Department of Chemistry, Rice University, 6100 Main St, Houston,TX 77005-1892, USA1
Author for correspondence: Ronald J. Parry. Tel: +1 713 527 8101 ext. 2446. Fax: +1 713 285 5155. e-mail: parry{at}rice.edu
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ABSTRACT |
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Keywords: Streptomyces, antibiotic, resistance, valanimycin
The GenBank accession number for the sequence in this paper is AF148322.
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INTRODUCTION |
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METHODS |
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Cosmid library construction and screening.
Genomic DNA from S. viridifaciens MG456-hF10 was partially digested with Sau3AI. DNA of the correct size range (3540 kb) was obtained by digestion for 25 min at 37 °C with aliquots of serially diluted enzyme. The digests were monitored by gel electrophoresis using a FIGE mapper (Bio-Rad). The DNA was then dephosphorylated, and ligated to the two arms generated from digestion of pOJ446 with HpaI, followed by dephosphorylation and digestion with BamHI. The ligation mixture was packaged using MaxPlax Packaging Extract from Epicentre Technologies (Madison, WI, USA). The packaged phages were propagated in E. coli DH10B cells. For library screening, the vlmH gene was removed from plasmid pEIBAH (Parry et al., 1997 ) as a 1·1 kb NdeIXhoI fragment and radiolabelled with [
-32P]dCTP using the New England Biolabs NEBlot kit. The pOJ446 library was grown on NZYM agar plates (Sambrook et al., 1989
) and transferred to Hybond-N nylon membranes (Amersham) in accordance with the manufacturers instructions. The membranes were probed at 42 °C in 5xSSC and 5xDenhardts solution. The final wash was in 0·5xSSC, 0·5% SDS at 59·5 °C for 2 h. Cosmid DNA was isolated from the positive colonies using a QIAprep spin plasmid kit (Qiagen). Competent E. coli S17-1 cells were transformed with isolated cosmid DNA by standard methods (Sambrook et al., 1989
). Cosmids in E. coli S17-1 were introduced into S. lividans TK24 by the procedure of Bierman et al. (1992)
.
Analysis of cosmids for valanimycin production.
S. lividans TK24 strains containing cosmids recovered from the cosmid library by screening with the vlmH gene were fermented in valanimycin production medium according to the procedure of Yamato et al. (1986) . After approximately 40 h, the mycelium was removed by centrifugation, and the fermentation broth was acidified to pH 3. The acidified broth was extracted twice with ethyl acetate. The combined ethyl acetate extracts were concentrated to a small volume in vacuo and examined for the presence of valanimycin by TLC (silica gel; chloroform/methanol/acetic acid, 200:40:1), with UV detection. Several cosmid-containing strains appeared to produce valanimycin in varying amounts, while the fermentation broth derived from S. lividans containing pOJ446 failed to show the presence of valanimycin. The highest producing strain, which contained pVal38, was selected for further study. The fermentation of S. lividans containing pVal38 was carried out on a larger scale and the putative valanimycin was isolated by ethyl acetate extraction and purified by chromatography on Dowex 1X8 (Cl-) (Yamato et al., 1986
). Since purified valanimycin is somewhat unstable, the solution of valanimycin in ethyl acetate obtained after purification was taken to dryness in vacuo below room temperature. The residue was immediately dissolved in CDCl3 and the solution was analysed by 1H NMR spectrometry. The proton NMR spectrum verified that valanimycin had been produced (Table 2
).
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Sequence analysis.
DNA sequence assembly was performed with Sequencer, Macintosh version 3.0. BLAST searches were performed at the National Center for Biotechnology Information website. SmithWaterman searches were performed at the European Bioinformatics Institute website. The DNA sequence was analysed with the University of Wisconsin Genetics Computer Group (GCG) package, version 8.1-UNIX, including CODONPREFERENCE, BESTFIT, COMPARE, PILEUP and DOTPLOT. Protein alignments were derived from the GCG PILEUP program and from CLUSTALX. The alignments were displayed with SeqVu, Macintosh version 1.01. Hydropathy plots were based on the amino acid hydropathy values of Kyte and Doolittle (Kyte & Doolittle, 1982 ). The DNA Strider program was occasionally used for sequence analysis.
Preparation of valanimycin and valanimycin-containing NE agar.
Streptomyces viridifaciens MG456-hF10 was fermented according to the published procedure. The antibiotic was purified by anion-exchange chromatography on Dowex 1X8 (Cl-). The solution of valanimycin in ethyl acetate obtained after purification was taken to dryness in vacuo below room temperature in a tared flask, and the weight of the valanimycin was determined immediately after removal of traces of solvent under high vacuum. The residue was then immediately dissolved in 10 mM sodium phosphate solution, pH 7·0, and aliquots of the resulting solution were added to warm NE agar before pouring plates.
Determination of MICs of valanimycin and other antibiotics.
E. coli cells and S. lividans spores were inoculated onto NE agar plates containing varying amounts of valanimycin, tetracycline or puromycin. S. lividans containing pVal2 was grown at 30 °C in the presence of thiostrepton (5 µg ml-1) to induce vlmF expression. E. coli NovaBlue(DE3)(pVal3) was grown at 37 °C in the presence of 1 mM IPTG to induce vlmF expression. Growth was scored after overnight incubation with E. coli and after 3 d with S. lividans.
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RESULTS |
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Cloning and sequencing of the valanimycin-resistance gene (vlmF)
S. lividans TK24 containing pVal38 was screened for valanimycin resistance on NE agar. This clone was found to be more resistant to valanimycin than S. lividans TK24 and S. lividans TK24 carrying the parent vector pOJ446 (Table 3). Analysis of pVal38 by digestion with several restriction endonucleases revealed that a 15 kb fragment internal to the cosmid insert could be generated by treatment with EcoRI (Fig. 2
). This fragment was subcloned into the E. coliStreptomyces shuttle vector pKC1218 (Bierman et al., 1992
) to give pVal15. Introduction of pVal15 into S. lividans TK24 conferred the same level of valanimycin resistance as did pVal38 (Table 3
), a result suggesting that the valanimycin-resistance gene resides on the 15 kb EcoRI fragment. Digestion of the 15 kb fragment with EcoRV yielded a 12 kb fragment that was cloned into pKC1218 to give pVal12. Introduction of pVal12 into S. lividans conferred the same level of valanimycin resistance as pVal38 and pVal15 (Table 3
). In order to locate the valanimycin-resistance gene(s), the insert in pVal12 was sequenced. Sequencing revealed the presence of vlmH and vlmR as well as several potential ORFs. The translated amino acid sequence of one of these (vlmF) exhibited similarities to known antibiotic-resistance determinants. The vlmF gene was located approximately 6·4 kb downstream of vlmH and was transcribed in the same direction. vlmF was found to consist of 1206 bp and to encode a polypeptide of 402 amino acids. The vlmF gene exhibited an overall G+C content of 70·4 mol%, with a mean G+C content at the third codon position of 90·0 mol%. These values are typical for Streptomyces genes (Wright & Bibb, 1992
). The sequence CGAGGA was found about seven nucleotides 5' to the first codon of the vlmF gene. This sequence exhibits a significant degree of complementarity to the 3' end of 16S rRNA of S. lividans and it could serve as a ribosome-binding site (Bibb & Cohen, 1982
). The calculated molecular mass of the vlmF gene product was 41015 Da, and the calculated isoelectric point was 10·1.
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Sequence homology studies
GAP, PILEUP and CLUSTALX analyses of vlmF revealed a significant degree of similarity between VlmF and the tetracycline [TetA(H)] and puromycin (Pur8) resistance proteins as well as a number of putative efflux proteins (Fig. 3). A hydropathy plot of VlmF using the amino acid hydropathy values of Kyte and Doolittle (Kyte & Doolittle, 1982
) indicated that VlmF is a transmembrane protein (Fig. 4
). A DOTPLOT comparison of VlmF with TetA(H) and Pur8 indicated that the greatest similarity between these proteins lies within their N-terminal regions (data not shown).
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DISCUSSION |
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It is now well established that antibiotic-producing micro-organisms carry antibiotic self-resistance genes in order to avoid the lethal effects of their own toxins and, further, that the resistance genes are often closely associated with the corresponding biosynthetic genes. S. lividans containing cosmid pVal38 was therefore evaluated for resistance to valanimycin. The recombinant strain was found to be resistant to valanimycin at twice the concentration of the host organism (Table 3). In a control experiment, the presence of cosmid vector pOJ446 did not affect the level of resistance exhibited by the host. Subcloning experiments combined with resistance screening localized the valanimycin-resistance determinant to a 12 kb EcoRV fragment. Sequencing of this fragment revealed the presence of an ORF (vlmF) exhibiting similarities to known antibiotic-resistance genes and to several hypothetical efflux proteins (see below). When vlmF was cloned into the E. coli and Streptomyces expression vectors pRSETB and pWHM1109, the resulting constructs (pVal3, pVal2) conferred valanimycin resistance on E. coli and S. lividans in the presence of the inducers IPTG and thiostrepton, respectively. In the case of E. coli, pVal3 conferred a level of valanimycin resistance that was at least 10 times greater than that tolerated by the same E. coli strain carrying pRSETB (Table 3
). S. lividans carrying pVal2 was resistant to levels of valanimycin at least four times greater than those acceptable to S. lividans containing pWHM1109 (Table 3
).
GAP, PILEUP and CLUSTALX analyses of vlmF revealed a significant degree of similarity between VlmF and the tetracycline [TetA(H)] and puromycin (Pur8) resistance proteins as well as between VlmF and a number of putative efflux proteins (Fig. 3). A hydropathy plot of VlmF using the amino acid hydropathy values of Kyte and Doolittle indicates that VlmF is a transmembrane protein with 12 putative transmembrane-spanning domains (Fig. 4
). These properties suggest that VlmF is a member of the DHA12 family within the major facilitator superfamily of transport proteins (Pao et al., 1998
). This assignment is supported by the absence in VlmF of the H motif that is found in the DHA14 family of transport proteins (Paulsen et al., 1996
). It is of interest that VlmF exhibits similarities to both TetA(H) and Pur8, since TetA(H) is a member of the DHA12 family, while Pur8 is a member of the DHA14 family (Paulsen et al., 1996
; Pao et al., 1998
). VlmF contains part of the ß-turn motif [RK]-X-G-R-[RK] which is found between transmembrane domains 2 and 3 in most eukaryotic and prokaryotic transport proteins (shaded in Fig. 3
) (Henderson & Maiden, 1990
). It also contains the SerAsp dipeptide (shaded in Fig. 3
) that is conserved in all known tetracycline antiporter proteins (Yamaguchi et al., 1990
). In addition, VlmF contains a conserved glycine residue (Gly76) that is found in all tetracycline/H+ antiporters and sugar transporters (Yamaguchi et al., 1992
). While relatively little is known about the mechanism of action of valanimycin, the available information suggests that it damages DNA (Yamato et al., 1987
). The structure of valanimycin suggests it may be a DNA alkylating agent since the antibiotic contains an electrophilic dehydroalanine moiety. An additional mechanism of action might involve reaction of the dehydroalanine moiety of valanimycin with protein cysteine residues (Chiu et al., 1996
). A drug efflux protein such as VlmF may be the simplest way for an organism to protect its DNA or proteins against an electrophilic agent such as valanimycin.
Our investigations have shown that VlmF does not confer resistance to either tetracycline or puromycin on S. lividans, even though it exhibits similarities to TetA(H) and Pur8. This is consistent with other studies that have shown that the resistance phenotype conferred by Pur8 on S. lividans is specific for puromycin (Tercero et al., 1993 ). An inspection of the multiple sequence alignment shown in Fig. 3
reveals that most of the similarities between VlmF and other efflux proteins lie in the N-terminal region. This is confirmed by a COMPARE/DOTPLOT analysis (data not shown). The lack of strong similarity between the C-terminal regions of VlmF, TetA(H) and Pur8 as revealed by the DOTPLOT has been observed for other antibiotic efflux proteins (Tercero et al., 1993
). It may indicate that the C-terminal end of these proteins confers drug-binding specificity (Paulsen et al., 1996
).
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ACKNOWLEDGEMENTS |
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Financial support from the National Institutes of Health (grant GM53818) and the Robert A. Welch Foundation (grant C-0729) is gratefully acknowledged.
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Received 13 July 1999;
revised 21 September 1999;
accepted 20 October 1999.
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