Centro Nacional de Biotecnologia, Campus de la Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain1
Author for correspondence: Francisco Malpartida. Tel: +34 91 5854548. Fax: +34 91 5854506. e-mail: fmalpart{at}cnb.uam.es
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ABSTRACT |
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Keywords: actII-ORF4, actinorhodin, antibiotic, heterologous complementation, regulation
Abbreviations: PKS, polyketide synthase
The GenBank accession number for the sequence reported in this paper is Y19177.
a Present address: Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
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INTRODUCTION |
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The clusters encoding different metabolic steps for a particular biosynthetic pathway usually include specific regulatory genes. In most cases such regulatory genes are positive regulators, such as those for actinorhodin (actII-ORF4; Fernández-Moreno et al., 1991 ), undecylprodigiosin (redD; Narva & Feitelson, 1990
), daunorubicin (dnrI; Stutzman-Engwall et al., 1992
) and mithramycin (mtmR; Lombó et al., 1999
), among others. These pathway-specific regulators have been recently grouped within the SARP family (Wietzorrek & Bibb, 1997
), a group of Streptomyces regulatory proteins involved in the transcriptional regulation of antibiotic biosynthetic genes. The pathway-specific regulator is also interconnected with other aspects of Streptomyces physiology, such as morphological differentiation or carbon metabolism (Bibb, 1996
; Chakraburtty et al., 1996
; Martínez-Costa et al., 1996
). The mechanisms to globally regulate these complex processes in Streptomyces have been a topic for extensive analysis in several laboratories, giving rise to the isolation of several structurally different genes, such as bldA (Lawlor et al., 1987
), the two-component systems afsR/afsK (Hong et al., 1991
) and afsQ1/afsQ2 (Ishizuka et al., 1992
), and abaB (Scheu et al., 1997
).
The characterization of genes encoding polyketide biosynthesis is interesting for two reasons: on the one hand, the cloned biosynthetic genes can be useful to make libraries for combinatorial biosynthesis (Hopwood, 1997); on the other hand they might provide tools to overcome one of the limiting steps for antibiotic production, such as the control gene(s) for antibiotic biosynthetic pathways (Strauch et al., 1991 ). This paper describes the isolation and some preliminary characterization of a cluster of polyketide biosynthetic genes from Streptomyces antibioticus and a transcriptional regulatory gene linked to the cluster. We believe that these studies on the understanding of the basis of the regulation of antibiotic production in Streptomyces could contribute to improve our knowledge of this complicated control network.
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METHODS |
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The Streptomyces plasmid vector was the low-copy-number SCP2 derivative pIJ941 (Lydiate et al., 1985 ); its derivatives were those depicted in Fig. 1
. pVR22 was constructed in several steps. First the EcoRI (3)BamHI (5) fragment was cloned in pUC19, the 264 bp internal fragment was removed by digesting the resulting plasmid with EspI and BstEII, end-filled with the Klenow fragment of DNA polymerase and finally ligated. In the second step, the entire chromosomal insert of ant1 was rescued by partial digestion with EcoRI and cloned into the EcoRI site of pUC18; its XbaI (4)XbaI (polylinker fragment of pUC18) sequence was used to replace the XbaI (4)BamHI (5) fragment of the construction obtained in the first step, giving rise to a final recombinant plasmid carrying the truncated ORF0 and ORFs 14. This plasmid was then cloned into pIJ941.
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DNA sequencing.
DNA sequencing was carried out by using the dideoxy-chain termination method (Sanger et al., 1977 ). We used the 7-deaza-dGTP reagent kit from USB using the conditions recommended by the manufacturer. Convenient DNA fragments were previously cloned in either M13mp18 or M13mp19 vectors from suitable restriction fragments. Identification of DNA sequence in S1 protection experiments was carried out by the Maxam & Gilbert (1980)
method.
Computer analysis of sequences.
The DNA sequence was analysed using the software programs of the University of Wisconsin Genetic Computer Group (Devereux et al., 1984 ). For analysis of ORFs we used CODONPREFERENCE with a codon usage table made from 100 Streptomyces genes (Wright & Bibb, 1992
). Comparisons of sequences were made against the EMBL nucleic acid database and the SWISS-PROT database by using FASTA and TFASTA.
DNA and RNA manipulations.
For isolation, cloning and manipulation of nucleic acids, the methods of Hopwood et al. (1985) were used for Streptomyces spp. and of Maniatis et al. (1982)
for E. coli. For high-resolution S1 mapping, the method of Murray (1986)
was used. The growth medium was liquid SY inoculated with pre-germinated spores (Hopwood et al., 1985
); RNA preparations were made at several time points as indicated.
Expression of ORF0 in E. coli.
ORF0 protein was overproduced in E. coli by using the pAZe3ss expression system (Zaballos et al., 1987). The translation start codon was changed by using PCR. PCR amplifications (Saiki et al., 1988 ) were carried out with an oligonucleotide carrying an NcoI site at the ATG translation start site and Thermostase (Promega) using the conditions suggested by the manufacturer. The engineered ORF0 gene was cloned into pAZe3ss to generate pVE3 and transformed into the recipient E. coli strain (Zaballos et al., 1987
). Overnight cultures grown at 30 °C in LB medium were used to inoculate fresh medium at a dilution rate of 2%. After 1 h the cultures were induced by shifting the temperature to 42 °C and continuing growth for 23 h. Almost all of the ORF0 protein from the induced culture (estimated to be nearly 20% of the total protein) was found in inclusion bodies. The cells were harvested by centrifugation and washed with a solution containing 10 mM EDTA, 100 mM NaCl, 0·5 mM PMSF, 10% glycerol, 1 mM mercaptoethanol and 50 mM Tris/HCl, pH 8. The cells were resuspended in the same buffer and disrupted by sonication. Unbroken cells and heavy materials were fractionated by a preliminary centrifugation at 1000 g. The insoluble material was recovered from the supernatant by centrifugation at 12000 g. The pellet was washed once with a solution containing 1·5 M urea, 0·1 M Tris/HCl buffer, pH 8, and finally solubilized with a solution containing 4·5 M urea and 0·1 M Tris/HCl, pH 8. Most of the solubilized protein was ORF0, which was renatured by dialysis against a solution containing 1 M NaCl, 0·005 M DTT, 10% glycerol and 0·05 M Tris/HCl, pH 8. This material was used for further analysis.
Gel retardation assays.
The probes were end-labelled with [-32P]dCTP and Klenow fragment. Labelled DNA (2000 c.p.m.) was incubated for 15 min at room temperature with the purified ORF0 protein. The incubation mixtures contained (in a total volume of 20 µl) 0·3 M NaCl, 0·015 M Tris/HCl, pH 8, 0·0015 M DTT, 1 µg poly-dIdC and 35% glycerol with the indicated amount of ORF0 protein. ProteinDNA complexes and free DNA were resolved on 5% polyacrylamide gels. After electrophoresis, the gels were dried and analysed by autoradiography.
DNase I footprinting.
The EcoRI (3)RsaI (3.2) fragment was used as probe. The fragment was previously cloned in M13mp18 and M13mp19 to obtain both strands. The direct primer (USB) was labelled with [-32P]ATP and with the cold reverse primer used to amplify by PCR. The reactions were carried out in 60 µl of the mixture described above for the gel retardation assays. After incubation, 50 ng DNase I was added to each reaction mixture. The DNA was precipitated and applied along with the dideoxy DNA sequencing ladders to a 6% polyacrylamide gel. Sequencing reactions on the same probes were also applied to the gels. After electrophoresis, the gels were dried and analysed by autoradiography.
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RESULTS |
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Comparison of the deduced products of the ORFs with sequences in the databases showed high similarities with proteins known to be involved in the early steps of polyketide biosynthesis. ORF1, ORF2 and ORF3 show strong similarities with gene products related to either fatty acid or polyketide biosynthesis from different organisms, and could correspond to ketosynthase (or KS), chain length factor (or KSß) and acyl carrier protein, components of a minimal PKS (McDaniel et al., 1994
). The best scores were obtained with the gra (87, 61 and 65% identity to ORF1, ORF2 and ORF3, respectively; Sherman et al., 1989
), act (76, 59 and 50% identity, respectively; Fernández-Moreno et al., 1992
) and tcm (69, 50 and 46% identity, respectively; Bibb et al., 1989
) homologues. ORF2 and ORF3 contain conserved motifs (encoding cysteine and glutamine, respectively) that have been reported as active site motifs in homologous ketosynthase and chain length factors (Bisang et al., 1999
). The translated product of ORF4 shows end-to-end similarities with Gra-ORF4 (60% identity) and ActI-ORF4 (49% identity; aromatases) (Sherman et al., 1991
; Fernández-Moreno et al., 1992
). The N-terminal sequence of ORFA also shows similarities to the dehydrase domain of the same aromatases. The translated product of ORF5 revealed strong similarities with the products of the actIII (72%; Hallam et al., 1988) and gra-ORF5 (84%; Sherman et al., 1989
) genes and is thus likely to encode a ketoreductase (Hallam et al., 1988
; Bartel et al., 1990
).
The deduced product of ORF0 has significant similarities to members of the LysR family, known as transcriptional regulators (Schell, 1993 ). The members of this family have high similarities within the N-terminal domain they contain a characteristic helixturnhelix DNA-binding domain (which is present in ORF0) but lower, if any, similarities along the C-terminal domain (involved in the binding to different ligands). Nevertheless, ORF0 shows 30% identity end-to-end to NodD proteins, which are involved in the recognition and response to flavonoids (members of the polyketide family; Downie & Johnson, 1986
).
Expression and complementation analysis
To characterize the putative function of the cloned genes, the DNA fragments from the EMBL4 vector, ant1 and ant3, were cloned on the low-copy-number vector pIJ941 to produce pMH4 and pMH9, respectively. These plasmids were introduced by transformation into several S. coelicolor act mutants and the transformants were tested for actinorhodin production. pMH4 restored actinorhodin production in mutants TK17 (actI-), B-140 (actVII-) and JF4 (actVA-), but not in TK18 (actIII-) and JF1 (actII-). pMH9 restored actinorhodin production in mutants TK17 (actI-) and TK18 (actIII-), but not in B-140 (actVII-), JF4 (actVA-) and JF1 (actII-). The observed complementation with plasmids, after transformation of the corresponding S. coelicolor mutants, suggests that the cloned genes are involved in an unknown polyketide biosynthetic pathway.
For further characterization of the involvement of ORF0 in the expression of the cloned polyketide biosynthetic genes, several clones were constructed (see Fig. 1). The fragment containing ORF04 was subcloned into the low-copy-number vector pIJ941 to generate pVR20; pVR22 was generated by deleting from pVR20 the 264 bp EspI (3.6)BstEII (3.7) fragment which is internal to ORF0 (see Methods). Both constructs were used to transform the S. coelicolor actI mutant MAFM0195 (see Table 1
). Only pVR20 could restore actinorhodin production, suggesting that ORF0 is needed for expression of the cloned PKS, at least under heterologous conditions.
Transcription of the cloned genes
The transcriptional analysis of the cloned ORFs was determined by high-resolution S1 nuclease protection experiments. For ORF0 and ORFA determination, total S. antibioticus RNA was hybridized with the 318 bp EcoRI (3)DdeI (3.3) fragment (Fig. 1) labelled at either end. When the EcoRI (3) site was labelled, a 110 bp protected fragment was seen after S1 digestion (data not shown), while a 185 bp fragment was protected when the DdeI (3.3) site was labelled. These results show that two divergent transcripts could be generated in this region, one beginning 30 nt upstream of the ORFA start codon and the other 81 nt upstream of ORF0. This arrangement is a typical feature which has been described for many other LysR-type transcriptional activators (Schell, 1993
).
The transcriptional start point of ORF1 was determined by hybridizing total RNA with the 273 bp SacI (3.10)BclI (4.1) fragment, labelled at the 5' BclI (4.1) site. A 185 bp protected fragment was obtained after S1 digestion, indicating the presence of a transcript initiating 70 nt upstream of the ORF1 start codon Fig. 2(a).
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Characterization of the ORF0 protein
From the previous experiment it was clear that ORF0 was needed for expression of the cloned PKS genes. It was interesting to analyse the mechanism involved in the control of PKS expression more deeply. Thus, the ORF0 protein was purified from E. coli cultures to test its functional activity. The protein, purified from inclusion bodies, was analysed for its possible DNA-binding activity by gel retardation assays. The ORF0A intergenic region was chosen as the target sequence because most of the LysR regulators interact with the DNA region of their own promoter and thus regulate their own transcription. A set of overlapping DNA fragments was labelled as indicated in Methods and tested for interaction with the ORF0 protein. As shown in Fig. 3 a band-shift is clearly detected, suggesting that the intergenic region could be a target for the ORF0 protein and that the protein is functional and probably correctly folded even after renaturation from the urea solubilization step.
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To explore this possibility, the DNA-binding activity of the ORF0 protein was tested within the promoter region of the actII-ORF4 gene. The gel mobility shift assays were carried out with a 463 bp end-labelled DNA fragment and different amounts of the ORF0 protein (Fig. 5a). The results clearly show that the ORF0 protein interacts with this region, suggesting that this might be another target for the ORF0 protein. Interestingly, immediately upstream of the -35 region of the actII-ORF4 promoter (Fig. 5b
) there is a region with partial homology to the target region of the ORF0 promoter (see Fig. 5c
) which was previously determined in the DNase foot-printing assays. These sequences are not present within the PKS promoter. These results clearly suggest that an additional target for the ORF0 protein would be located immediately upstream of the actII-ORF4 promoter and thus would result in activation of actII-ORF4 transcription. It is noteworthy that ORF0, cloned in pIJ941, increased actinorhodin production in S. coelicolor J1501 by a factor of about two (data not shown).
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DISCUSSION |
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The work presented in this paper clearly shows that, in S. antibioticus, transcription of the PKS genes is temporally regulated, a feature similar to other PKS genes (Bibb, 1996 ). The PKS genes from S. antibioticus seem to be regulated by a LysR-type transcriptional regulator; the evidence presented in this paper strongly suggests that, in addition to this regulator, the control of PKS expression is also mediated by some other transcriptional regulator of the SARP family (Wietzorrek & Bibb, 1997
), similar to actII-ORF4, because the ORF0 protein recognizes the actII-ORF4 promoter region as a target. A similar LysR-type transcriptional regulator was found adjacent to the act cluster in S. coelicolor (Martínez-Costa et al., 1999
). It was shown to negatively control actinorhodin production in S. lividans, but unlike the ORF0 protein, the LysR-type regulator of S. coelicolor does not bind to the actII-ORF4 promoter region.
An interesting feature of LysR transcriptional regulators is that some of them behave as sensors of physiological changes and can bind directly to different kind of molecules. The strong similarities between the C terminus of the ORF0 protein and NodD domains in Rhizobium that interact with flavonoids (members of the polyketide family; Downie & Johnson, 1986 ) lead us to suggest, as an attractive hypothesis, a putative role of the encoded polyketide in the autoregulation of the ORF0 gene and the entire cluster. Further characterization of the cloned genes and the surrounding region will be an interesting issue for the future to clarify the complex network of regulatory signals in antibiotic regulation.
In this context this biosynthetic cluster might offer an excellent opportunity for exploring the intricate mechanisms that modulate gene expression leading to secondary metabolite production in Streptomyces.
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ACKNOWLEDGEMENTS |
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Received 15 December 2000;
revised 21 May 2001;
accepted 23 July 2001.
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