation (19 h) and aerial mycelium during sporulation (36 h). E. coli tRNA was used as a control (lane C). (b) Control S1-nuclease mapping experiments with the same RNA samples and a DNA probe for the hrdBp2 promoter (Kormanec & Farkasovsky, 1993). The RNA-protected DNA fragments were analysed on DNA sequencing gels together with G+A (lane A) and T+C (lane T) sequencing ladders derived from the end-labelled fragments (Maxam & Gilbert, 1980). All S1-nuclease mapping experiments were performed twice with independent sets of RNA with similar results.

 
Binding of Aur1P to the aur1Ap promoter region
To check whether the dependence of the aur1Ap promoter upon aur1P is direct, the aur1P gene was overexpressed in the E. coli T7 RNA polymerase expression system (Methods), and the purified N-terminal His-tagged Aur1P was examined for its binding to the aur1Ap promoter region. Total protein extracts of E. coli transformed with the plasmid pET-aur1P and the cloning plasmid pET28a after induction with IPTG at 30 °C were examined by SDS-PAGE. A prominent band was clearly visible after induction with IPTG (Fig. 6a). This protein was partially present in the soluble fraction of the E. coli cell extracts, and was purified by native Ni2+-affinity chromatography, as described in Methods. The estimated molecular mass of the purified His-tagged Aur1P on SDS-PAGE (33 kDa) corresponded closely to the theoretical Mr, 32 454, calculated by adding the Mr of the His-tag fusion (2181) to the predicted Mr from the Aur1P amino acid sequence (30 273). The purified protein was used in a gel mobility-shift assay with the 32P-labelled 200 bp aur1Ap promoter DNA fragment (positions –167 to +32 bp, in relation to the TSP of the aur1Ap promoter, Figs 1a and 6c). As shown in Fig. 6b, two retarded bands were clearly visible, which may correspond to two different complexes. The specificity of the interaction was demonstrated by the competitive binding of the unlabelled fragment (Fig. 6b, lane 5). These results indicate that Aur1P is capable of binding to the aur1Ap promoter region. To locate this Aur1P binding site in the aur1Ap promoter region, DNase I footprinting assays were carried out using the same 200 bp aur1Ap promoter fragment. As shown in Fig. 6c, the region from –134 to –46 bp upstream of the TSP was protected. The position of the binding site is indicated in Fig. 6d. The results of the binding studies indicated that the dependence of the aur1Ap promoter upon aur1P is direct and that Aur1P is a transcriptional activator that binds to the aur1Ap promoter and activates its expression.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Owing to the exact timing of production and the potential toxicity to their producers, antibiotic biosynthesis in streptomycetes must be tightly controlled on several levels. Regulation at the lowest level is represented by the pathway-specific transcriptional activators which are genetically linked to biosynthetic gene clusters and activate transcription of the corresponding antibiotic biosynthesis genes. The genes encoding these pathway-specific regulators have been identified in many antibiotic gene clusters, and they appear to constitute several families of homologous transcriptional regulators. Most of them belong to the growing family of Streptomyces antibiotic regulatory proteins (SARPs) that are characterized by the N-terminal-located DNA binding domain of the OmpR family of winged-helix transcription factors (Wietzorreck & Bibb, 1997). In several cases, the activity of the pathway-specific genes has been shown in turn to be regulated by genes at a higher level in the regulatory hierarchy, including global regulatory genes mediating environmental, nutritional and growth-rate effects. Several of these global regulators that govern expression of the Streptomyces pathway-specific antibiotic regulators have been shown to belong to the widespread response-regulator family of bacterial two-component signal transduction systems (Hutchings et al., 2004; Stock et al., 2000). These homologues of response regulators include DnrN, which directly governs expression of the SARP gene dnrI from the Streptomyces peucetius daunorubicin gene cluster (Furuya & Hutchinson, 1996), and RedZ, which affects the expression of the SARP gene from the S. coelicolor A3(2) undecylprodigiosin gene cluster, redD (Guthrie et al., 1998). Interestingly, these genes are not typically linked with genes encoding the corresponding sensor histidine kinases. Sequence analysis of their proposed C-terminal DNA-binding effector domains indicates that they both belong to the NarL subfamily of response regulators, which contain a fold with the typical helix–turn–helix motif (Stock et al., 2000). The NarL subfamily also includes the response regulator AbsA2, which, together with its sensor histidine kinase, AbsA1, has been shown to repress antibiotic biosynthetic genes in S. coelicolor A3(2) (Brian et al., 1996).

In the present paper, we have described a gene, aur1P, encoding a homologue of response regulators that, in contrast to the examples described above, directly and positively affected the expression of biosynthetic genes for the angucycline-like polyketide antibiotic auricin in S. aureofaciens CCM 3239, thus clearly representing the pathway-specific transcriptional activator for the aur1 antibiotic gene cluster. This conclusion is supported by the following experimental data: (i) the S. aureofaciens CCM 3239, aur1P : : tsr mutant did not produce auricin, and its production was restored by a copy of the aur1P gene including its promoter in trans in this mutant; (ii) transcription of the aur1Ap promoter, directing expression of the first gene, aur1A, encoding a putative oxygenase in the aur1 cluster, was dramatically decreased in the aur1P mutant; (iii) this dependence was shown to be direct by confirmation of binding of Aur1P to the aur1Ap promoter by in vitro DNA-binding assays. Thus, this study revealed that the new transcriptional regulator Aur1P is essential for production of auricin and directly controls the activity of the aur1Ap promoter that governs transcription of the first gene aur1A of the auricin polyketide synthase gene cluster.

While the gene was identified using our previously established E. coli two-plasmid system (Novakova et al., 1998) as being dependent upon a homologue of the principal sigma factor gene, hrdA (Kormanec et al., 1993), and the position of the putative hrdA-dependent promoter, aur1Pp, located in the E. coli system was identical in S. aureofaciens CCM 3239, the transcriptional analysis in S. aureofaciens CCM 3239 wild-type and the hrdA mutant did not confirm this dependence. It is likely that the aur1Pp promoter is recognized by the principal sigma factor HrdB or one of its homologues, such as HrdD or HrdE. All Hrd sigma factors are highly similar in the regions 2·4 and 4·2 which are responsible for interaction with cognate promoters (Kormanec et al., 1992). Thus, they can all recognize very similar promoters. In fact, using our E. coli two-plasmid system (Novakova et al., 1998) with other hrd homologues, we have found that the putative hrdA-dependent promoter aur1Pp is also active with the other homologues, HrdB and HrdD, indicating some cross-recognition of the aur1Pp promoter with additional Hrd sigma factors. However, similar S1-mapping experiments in the S. aureofaciens hrdD mutant revealed that expression of the aur1Pp promoter is not affected in this mutant (J. Kormanec, unpublished results). The aur1Pp promoter has been found to be induced at the beginning of stationary phase, which is typical for expression of pathway-specific transcriptional activators (Chater & Bibb, 1997). Considering this time-course expression of aur1Pp, it is unlikely that aur1Pp could be controlled by HrdA or HrdE, as the hrdE gene is not expressed under these conditions and the hrdA gene is expressed later during sporulation (Kormanec & Farkasovsky, 1993). As only hrdD and hrdB are expressed in substrate mycelium (Kormanec & Farkasovsky, 1993), it is likely that the aur1Pp promoter is recognized by both HrdB and HrdD in S. aureofaciens CCM 3239.

The Aur1P protein was related to several recently characterized Streptomyces response-regulator-like proteins that act as pathway-specific transcriptional activators in several, mainly angucycline, biosynthetic gene clusters (Fig. 2a). They all are distinct from the members of the SARP family in that they belong to the family of response regulators of bacterial two-component signal transduction systems and contain a winged-helix DNA-binding domain of the OmpR family in their C-terminal region, while a similar DNA-binding domain is found in the N-terminal region in the SARP family (Wietzorreck & Bibb, 1997). Moreover, all these proteins are dissimilar to two previously characterized Streptomyces response regulators of the NarL family, DnrN (Furuya & Hutchinson, 1996) and RedZ (Guthrie et al., 1998), which have been shown to govern the expression of SARP genes (13 and 15 % identity to Aur1P, respectively). Thus, it seems that this specific group of mainly angucycline-related transcriptional regulators constitutes a new, separate branch of the SARP family. Interestingly, as for redZ and dnrN, all the response-regulator-like genes of this family are also not typically linked with genes encoding the corresponding sensor histidine kinases.

In addition to Aur1P, only three members of this emerging family have been characterized in more detail and shown to be essential for antibiotic production. The first characterized, JadR1, from the S. venezuelae ISP5230 jadomycin B biosynthesis gene cluster, has been proved to be essential for biosynthesis of this antibiotic. Together with a deduced protein product, JadR2, encoded by a divergently expressed gene, this JadR1/JadR2 regulatory pair has been suggested to represent a novel two-component system linking antibiotic synthesis to stress. It has been suggested that jadR1 is not expressed under unstressed conditions, and that this absence is due to repression exerted by a proposed repressor, JadR2. However, this conclusion has not been corroborated by any transcriptional analysis (Yang et al., 2001). Interestingly, a gene highly homologous to jadR2 and similarly organized was identified upstream of aur1P (Figs 1a and 2b). Its product, Aur1R, may have a similar function to that of its homologue in the jadomycin B gene cluster. However, we could not detect any increase of auricin production after the application of different stress conditions (J. Kormanec, unpublished results). Therefore, further studies will be needed to investigate their potential stress dependence.

Two other members of this family have been identified in two landomycin gene clusters: LndI and LanI from S. globisporus 1912 and the S. cyanogenus S136 landomycin E and landomycin A gene clusters. Both have been shown to be essential for biosynthesis of the corresponding antibiotic and have been shown to be interchangeable (Rebets et al., 2003). In the course of writing our paper, LndI was confirmed to be a DNA-binding protein. By gel mobility-shift assay, LndI was shown to bind to its own promoter and to the promoter located upstream of the oxygenase gene lndEp (the homologue of aur1A in the auricin cluster: its position in the cluster is also similar). Using the EGFP reporter system, transcription of the lndIp promoter was similarly induced later in growth and spatially in the substrate mycelium (Rebets et al., 2005). However, comparison of the lndEp and aur1Ap promoters has not revealed any significant similarity (data not shown).

The in vitro binding experiments clearly indicated that Aur1P binds directly to the aur1Ap promoter region, upstream of the proposed –35 region of the promoter (Fig. 6c). This type of binding is typical for transcriptional activators. Interestingly, gel-retardation analysis of the aur1Ap promoter fragment with increasing concentrations of Aur1P resulted in two complexes (Fig. 6b). These results indicate that Aur1P may bind to two independent binding motifs present in the aur1Ap promoter region. Thus, at low concentration, Aur1P may randomly recognize and bind to one binding motif, and at saturated concentration, Aur1P may bind to two motifs. The members of the OmpR family for which DNA recognition sites have been determined appear to bind to direct repeat DNA sequences. However, there is variation in the arrangement of sites, both with respect to the number of recognition sites and the spacing between them (Martinez-Hackert & Stock, 1997). Inspection of the DNase I-protected region in the aur1Ap promoter has not revealed any similarity to the previously published binding motifs of several members of OmpR family, including Streptomyces SARP (data not shown). However, sequence analysis of this protected region has revealed a tandem repeat sequence TCCCTTG separated by a 24 bp spacer region. Moreover, both motifs were accompanied by partially similar sequence regions (CCTTG and CCT) in their vicinity (Fig. 6c). Thus, these regions might serve as binding sites for Aur1P. However, further experiments are needed to prove this hypothesis.

In conclusion, we have characterized a gene, aur1P, in the polyketide gene cluster aur1 for the angucycline-like antibiotic auricin of S. aureofaciens CCM 3239, which is essential for biosynthesis of this antibiotic. Its deduced protein product, Aur1P, strongly resembles members of the OmpR subfamily of response regulators of bacterial two-component signal transduction systems. Transcription of aur1P is induced at the onset of stationary phase. Aur1P is essential for expression of the first promoter, aur1Ap, in the aur1 gene cluster governing expression of the oxygenase gene aur1A, and it specifically binds to the promoter. DNase I footprinting analysis indicates an Aur1P-binding region from –134 to –46 bp upstream of the TSP of aur1Ap. The results indicate that Aur1P is a pathway-specific transcriptional activator for the auricin gene cluster in S. aureofaciens CCM 3239.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Mark Buttner for providing us with plasmid pPM927, and to Dr M. K. Winson for plasmid pSB40. We would like to thank Mrs Renata Knirschova for excellent technical assistance. This work was supported by grant 2/3010/23 from Slovak Academy of Sciences.


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DISCUSSION
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Received 10 March 2005; revised 18 May 2005; accepted 27 May 2005.



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