UNIGEN Center for Molecular Biology and Department of Biotechnology, NTNU,N-7489 Trondheim, Norway1
Nord-Trondelag College, Dept of Engineering,N-7600 Levanger, Norway2
SINTEF Applied Chemistry, SINTEF, N-7034 Trondheim, Norway3
Author for correspondence: Sergey Zotchev. Tel: +47 73 59 86 79. Fax: +47 73 59 87 05. e-mail: sergey.zotchev{at}chembio.ntnu.no
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ABSTRACT |
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Keywords: Streptomyces noursei, gene cluster, macrolide antibiotic
Abbreviations: ABC, ATP-binding cassette; PKS, polyketide synthase; TDP, deoxythymidyl diphosphate; WT, wild-type
The GenBank accession numbers for the sequences reported in this paper are AF071512 for ORF1, AF071513 for ORF2, AF071514 for ORF3, AF071515 for ORF4, AF071516 for ORF5, AF071517 for ORF6, AF071518 for ORF7, AF071519 for gdhA, AF071520 for ORF8, AF071521 for ORF9, AF071522 for ORF10 and AF071523 for ORF11.
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INTRODUCTION |
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A large number of antibiotics used in medical and veterinary care, and agriculture are of a polyketide origin. The polyketide backbone of these antibiotics is synthesized by polyketide synthase (PKS)-assisted condensation of the building units represented usually by acetyl-, propionyl- and (methyl)malonyl-CoA (Hopwood, 1997 ). Both the sequential manner in which the backbone is assembled and the fact that specific domains within a PKS are responsible for a given biosynthetic step provides an excellent opportunity for protein engineering. Indeed, several research groups have reported successful manipulations of PKS genes which resulted in biosynthesis of new compounds (for example, Kuhstoss et al., 1996
; Ruan et al., 1997
). However, the polyketide backbone by itself usually has little, if any, biological activity. Modifications of this backbone by means of hydroxylation, methylation, acylation and glycosylation are usually required for achieving full potency, and the genes providing these activities are often found in the proximity of the PKS genes (Katz & Donadio, 1995
). This fact further expands the possibilities for manipulations of the biosynthetic pathways, potentially leading to overproducing strains or new antibiotics (Sezonov et al., 1997
; Solenberg et al., 1997
).
We have initiated molecular genetic studies on Streptomyces noursei ATCC 11455, the producer of the antifungal polyene antibiotic nystatin. The latter is currently being used for the treatment of many fungal infections in humans. The structure of nystatin predicts the involvement of type I (modular) PKS enzymes in biosynthesis of its polyketide backbone, while a mycosamine moiety attached to the aglycone implies involvement of deoxysugar biosynthesis genes. Using a PCR-derived probe for the deoxythymidyl diphosphate (TDP)-glucose dehydratase gene, we have identified part of the gene cluster apparently governing the biosynthesis of a macrolide antibiotic. Gene disruption and replacement experiments showed that this gene cluster is not required for nystatin biosynthesis, but that it governs the synthesis of an antibacterial agent in S. noursei ATCC 11455.
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METHODS |
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Molecular genetic techniques.
Plasmid, phage and total DNA preparations, endonuclease digestions, ligations and fractionation were performed as described previously (Sambrook et al., 1989 ; Hopwood et al., 1985
). Modification of plasmid DNA in vitro was done according to Matsushima & Baltz (1994)
. DNA fragments were isolated from agarose gels using the Qiagen QIAEX kit, labelled by the use of the HighPrime kit from Boehringer Mannheim, and used for Southern blot analysis. PCR amplification of the gdh fragment was done according to Decker et al. (1996)
. DNA sequencing reactions were performed with a DNA sequencing kit from Applied Biosystems and analysed with an automated DNA sequencer from the same supplier. DNA sequences were analysed using the GCG program package (Devereux et al., 1984
) and deposited in GenBank.
Analysis of secondary metabolites.
For nystatin production, S. noursei strains were grown for 120 h in 50 ml SAO-23 medium (g per l: glucose, 45; NH4NO3, 2·5; corn meal, 3; MgSO4 . 7H2O, 0·4; KH2PO4, 0·2; CaCO3, 5) in shake flasks at 28 °C (220 r.p.m.). Cells were then pelleted and extracted with DMSO, and extracts analysed by HPLC. For production of antibacterial compounds, S. noursei was grown for 120 h in 50 ml MP1 medium (g per l: glucose, 40; yeast extract, 1·5; NH4NO3, 2·5; MgSO4 . 7H2O, 0·5; KH2PO4, 0·5; CaCO3, 3) in shake flasks at 28 °C (220 r.p.m.). Culture supernatants were then extracted with equal volumes of ethyl acetate, and extracts were evaporated under vacuum. The pellets were dissolved in small amounts of ethyl acetate and subjected to TLC analysis. TLC plates (Merck) were developed in ethyl acetate/methanol/water (100:2·5:1, by vol.). For antibacterial agent bioassay, TLC plates were placed for 30 min on the surface of LB agar plates inoculated with M. luteus ATCC 10240. The agar plates were then incubated for 16 h at 35 °C and screened visually for growth-inhibition zones.
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RESULTS |
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Although a method for efficient protoplast formation and regeneration for S. noursei ATCC 11455 was established (see Methods), all our attempts to achieve S. noursei protoplast transformation with vectors pKC1218 and pSET152 using both standard and modified techniques failed. To extend the choice of vectors, the new plasmids pSOK101 (pIJ101 replicon), pSOK201 (pSG5 replicon) and pCHZ101 (pHZ1351 replicon) were constructed (see Fig. 2 and Table 1
). However, transformation of S. noursei protoplasts with the above vectors failed to give any transformants. All five plasmids used in these transformation experiments contain the origin of transfer, oriT, from plasmid RP4 allowing mobilization of the construct during conjugation from an appropriate E. coli strain into Streptomyces sp. (Mazodier et al., 1989
). The plasmids could easily be introduced into S. lividans 1326 via both protoplast transformation and conjugation from E. coli ET12567(pUZ8002). Conjugation experiments with S. noursei (Table 3
) using pSOK101, pKC1218 and pCHZ101 were unsuccessful. For pSOK201, a relatively high number of small apramycin-resistant colonies appeared on the conjugation plates. However, these putative transconjugants stopped growing after 34 d incubation and did not develop into normal colonies. Attempts to transfer these small colonies to fresh selective media resulted in slow-growing and poorly sporulating colonies from which no plasmid DNA could be isolated. About 12 colonies per plate were isolated when plasmid pSET152 carrying the site-specific integration system from streptomycete phage
C31 was used for conjugation. Southern blot analysis of two such S. noursei (pSET152) clones with labelled pSET152 DNA indicated integration of the plasmid into a specific site on the chromosome (data not shown).
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Finally, inactivation of ORF11, presumably encoding a type I PKS enzyme in S. noursei ATCC 11455, was made by gene disruption with pND2. pND2 was constructed by cloning the internal 1·3 kb BamHIEcoRI fragment of ORF11 from KH16 into a mobilizable suicide vector (see Fig. 1a and Table 1
). Integration of pND2 into the chromosome via a single crossover upon intergeneric conjugation resulted in strains ND21 and ND22. The integration of pND2 in these strains was also confirmed by Southern blot analysis of their total DNA (data not shown). The genotypes of all the recombinant strains constructed are presented in Fig. 1(b)
.
Analysis of secondary metabolites produced by the wild-type strain and knock-out mutants
S. noursei strains RN413, RN803 and ND21 were tested for nystatin production by shake flask experiments in the semi-defined medium SAO-23. All the mutants constructed produced nystatin in amounts similar to the wild-type (WT) strain ATCC 11455. Thus, it was concluded that the identified gene cluster is not involved in the biosynthesis of nystatin. From the information we had on the putative genes within the cluster, it was logical to assume that the cluster governs the biosynthesis of some macrolide antibiotic which is exported out of the cell (presence of gene for a putative ABC transporter). Thus, we focused our attempts on isolation of this unknown antibiotic. The shake flask fermentations were carried out with the mutant and the WT strains in the semi-defined medium MP1. Ethyl acetate extracts of the supernatants from 120 h cultures were analysed by TLC followed by a bioassay against M. luteus ATCC 10240. A large growth inhibition zone corresponding to the TLC spot with RF 0·44 (A) was observed in the case of the WT strain (Fig. 4), while none of the mutants displayed a similar inhibition zone. We thus concluded that this zone corresponds to the compound (A) synthesized by the products of the putative genes within the identified gene cluster. In addition, the extracts from all strains contained another compound with antibacterial activity, as small inhibition zones corresponding to TLC spots with RF 0·12 (B) were also observed (Fig. 4
). Thus, the WT strain is capable of producing at least two antibacterial compounds, A and B, while the mutants fail to produce A due to the specific mutations within its biosynthetic gene cluster.
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DISCUSSION |
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We speculate that this new compound is a macrolide antibiotic on the basis of the observed absence of the specific antibacterial activity in the mutant ND21 with disrupted ORF11, presumably encoding a type I PKS. Sequence comparisons also suggest that the polyketide backbone of this compound might be modified by the means of hydroxylation (ORF5) and O-methylation (ORF6) during antibiotic synthesis. Five of the ORFs found within the cluster appear to be responsible for synthesis (gdhA, ORF8, ORF9 and ORF10) and attachment (ORF3) of deoxysugar(s) to the macrolide antibiotic precursor(s). It thus seems likely that at least one deoxysugar is attached to the polyketide moiety. Interestingly, two putative genes, gdhA and ORF9, both presumably encoding Gdh, are present in this cluster. It is possible that the two putative gdh genes are responsible for synthesis of two different deoxysugars, even though to our knowledge there is no precedent for finding two gdh genes within one cluster. Alternatively, one of the identified gdh genes may not encode a true dehydratase, but another as yet undescribed enzyme involved in deoxysugar formation. The modifications of the polyketide moiety seem to be crucial for the antibiotic activity, as a deletion affecting ORFs 58 and gdhA abolishes the specific antibacterial activity in mutant RN413. However, due to the limited DNA sequence data, we cannot exclude the possibility of a regulatory gene being affected by the deletion.
The two ORFs found within the cluster might be responsible for the resistance of S. noursei to the macrolide antibiotic identified. ORF1 seems to encode an ABC transporter homologous to the Streptomyces antibioticus OleB ATP-binding protein involved in the efflux of oleandomycin from the producing organism (Olano et al., 1995 ). Another putative resistance gene might be represented by ORF2, which displays similarity to the Streptomyces glaucescens streptomycin 6-kinase responsible for the inactivation of streptomycin by means of its phosphorylation (Vogtli & Hutter, 1987
).
Finally, the putative ORF4 product resembles transposases from the Rhizobium japonicum insertion element (Kaluza et al., 1985 ) and insertion elements from some other bacteria. There is at least one other reported case of an insertion element being present in the antibiotic biosynthesis gene cluster; IS1136 was found between the PKS-encoding genes in the erythromycin biosynthesis gene cluster of Saccharopolyspora erythraea (Donadio & Staver, 1993
).
So far, the production of two antifungal antibiotics of polyketide origin by S. noursei, cycloheximide and nystatin, had been documented (Roszkowski et al., 1972 ). Although Brown & Hazen (1953)
reported isolation of the antibacterial antibiotic phalamycin from a spontaneously obtained morphological variant of S. noursei, this strain was apparently a nystatin non-producer and phalamycin was not produced by the wild-type S. noursei. Thus, it is not clear whether the phalamycin-producing strain was really S. noursei. We therefore suggest that the antibacterial compound identified in this study and the putative genes for its biosynthesis are novel. Further characterization of this antibiotic aimed at its purification, and elucidation of its chemical structure and potency, is under way.
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ACKNOWLEDGEMENTS |
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Received 10 November 1999;
accepted 8 December 1999.