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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY956334.
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INTRODUCTION |
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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), its purification by HPLC for structural analysis has been hampered by very low yields (J. Kormanec, unpublished results).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.
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METHODS |
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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 [
-32P]ATP (ICN, 1·665x1014 Bq mmol1) 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 NdeIHindIII 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 (0·51·2 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 [
-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
coding region and the primer mut80 (5'-GGGTTCCGCGCACATTTCCCCG-3') from the 5' region flanking the polylinker of pSB40; probe 2 was a 550 bp BamHIINotI fragment uniquely labelled on the 5' end at the BamHI site; probe 3 was a 970 bp BsiWIBamHI 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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The plasmid pPMaur1P, used for complementation of the aur1P mutation, was prepared by inserting a 1·2 kb BclIMluI (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 NdeIEcoRI 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 ml1 and 40 µg kanamycin ml1 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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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.
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RESULTS |
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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 TTGACAN18TATCTT, which is similar to the consensus sequence (TTGACNN1618TAGAPuT) 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
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 (24 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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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
-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 NcoISacI 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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 10 March 2005;
revised 18 May 2005;
accepted 27 May 2005.
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