Cloning and sequence of a gene encoding macrotetrolide antibiotic resistance from Streptomyces griseus

Gene. 112 (1992) 117-122 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00

117

GENE 06351

Cloning and sequence o f a gene encoding macrotetrolide antibiotic resistance from S t r e p t o m y c e s g r i s e u s (Recombinant DNA; polyketide; esterase; biosynthesis; nonactin; tetranactin)

Richard Plater and John A. Robinson Institute of Organic Chemistry, University of Zarich, CH-8057 Z~rich (Switzerland) Received by K.F. Chater: 20 September 1991 Revised/Accepted: 18 December/20 December 1991 Received at publishers: 7 January 1992

SUMMARY

A gene (nonR) conferring tetranactin resistance on the macrotetrolide-sensitive strain, Streptomyces lividans TK64, was isolated during a shotgun cloning experiment, in which chromosomal fragments from Streptomyces griseus were ligated into the vector pIJ699 and then introduced by transformation into S. lividans TK64. The sequence (3326 bp) of the cloned DNA revealed three complete open reading fra.,nes (ORFs) and one incomplete ORF encoded on one strand of the DNA. The nonR gune (designated here ORFA) encodes a polypeptide of 279 amino acids (Mr 30610) and contains a putative active site motif, GXSXG, characteristic of serine proteases and esterases. A functional role for the nonR gene product may involve the inactivation of the antibiotic through hydrolysis of one or more ester linkages in the macrotetrolide ring. The deduced product of the incomplete ORFX lying adjacent to ORFA showed 27.9% sequence identity with the C-terminal region of rat mitochondrial enoyI-CoA hydratase, and is possibly a macrotetrolide biosynthetic enzyme.

INTRODUCTION

The macrotetrolidos are a small family ofpolyketide ionophore antibiotics produced by many species of Streptomyces (Keller-Schierlein and Gerlach, 1968). in common with other polyketide metabolites, it is likely that the carbon backbones of these antibiotics are assembled by a PKS multi-enzyme complex (Hopwood and Sherman, 1990). To gain access to the structural genes for this macrotetrolide PKS we set out to clone a macrotetrolide resistance gene

Correspondence to: Dr. J.A. Robinson, Organisch-Chemisches lnstitut, Universitat Z[lrich, Winterthurerstrasse 190, CH-80~7 Zfirich (Switzerland) Tel. (41-1)257-4242; Fax (41- I) 361-9895.

Abbreviations: aa, amino acid(s); bp, base pair(s); HPDA, 2-hydroxy-6oxo-6-phenylhexa-2,4-dienoicacid; kb, kilobase(s)or 1000 bp; nonR, gene encoding Ta resistance; nt, nucleotide(s); ORF, open reading frame; R, resistance/resistant; RBS, ribosome-binding site(s); R2YE, R2 regeneration medium with yeast extract; Ta, tetranactin; Ts, thiostrepton.

from the producing organism S. griseus A7796, since biosynthetic and regulatory genes for antibiotic production are usually clustered in the genome of antibiotic-producing streptomycetes along with one, and sometimes several, resistance genes (Martin and Liras, 1989).

EXPERIMENTAL AND DISCUSSION

(a) An assay for detecting resistance to tetranactin The growth of S. lividans TK64 on glucose minimal medium or R2YE was severely inhibited by Ta (10 #g/ml) in the presence of elevated levels of KCI. The KC! enhances the inhibitory effect of Ta on the assay plates. Thus 200 mM KCI and 10/zg Ta/ml are sufficient under these conditions to completely inhibit growth of S. lividans TK64, whereas S. griseus ETHA7796 was resistant to at least 500/~g Ta/ ml in the presence of 300 mM KCI. When S. lividans TK64 containing only the plasmid cloning vector pIJ699, which confers resistance to Ts (Kies,~r and Melton, 1988), was plated on R2YE medium supple-

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mented with 200 mM KCI/10/~g Ta per ml/50/~g Ts per ml, 75~/oof the plates spores survived, grew and sporulated, whereas under the same conditions, but in the absence of Ts, growth was again completely inhibited by the Ta. Thus the presence of Ts induces a partial resistance to the structurally unrelated macrotetrolide Ta. The reasons for this are unknown, although Ts is known to induce the expression of several genes in S. iividans (Murakami et al., 1989). When screening for Ta resistance, Ts was omitted from the assay medium. (b) Cloning a Ta n gene from Streptomyces gdseus Size-fractionated fragments (5-Skb) of Sau3Aldigested S. griseus genomic DNA were ligated between the Bglll sites of the purified pIJ699 vector DNA (Kieser and Melton, 1988), and the resulting ligation mixture was used to transform S. lividans TK64 protoplasts. About 80000 transformants were selected on R2YE plates containing 50 #g Ts/mi (Hopwood et al., 1985). Plasmids from several of these transformants all contained inserted fragments in the size range 4.0-6.5 kb. Spores were harvested from these primary transformation plates and distributed at a density of < 1000 per plate

onto 40 R2YE plates supplemented with 200 mM KCI/ 10 pg Ta per ml. After six days growth, 32 resistant colonies were isolated of which only 25 grew in a second screen on the same selective medium, and only 17 in a third round of screening. This was suggestive of clone instability (see section d).

(c) Isolation of plasmid DNA and retransformation of Streptomyces lividans Initial attempts to isolate plasmid DNA from the resistant clones by procedures routinely used for high-copy- or low-copy-number Streptomyces plasmids (Hopwood et al., 1985) gave only poor yields, and one of the final 17 resistant clones gave no plasmid DNA by either procedure. Digestion of the recombinant pIJ699 plasmids with HindIII should have released the insert from the vector fragment (Kieser and Melton, 1988). However, only five clones yielded plasmids which showed a restriction pattern consistent with this expectation, the others showed multiple bands, the relative intensities of which suggested that in some cases the HindIII fragments were not present in stoichiometric amounts with the vector fragment. Plasmid DNA from two of the resistant clones was used

Fig. 2. The nt and deduced an sequences of 3.4 kb of S, griseus DNA, The 3,4-kb Xbal.generated restriction fragment from pOCl2 i 5 (Fig. 1) was cloned into the XbaI site in MI3mpl8 (Yanisch-Perron et al., 1985). Stable clones were obtained with only one insert orientation. Nested deletions were made from this clone using exonuelease.l !1, by the method of H cnikoff (1984), To sequence the second strand the 3.4-kb region was subcloned into M I3mp 18 or M 13rap 19 as three smaller fragments; a 1.8-kb Xbol-Sacl fragment, a 1.6-kb Sacl-Xbal fragment, and a 1-kb H/nell fragment. After selecting the correct insert orientation, nested deletions were again generated from each subclone. M 13 clones were sequenced by the dideoxy method (Sanger et al., 1977) such that caeh nt was dctcrmined on each strand, on average a total of 4. I times. Sequenase (US Bioehemicals, Cleveland, OH) was used for all sequencing reactions. Sequence information was assembled using the programs SEQIN and ASSEMGEL in the PC/GENE package. Potential ORFs were identified using the program GCWIND. The restriction sites shown in Fig. 1 arc indicated in the sequence. Putative ribosome-binding sites are underlined and denoted by RBS. The pair of inverted repeats between ORFX and ORFA are indicated by arrows bclow and above the sequence. The nt sequence data reported in this paper will appear in GcnBank/EMBL/DDBJ data bases under accession No. M75853.

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120 to re-transform S. lividans TK64. Ts R spores were collected and subsequently replated at a known density on R2YE with and without Ta. Only about 50% of the spores, however, were resistant to Ta at 10 #g/ml. This incomplete Ta R upon retransformation, and the appearance of multiple restriction fragments described above, indicated that the cloned inserts in plJ699 were not stably maintained in S. iividans under Ts selection, a conclusion supported also by Southern-blot hybridisation experiments (described in section d below).

(d) Subeloning and Southern-blot hybridisation analysis The Hindlll inserts in the plasmids from six of the resistant clones were subcloned into the E. coli vector pBR329 (Covarrubias and Bolivar, 1982) to facilitate further characterisation. Further restriction mapping suggested that they contained overlapping nt sequences, representing in total 9 kb of S. griseus genomic DNA, with a central 3.4-kb region common to all of the clones (Fig. 1). To assess whether all 16 Ta n clones from which plasmid DNA could be isolated contained a common nt sequence, Southern blot hybridisation was performed. The H/ndIII insert in pOCI215 was used as a hybridisation probe against HindIII-digested plasmid DNA from each resistant clone. At high stringency, the probe hybridised with at least one fragment from each clone. Next, the same hybridisation probe was used in a Southern blot against separate BamHI and Bglll digests of S. griseus genomic DNA. The pattern ofhybridising bands was consistent with the restriction map of the isolated resistance locus (shown in Fig, 1), demonstrating that the probe hybridises specifically to itself at high stringency, and that a unique region of the genome had been isolated without major cloning-induced rearrangements. Further attempts to obtain a clone conferring Ta resistance stably o,a S. lividans were made by subcloning the 3.4-kb region from clone 11 (Fig. 1), as an Xbal-generated fragment, into the unique XbaI site in another multi-copy Streptomyces plasmid, pIJ385 (Hopwood et al., 1985), and in the SCP2* derived low-copy vector pIJ943 (Hopwood et al., 1985), but in each case the same signs of instability were observed (data not shown).

(e) Sequence analysis The 3.4-kb HindIII insert m pOCI215 was subcloned into M 13mp18 and overlapping sequence information was obtained on bo*.h strands using the dideoxy procedure (Sanger et al., 1977). The nt sequence shown in Fig. 2 ineludes 3326 bp of S. griseus DNA with a G+C content of 72.6~o, which is a typical value for Streptomyces DNA. Application of the program GCWIND (similar to the program FRAME; Bibb et al., 1984) revealed regions containing three complete ORFs, named ORFA, -B and -C, and

a fourth which was only partly represented, named ORFX (Fig. 1). These ORFs are encoded on the same strand, and each is preceded by a putative Streptomyces RBS (Hopwood et al., 1986). The relatively long interval between ORFX and ORFA may indicate that ORFX is part of a separate transcript. Consistent with this view is the occurrence of two perfect G+C-rich inverted repeats, one 14 bp the other 12 bp in length, situated between bp 398 and 451 (398-411 =417-431 and 419-430 =440-451) in Fig. 2, which could function as transcription terminators. The AG O of formation for the corresponding RNA stem-loop structures would be -29.7 kcal/mol and -24.9 kcal/mol (Turner et al., 1987) (or -45.4 kcal/mol and -37.4 kcal/mol using the rules of Tinoco et al., 1973). The 98-bp sequence between ORFB and ORFC has an unusual base composition comprising 80~0 C or T on the coding strand. The G+C content of this region is, however, only ,';lightly lower than normal at 64%. A similar stretch of strand-specific pyrimidine bias was noted between ORFs I and 11 of the whiE spore pigment biosynthetic gene cluster of S. coelicolor A3(2) (Davis and Chater, 1990). Potential ATG start codons for ORFA and ORFB, preceded by potential RBS, appear at nt positions 633-635 and 1488-1490, respectively. The ORFC start codon could not be assigned unambiguously, since three in-frame GTG codons were found in close proximity at nt positions 2087-2089, 2093-2095 and 2099-2101. The first of these overlaps the proposed RB S, while the second and third are spaced 5 bp and 11 bp

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downstream from it. Several Streptomyces genes for antibiotic resistance, antibiotic production and cell differentiation have been characterised where the mRNA start coincides with, or is very near to, the translational start codon (Li etal., 1990; Horinouchi etal., 1987;1989; L6pezCabrera et al., 19891 Hoshiko et al., 1988; Bibb et al., 1985;1986). The deduced aa sequences of ORFB (168 aa) and ORFC (346-348 aa; see above) showed no significant similarities to sequences listed in the NBRF-PIR (release 26) and SWISS-PROT (release 15) databases using the software DNASTAR (Madison, WI). However, the partial ORFXdeduced product aligned colinearly, and shared 27.9% aa identity, with the C-terminal 129 aa of rat mitochondrial enoyl-CoA hydratase (Minami-lshii et al., 1989). The deduced product of ORFA contains 279 aa, is not particularly hydrophobic, and showed a 24.7% sequence identity with the bphD gene product, a HPDA hydrolase (286 aa) involved in biphanyl degradation in Pseudomonas putida KF715 (Hayase et al., 1990) (Fig. 3). Of special interest is a conserved sequence motif, GXSXG (residues 112-116 in ORFA, and residues 110-114 in bphD), that is characteristic of the active sites of many serine proteases, esterases and lipases (Fig. 4; Brenner, 1988). (f) Identification of ORFA as a Ta n gene (nonR) The 3.4-kb region including the nonR gene can be subcloned as two BglII-SacI fragments (Fig. 1), the larger of which contains only an intact copy of ORFA, and the smaller of which encodes only an intact copy of ORFC. The relevant sequences were excised from MI3 clones by cleavage at the EcoRI (adjacent to the SacI site in the polylinker) and Bglll sites, purified, ligated between the unique EcoRI and BamHI sites of the low-copy Streptomyces vector pIJ922 (Lydiate et al., 1985), and introduced into S. lividans TK64. The Ts R transformants were then tested for Ta resistance in the usual way. Only those containing ORFA were Ta R. Furthermore, all the spores recovered were resistant to Ta up to at least 500/~g/ml, and the transformants yielded homogeneous plasmid DNA with the correct restriction pattern, indicating that the construct was now being stably maintained.

(g) Conclusions (1) The isolation of 17 clones from an S. griseus genomic library all containing the same Ta R gene (nonR) indicates that this sequence is either the sole Non R determinant in this macrotetrolide producer, or that other resistance genes are not fully expressed when cloned on small DNA fragments in S. iividans. (2) Some instability associated with the initially cloned Ta R gene in pIJ699 in S. lividans was mitigated by subeloning a smaller restriction fragment, encoding only the nonR ORFA, into the low-copy-number plasmid pIJ922. (3) The deduced product of the nonR gene (ORFA) is a protein of predicted Mr of 30610 showing 24.7% identity to HPDA hydrolase, from P. putida KF715 (Hayase et al., 1990). Of special interest is a conserved putative active site GXSXG motif, which is characteristic of many serine proteases, esterases and lipases (Brenner, 1988), suggesting that the nonR gene product may function in S. lividans and S. griseus as an esterase by cleaving one or more of the four inter-subunit ester linkages in the Ta macrocycle. Our attempts so far to demonstrate this have been frustrated by the very low solubility of Ta in aqueous solutions. (4) It is likely that the ORFB, ORFC and partial ORFX, identified in this work, are biosynthetic or regulatory genes for macrotetrolide biosynthesis in S. griseus, especially in view of the sequence similarity between the deduced product of the partial ORFX and a known enoyI-CoA hydratase (Minami-Ishii, 1989); an enzyme catalysing the addition of a hydroxyl group (rather than water) onto an enoyl-CoA or enoyl-S-ACP substrate should be required during macrotetrolide biosynthesis to form the five-membered oxygenheterocyclic rings (Ashworth et al., 1989). (5) The nonR gene confers Ta resistance on S. lividans only in the presence of elevated levels of K + ions, consistent with the fact that at low alkali-metal ion concentrations the biological effects of the macrotetrolides are reduced (Graven et al., 1967). However, Kanne and Z~hner (1976) showed that a macrotetrolide-negative mutant of S. griseus displayed a different specificity of K + accumulation compared to the wild-type strain. They speculated that the macrotetrolides may function in S. griseus in a cellular, ion-specific uptake system for K+. If the role of Ta in S. griseus is to facilitate K + uptake, then the nonR gene product conceivably may also be a component ofthis transport mechanism, perhaps to drive the release of K + from the ionophore once inside the cell.

ACKNOWLEDGEMENTS The authors are indebted to Dr. D. Hopwood for supplying the vectors and several of the strains used in this study, and for many valuable discussions, to Dr. H. Zahner

122 for pointing out the earlier work o n K + uptake by S. griseus A7796, and Dr. Ashley Birch for careful reading of the manuscript. We t h a n k also Dr. Denis Shields ( S o u t h a m p t o n University) for the p r o g r a m G C W I N D , a n d the Chugai pharmaceutical c o m p a n y for a gift of Ta. This study was supported by the S E R C , Pfizer Central Research ( C A S E award to R.P.), a n d the Schweizerische N a t i o n a l f o n d s u n d e r grant No. 31-25718.88,

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