Universität Tübingen, Pharmazeutisches Institut, Auf der Morgenstelle 8, D-72076 Tübingen, Germany1
Hoechst Marion Roussel Deutschland GmbH, Process Development, D-65926 Frankfurt, Germany2
3Medical University of South Carolina, Department of Pharmaceutical Sciences, 171 Ashley Avenue, Charleston, SC 29425-2303, USA3
Author for correspondence: A. Bechthold. Tel.: +49 7071 2975483. Fax: +49 7071 295250. e-mail: andreas.bechthold{at}uni-tuebingen.de
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ABSTRACT |
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Keywords: glycosyltransferase, oxygenase, angucycline, urdamycin A, Streptomyces
Abbreviations: PKS polyketide synthase
This paper is dedicated to Professor Heinz Floss, a pioneer in the field of antibiotics, on the occasion of his 65th birthday.
The GenBank accession numbers for the sequences reported in this paper are AF164960 and AF164961.
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INTRODUCTION |
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METHODS |
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General genetic manipulation and SDS-PAGE.
Isolation of E. coli plasmid DNA, digestion of DNA with restriction endonucleases and Southern hybridization were carried out according to the directions of the supplier of kits, enzymes and reagents (Amersham). Southern hybridization was performed with Hybond N nylon membranes (Amersham). Probes were labelled with digoxigenin by using a DIG (digoxigenin) labelling and detection kit (Boehringer Mannheim). Restriction mapping, other routine molecular biology methods and SDS-PAGE (Coomassie blue staining) were performed as described by Sambrook et al. (1989) . Protoplast formation, transformation, and regeneration of protoplasts from S. fradiae Tü2717 were carried out by standard procedures (Hopwood et al., 1985
).
Library construction and screening.
A complete genomic library of S. fradiae Tü2717 was prepared in cosmid pOJ446 as described by Westrich et al. (1999) . The cosmid library was screened for hybridization using an internal fragment of a dNDP-glucose 4,6-dehydratase gene as a probe. This fragment had been obtained by PCR amplification (Decker et al., 1996
).
DNA sequencing and computer-assisted sequence analysis.
DNA was sequenced by the dideoxynucleotide chain-termination method with thermosequenase. Universal and reverse primers were used. Sequencing reactions were performed on an automated sequencer (Vistra 725) from Molecular Dynamics and on an ABI sequencer from 4-base lab. DNA sequences were analysed using the DNASIS software package (version 2, 1995; Hitachi Software Engineering). BLASTX (Altschul et al., 1997 ) was used to search the GenBank CDC translations+PDB+SWISS-PROT+Spupdate+PIR, release 2.0 for matching sequences.
Generation of a chromosomal urdGT2 mutant of S. fradiae Tü2717.
For generation of a chromosomal urdGT2 mutant of S. fradiae Tü2717 by homologous recombination the plasmid pSP-urdGT2d was constructed. A 1·9 kb SalI DNA fragment containing the 3'-terminal part of a putative NDP-hexose 3,5-epimerase gene (urdZ1), the entire glycosyltransferase gene urdGT2 and the 5'-terminal part of an NDP-hexose synthetase gene (urdG) was ligated into pSK- to create pSK-urdGT2. As urdGT2 contained three internal StyI restriction sites, a 327 bp deletion within urdGT2 could be generated by restriction of pSK-urdGT2 with StyI and religation of the resulting 4·53 kb fragment. A 1·57 kb XbaIKpnI fragment carrying the deleted urdGT2 was subcloned into the corresponding sites of pSP1, generating pSP-urdGT2d. Protoplasts of S. fradiae Tü2717 were transformed by pSP-urdGT2d. Primary transformants were selected on HA agar medium plus erythromycin. For characterization of transformants by Southern hybridization, an internal 1·7 kb SmaI fragment of the ermE gene, a 207 bp StyI fragment containing parts of urdGT2 and the 1·9 kb SalI fragment were used as probes. After screening for erythromycin sensitivity a double cross-over mutant was obtained. Chromosomal DNA from this mutant was analysed by Southern hybridization using the 1·7 kb SmaI fragment, the 207 bp StyI fragment and the 1·9 kb SalI fragment as probes.
Generation of a chromosomal urdM mutant of S. fradiae Tü2717.
To disable urdM, a 5·5 kb EcoRIBamHI fragment carrying the gene was digested with NcoI followed by religation. This created an in-frame 498 nt deletion that removed codons 415580 of urdM. After alteration, the 5 kb fragment was transferred to plasmid pKC1132 to create pKC-urdMd. pKC-urdMd was introduced into S. fradiae Tü2717 by protoplast transformation. From several resulting apramycin-resistant transformants a strain in which the integrated plasmid had been excised was obtained by screening for loss of apramycin resistance. Chromosomal DNA from this sensitive strain was examined by Southern analysis, using a 3·4 kb DNA fragment as a probe. Results of the Southern hybridization were confirmed by PCR using oligonucleotide primers urdM1 (5'-TCCTTCTCGCCGGTGACGCCGCGC-3') and urdM2 (5'-AGCACCACCGAGACCTCCAGGGCG-3'). The conditions for PCR were similar to those described by Bechthold & Floss (1994) . Reactions were performed using a GeneAmp 2400 Genetic Thermal Cycler system (Perkin Elmer).
Overexpression of urdM in E. coli.
A NdeI restriction site spanning the ATG start codon, a BamHI site 5' to the start codon and a EcoRI site 3' to the termination codon were introduced into urdM, using PCR. The template for PCR was a 7·6 kb EcoRI fragment cloned into pUC19 (p2-10). The primer sequences were: (primer M1) 5'-AGAACAGGATCCGCATATGGTCGCGCCCTC-3' and (primer M2) 5'-GAGACCTCCAGGAATTCGATGAGCATGTTC-3' (the NdeI, BamHI and EcoRI restriction sites are underlined). The conditions for PCR were similar to those described above. The PCR product was restricted with BamHI and EcoRI and ligated into the expression vector pRSETb (Invitrogen) to create pRSETb-urdM. E. coli BL21(DE3)/pLysS (Tabor & Richardson, 1985 ) was transformed by pRSETb-urdM. Bacteria were grown as described by Sambrook et al. (1991). At the end of exponential growth, T7 RNA polymerase was induced by adding IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation (15 min, 10000 g). After resuspension in SDS gel loading buffer (Sambrook et al., 1989
) cells were disrupted by heating to 100 °C. The supernatant from centrifugation (1 min, 12000 g) was analysed by SDS-PAGE.
Detection of urdamycin A and products accumulated by the mutants.
S. fradiae Tü2717 and the mutants were grown in NL111V medium (Decker & Haag, 1995 ) for 6872 h at 28 °C. Cultures were extracted with ethyl acetate. Extracts were evaporated, redissolved in methanol, and investigated by TLC analysis on silica gel plates (Merck) with CH2Cl2/CH3OH (9:1, v/v) as solvent, and by HPLC on a Hewlett Packard 1090 Liquid Chromatograph with a diode-array detector and an HP-ODS-Hypersil 5Mm, 200x2 mm column. The detection wavelength was 260 nm. The solvent system was as follows. Solvent A, acetonitrile/(H2O/H3PO4 [99·9:0·1]), 5:95 (v/v); solvent B, acetonitrile/(H2O/H3PO4 [99·9:0·1]), 42:58 (v/v); nonlinear gradient, 0100% B in 43 min at a flow rate of 0·3 ml min-1. Urdamycin A was identified by comparison with an authentic sample. The structures of urdamycin I, urdamycin J and urdamycin K were elucidated using NMR spectroscopic methods as well as mass spectrometry (Künzel et al., 1999
). Rabelomycin, the principal product of the urdM deletion mutant of S. fradiae Tü2717 (see above) was identified by its 1H NMR spectrum (Rohr et al., 1993
), and by electrospray mass spectrometry (m/z 338).
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RESULTS |
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Identification of a chromosomal urdGT2 mutant of S. fradiae Tü2717
To generate a chromosomal urdGT2 mutant of S. fradiae Tü2717 by homologous recombination the plasmid pSP-urdGT2d was constructed (Fig. 3b). This was used to introduce an in-frame deletion into urdGT2. After transformation of S. fradiae Tü2717 by pSP-urdGT2d, several erythromycin-resistant colonies were obtained. SalI-digested chromosomal DNA of such mutants was probed with a 1·7 kb SmaI fragment from pSP1 containing parts of the erythromycin-resistance gene, and with the 1·9 kb SalI fragment containing urdGT2. In mutant BF-1 the 1·7 kb probe detected a hybridizing fragment. When the 1·9 kb SalI fragment was used as a probe, a 1·55 kb band was detected. Integration of pSP-urdGT2d by a single cross-over event can take place in two different ways (upstream or downstream of the deleted fragment). Both integrations should give hybridization signals at 1·9 kb and 1·55 kb after SalI digestion when probed with the 1·9 kb SalI fragment. The absence of the 1·9 kb band in mutant BF-1 is not in accordance with one of these usual single cross-over events but it can be explained by the unusual integration event depicted in Fig. 3(c)
. Mutant BF-1-1 was obtained from mutant BF-1 after screening for loss of resistance to erythromycin. When chromosomal DNA of BF-1-1 was digested with SalI and probed with the 1·7 kb SmaI fragment no signal was detected. Using the 1·9 kb fragment as a probe a 1·55 kb band was detected, instead of the 1·9 kb signal detected in the wild-type strain (Fig. 4
). Using as a probe the 207 bp StyI fragment that had been deleted in pSP-urdGT2d no signal was detected whereas a 1·9 kb fragment was detected in the wild-type strain. These results confirmed that the expected deletion had occurred in the chromosome of BF-1-1 (Fig. 3d
). To investigate the structure of intermediates produced by mutant BF-1-1, compounds accumulated by cultures were isolated and their structures were elucidated by NMR spectroscopy (data not shown). Mutant BF-1-1 accumulated one major product, urdamycin I, and minor amounts of urdamycin J and urdamycin K (Fig. 1
) (Künzel et al., 1999
).
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Identification of a chromosomal urdM mutant of S. fradiae Tü2717
When S. fradiae Tü2717 was transformed by pKC-urdM, several apramycin-resistant colonies were obtained. Integration of pKC-urdM into the chromosome at the expected position was verified by Southern hybridization. To allow for the second recombination event, integrants were screened for apramycin sensitivity. The chromosomal mutation in mutant BF-2-1, which showed apramycin sensitivity, was analysed by Southern hybridization. A 3·4 kb SacI fragment, carrying the 3'-terminal portion of urdM, was used to probe SacI-digested chromosomal DNA. Analysis of the wild-type S. fradiae showed the expected 3·4 kb fragment after hybridization. When chromosomal DNA from clone BF-2-1 was treated similarly, one 2·9 kb fragment was detected, verifying the deletion of urdM. To confirm the presence of the deletion within the chromosome of BF-2-1 the mutant strain and the wild-type strain were individually subjected to PCR analysis. The size of the amplified fragment (0·8 kb) detected in the mutant strain was identical to the one generated with the plasmid pKC-urdMd, while the wild-type strain gave a PCR fragment of the expected higher mobility (1·3 kb). Analysis for antibiotic production showed that strain BF2-1 produced predominantly rabelomycin instead of urdamycin A (Fig. 1).
Complementation of mutant BF-2-1 with urdM and expression of urdM in E. coli
urdM, on a 7·1 kb fragment, was ligated into pUWL201 (U. Wehmeier & W. Piepersberg, personal communication) under the control of the ermE-up promoter to create pUWL-urdM. When pUWL-urdM was expressed in mutant BF2-1, production of urdamycin A should be restored, provided that no other gene of the urdamycin cluster was influenced by the deletion introduced into urdM.
UrdM was also expressed in E. coli by using an inducible T7-RNA-polymerase-dependent expression system. Before induction with IPTG, T7 lysozyme, provided by E. coli BL21(DE3)/pLysS, inhibits the T7 RNA polymerase and decreases production of UrdM. Induction with IPTG increases the amount of T7 RNA polymerase and thereby the amount of the expressed protein. SDS-PAGE was used to monitor the expression of UrdM before and after induction with IPTG (Fig. 5). Upon induction, the level of a 70·5 kDa protein in extracts of E. coli BL21(DE3)/pLysS/pRSETb-urdM increased substantially. This prominent band was not detectable in extracts of E. coli BL21(DE3)/pLysS/pRSETb, either before or after induction.
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DISCUSSION |
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Once a cyclized polyketide-derived aglycone moiety is available, two molecules of D-olivose and two of L-rhodinose must be added to it at specific sites. As aquayamycin is a central intermediate in urdamycin A biosynthesis, attachment of a D-olivose moiety at position C-9 should precede the addition of L-rhodinose at position C-12b (Rohr et al., 1993 ). The conclusion that urdGT2 encodes a glycosyltransferase was first based upon sequence similarities between its product and authentic bacterial glycosyltransferases including GraOrf14 and LanGT2. A targeted in-frame deletion of urdGT2 has now been accomplished and confirmed by Southern analysis. By introducing an in-frame deletion into urdGT2, negative effects on genes located downstream of urdGT2 could be avoided. An urdGT2 mutant accumulated urdamycins I, J and K, which are tetracyclic angucyclinones to which no C-glycosidic moiety is attached, thus implying that UrdGT2 catalyses the earliest glycosyltransfer step in the urdamycin biosynthetic pathway. This step is the C-glycosyltransfer of an activated D-olivose, since this precedes all other glycosylation steps. The structure of urdamycin I excludes its being the real substrate for the glycosyltransferase UrdGT2, and indicates that it is a shunt product derived from a hypothetical intermediate (Künzel et al., 1999
). This is further converted into urdamycin I through the influence of oxidoreductases. GraOrf14 was assigned to the transfer of L-rhodinose to granaticin and not to the transfer of D-olivose to the aglycone during granaticin B biosynthesis. GraOrf14 and UrdGT2 are indeed very similar, indicating similar functions. Therefore we might speculate that GraOrf14 is responsible for attaching D-olivose to the aglycone or may be involved in both glycosyltransfer steps.
In conclusion, the work described here has allowed the unambiguous assignment of the functions of the gene products of urdGT2 and urdM.
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ACKNOWLEDGEMENTS |
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Received 26 July 1999;
revised 14 September 1999;
accepted 22 September 1999.