Construction of a series of pSAM2-based integrative vectors for use in actinomycetes

Gene. 94 (1990) 53-59 Elsevier

53

GENE 03728

Construction of a series of pSAM2-based integrative vectors for use in actinomycetes (Recombinant DNA; tipA promoter; Escherichia coli-Streptomyces conjugation; gene expression) T a m a r a S m o k v i n a ' , Philippe Mazodier b, F r~ l ~ ri c Boccard**, Charles J. Thompson b and M i c h e l Gu~rineau* ° Laboratoire de Biologie et Gdndtique Moldculaire, Universit~ Paris-Sud, F-91405 Orsay Cedex (France) TEL(33)169416917, and b Unit~ de Gdnie M~robiologique, Instimt Pasteur, F-75015 Paris (France) Tel. (33)45688842

Received by K.F. Chater: 10 April 1990 Revised: 15 June 1990 Accepted: 18 June 1990

SUMMARY

We have developed vectors which allowed integration of cloned DNA at a single site in the chromosome of Streptomyces lividans 66. These vectors made use of(l) an Escherichia coli replicon, (2) a thiostrepton (Th)- and a streptomycin/spectinomycin-resistance gene for selection in Streptomyces, (3)a 3.5-kb fragment of the Streptomyces integrative plasmid pSAM2 containing its xis and int genes as well as its attachment site, attP, to direct the integration of the vectors at the chromosomal pSAM2 attachment site attB, (4)the origin of transfer of the IncP broad-host-range plasmid RK2 which allowed the mobilization ofthe vectors from E. coil to S. iividans, and (5) the Th-inducible tipA promoter to permit regulated transcription of cloned genes. We demonstrated that pPM927, aplasmid which contained all of these elements, was able to transfer cloned fragments from E. coil to S. iividans by conjugation, stably integrate into the chromosome, and express cloned genes from the tipA promoter. Furthermore, since pPM927 contained the pBR322 replicon, cloned fragments could be conveniently recovered from the S. lividans chromosome for analysis in E. coil by cleavage of genomic DNA isolated from transformed strains, intramolecular iigation and transformation. Since we have shown that the pSAM2 attB site forms part of a conserved prokaryotic tRNA gene, these integrative vectors are potentially useful tools for analysis and expression of genes in diverse bacteria.

INTRODUCTION

The development of basic cloning systems for Streptomyces (Hopwood et al., 1985) has been motivated not only out ofinterest in the biology of this differentiating prokaryote Correspondence to: Dr. M. Gu6rinean, Laboratoire de Biologic et G6n6tique Mol~culaire, U.R.A.C.N.R.S. D 1354, Bftt. 400, Universit6 Paris-Sud, F.91405 Orsay Cedex (France) Tel. (33) 169416917; Fax (33) 169417296. * Present address: The Sainsbury Laboratory, John lnnes Institute and A.F.R.C. Institute of Plant Science Research, Colney Lane, NR4 7UH Norwich (U.K.) Tel. (44)603 52571.

Abbreviations: Ap, ampicillin; amp, gene encoding ApR; attB, bacterial attachment site; attP, pSAM2 attachment site; attL, attR, left and right boundaries, respectively, of pSAM2 with chromosomal DNA; Bm, 0378-1119/90/$03.50 © 1990ElsevierSciencePublishersB.V. (BiomedicalDivision)

but also by their industrial importance for antibiotic production. High- and low-copy-number plasmids as well as temperate actinophage vectors have been constructed which employ streptomycete antibiotic-resistance genes as markers for selection of transformants (Hopwoo~ et al., bleomycin; ble, T ~ gene encoding BmR; bp, base pair(s); int, integrase gene; kan, Tn.5 gene encoding KmR; kb, kilobase(s) or 1000 bp; Kin, kanamycin; neo, gene encoding NmR; NI, nalidixic acid; Nm, neomycin; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; oriR, origin of E. coil DNA replication; oriT, origin E. coli of DNA transfer, ptipA, Th.inducible promoter (Murakami etal., 1989); R, resistance/resistant; s, sonsitivity/sensitive; Sin, streptomycin; Sp, spectinomycin; stm/spc genc~encoding SmR and SpR; ter, terminator; To, tobramycin; Th, thiostreFton; tsr, gene encoding Th R (Thompson et al., 1980); Tn, transposon; tRNA, transfer RNA; x~, gene encoding excisase; O, interposon (Prentki and Krisch, 1984); [ ], denotes plasmid.carrier state.

54 1985; 1986; Chater, 1986; Rao et al., 1987). Although these vectors have proven to be reliable tools for gene cloning experiments and are used routinely by many laboratories to study gene expression in diverse Streptomyces species, recent advances described below allow for their further refinement. The use of plasmids as cloning vehicles for the study of gene regulation is sometimes limited by the fact that recombinant plasmids can be unstable and that regulation of genes normally located on the chromosome can be altered when cloned on replicative plasmids. These potential problems associated with the maintenance ofchromosomal genes on plasmids may result from an increase in copy number or from conformational changes in DNA (Zuber et al., 1987; Omer et al., 1988). Integrative vectors can overcome these problems by allowing stable mainter.ance of single-copy genes in the chromosome without selective pressure. pSAM2 is an l l-kb element able to integrate sitespecifically in the genome of different Streptomyces species including S. ambofaciens, S. griseofuscus, S. lividans (Boccard et al., 1988; 1989a; Kuhstoss et al., 1989) and S. albus (unpublished data). A 3.5-kb fragment of this plasmid is required to promote the integrative recombination event (Smokvina et al., 1990) which is catalyzed by a site-specific recombinase of the integrase family (Boccard et al., 1989b) and occurs by recombination within a 58-bp sequence present both in pSAM2 (attP) and in the chromosome (attB) (Boccard et al., 1989a; Kuhstoss et al., 1989). The attB site overlaps a putative tRNA sequence which is conserved among diverse bacteria (Mazodier et al., 1990). The practice of genetically manipulating cloned genes in E, coil and studying their function in 5treptomyces has been somewhat limited by the fact that transformation of Strepto. myces is not always convenient and is sometimes not easily applicable to important antibiotic-producing species. Recently it has been reported (Mazodier et al,, 1989) that insertion of the RK2 origin of transfer to a bifunctional E. coli/Streptomyces replicon allowed conjugative transfer from E. coli to Streptomyces when the E. coil donor supplied the conjugative functions of plasmid RP4 in trans. A recently characterized promoter, ptipA, is well-suited for the controlled expression of cloned genes. It is a tightly regulated, relatively strong promoter which can be induced either during growth or stationary phase by the addition of Th (Murakami et al., 1989). The aim of the present study wa~. to construct and test the utility of vectors which make use of the pSAM2 integrative, RK2 (RP4) conjugative, and ptipA transcriptional regulatory functions.

RESULTS AND DISCUSSION

(a) The integrative vector pTS55 Integrative vectors in E. colihave proven extremely useful for the stable maintenance of cloned DNA. Similarly, an integrative vector derived from the S. coelicolor SLP1 element has been constructed and used successfully in S. lividans (Omer et ai., 1988). In this section, we describe the construction of pTS55 (Fig. 1), a vector which can replicate in E. coli and also allows the stable integration of cloned DNA in Streptomyces. The main features of this vector are: (1) the pBR329 replicon (which can be removed with EcoRI, if necessary, to comply with recombinant DNA guidelines), (2)a 3.5-kb EcoRI-BamHl fragment of the S. ambofaciens pSAM2 element containing integration but not replication functions, (3)the tsr antibiotic resistance gene for selection in Streptomyces, and (4) six unique restriction sites (BamHl, SstI, Kpnl, XbaI, XhoI, Hindlll) available for the insertion of DNA. The frequency and site of pTS55 integration in the S. lividans chromosome after transformation was identical to that previously observed with pSAM2 (Boccard et al., 1988; 1989a). Compared to replicative shuttle plasmids, the frequency of 'integrated' transformants was reduced by at least a factor of 100. Southern-blot analysis using various probes including pTS55, the 7.5-kb Pstl fragment of S. lividans containing the chromosomal integration zone (Boccard et al., 1988), or a 40-mer oligo included in the att site (Boccard et al., 1989b), demonstrated that the integrative recombination event occurred at the art sites and that no other integrated or replicative forms were present in the transformants (data not shown). DNA fragments indicating excision by a reversal of the integration event could not be detected even on overexposed films. Having shown that pTS55 was stably maintained without selection pressure, it was used to construct the more specialized vectors described below, (b) pPM927, an integrative, conjugative expression vector The integrative vector pTS55 was modified to allow transfer of DNA by conjugation from E. coli to Streptomyces as well as controlled expression ofcloned genes from the tipA promoter (Fig. 2). In a previous report, it had been demonstrated (Mazodier et al., 1989) that DNA transferred by conjugation could be maintained in Streptomyces recipients as a plasmid whose maintenance was dependent on plJ101 replication functions. Similarly, insertion of the origin of transfer of plasmid RK2 in pTS55 (pPM917) allowed conjugational transfer from E. coli S17.1 (Simon et al., 1983) to Streptomyces. However, with pSAM2-based vectors described here, transferred DNA was replicated as a part of the chromosome. Compared to replicative shuttle plasmids, the recovery of 'integrated' transconjugants was

55

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kml Fig. i. The construction ofpTS55. E. coli strains used for cloning (Maniatis et al., 1982) were HBI01 (Boyer and Roulland-Dussoix, 1969) and JMI01 (Messing et al., 1981). pBR329 (Covarrubias and Bolivar, 1982) was used as cloning vector in E. coil pSAM2 was isolated from S. ambofaciens JI3212 (Pernodet et al., 1984). The 7.$-kb BamHl fragment of pSAM2 was cloned in the BamHl site of pBR329. The resulting plasmid, pOS210, was then digested by EcoRi and religated to generate pOS210A. The insertion ofa 1.8-kb BamHl fragment ofS. azureus DNA containing the tsr gene (Thompson et al., 1980) into the unique BamHl site of pOS210A generated pTS53, pTS54 (not shown) was obtained by filling-in the BarnH! site closest to the pSAM2-derived fragment ofpTS53. A polylinker containing seven unique cloning sites, BamHl, Sstl, Kpnl, Xbai, Xhol, Hindlll and EcoRl, was derived from reannealed oligos (Lathe et al., 1984; 5'-GATCCGAGCTCGGTACCTCTAGACTCGAGAAGCTTGAATTCA-Y, $'-GATCTGAATr. CAAGCTTCTCGAGTCTAGAGGTACCGAGCTCG-Y ) made on an Applied Biosystems 381A DNA synthesizer and inserted into the unique BamHl site of pTS54 to generate pTS55. Restriction sites cleaved for the subsequent cloning step are underlined. Single and double asterisks are used to differentiate the two BamH! sites in pTS53.

reduced by at least a factor of 100; approx. 20 colonies were recovered on each plate. The stm/spc gene was inserted into pPM917 to provide an alternative selectable marker. It can be removed with HindIII and replaced with other antibiotic resistance genes. The bacteriophage fd transcription terminator (whose activity has been demonstrated in Streptomyces, Ward et al., 1986) was inserted adjacent to Q to generate a plasmid (pPM925) containing a transcriptionally inactive region with four unique sites available for the incorporation ofpromoters. We inserted ptipA here to generate the expression vector pPM927.

(c) Expression of genes integrated in the Streptomyces liddans chromosome To test the ability of pPM927 to express cloned genes from ptipA, a 1.5-kb BamHI-Bglll fragment which contained/can and ble genes ofTn5 (subcloned from pPM 111;

Mazodier et al., 1985) was inserted into the BamHl site of pPM927 in the appropriate orientation with respect to ptipA to generate pPM930 (map not shown). S. lividans was transformed with pPM930 and Sp a transformants were assayed for Th-inducible expression of kan and ble genes, The antibiotic sensitivity test plate presented in Fig. 3 demonstrated that expression of kan and ble was induced by Th in S. lividans containing pPM930. When the same fragment was cloned in pPM925 for use as a negative control, neither kan nor ble expression were induced by Th (data not shown). pSAM2-based vectors can also be used to express cloned genes from their endogenous promoters. M.-J. Virolle (personal communication) has used a pTS55 derivative to study the regulation ofthe S. iimosus a-amylase gene (Long et ai., 1987). After insertion into the S. iividanschromosome this gene was transcribed from its own promoter and was regulated by trans-acfing factors.

56

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Fig. 2. The construction of pPM927, pPM906 was obtained by cloning the 4$0-bp 5phl fragment ofpRS1105 (Schauer et al., 1988)containing tar in the 5phi site ofpUCI9. The Hindlll.Xbal fragment containing tar was cloned in pTS55 and the resulting plasmid was designated pPMgl0. Plasmid pPM917 was obtained after the pBR329 EcoRl fragment was replaced by an EcoR! fragment of pATI87 (Trieu-Cuot et al., 1987) containing pBR322, the origin oftransfer of RK2 and neo. The Hlndlll fragmentcontaining neo was replaced by the Ocassette (from pHP450; Prentki and Krisch, 1984)and the resulting plasmid was designated pPM925. To introduce the tlpA promoter into pPM925, it was first excised from pAKII3 (Murakami et al., 1989) with Sinai + BamHl. After filling.inthe single-stranded BamHl end, the blunt-ended fragment was cloned into the Sinai site ofpUCI9 to generate pMIPlS. Plasmid pPM927 was made by ligating the pMIPl5 Kpnl.BamHl fragment containing the tipA promoter to pPM925. Restriction sites cleaved for the subsequent cloning step are underlined.

(d) Stability of pPM927 derivatives carrying Ntreptomyces lividans DNA fragments To examine the use ofpPM927 as a stable cloning vector, total DNA of S. lividans cleaved with BamHl was ligated to BamHl-cleaved pPM927. After transformation ofE. coli, four recombinant plasmids (pPM927-4, pPM927-5, pPM927-8, pPM927-I0) were chosen for further analyses. Each plasmid was transferred to S. lividans JT46 (Tsai and Chen, 1987) by conjugation using either Sp or Th for selection and purification. Transconjugants representing three of the four recombinants tested were I00~ stable in the absence of selective pressure, however pPM927-5 could only be stably maintained in the presence of Sp. Exposure

Fig.3. Thiostrepton-induced expression of genes cloned in pPM927. Discs containing40/Jg Th (I), 30 pg Km (2), I00 pg Bm (3) were applied

to a lawn of $. iiv~lam[pPM930] growing on HT agar. Plates were incubated for two days at 30°C. Bm and Km zones of inhibition were distorted by diffusionof Th from the adjacent disc resultingin expression of/can and ble.

57 to Th resulted in dramatic loss of the Sp R and Th R phenotypes in strains representing all four recombinant plasmids (Table I). This was presumably related to tipA-induced transcription of the cloned S. iividans DNA since neither the vector alone, pTS55, nor pPM930 exhibited this instability. The most likely explanation for this apparent toxicity is that pupA-activated transcription of many S. lividans chromosomal fragments results either in over-expression of a cloned gene or, alternatively, antisense transcription could block expression of chromosomal genes homologous to those cloned. A

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na nd nd 169 40 70 190 126

na nd nd 169 40 70 190 126

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a The genetic stability of pSAM2-derived plasmids in S. lividans was tested after growth under selective or nonselective conditions, pTS$$ transformants (transformation carried out according to Thompson et al., 1982) were first selected on plates containing 25 ml P.,2YE by flooding with one ml of water containing ~ (100 tag)and then purified on R2YE containing Th (10/~g/ml). All remaining plasmids were introduced to S. lividans by conjugation from E. coli (Mazodier et al., 1989), purified once on HT ngar containing either Th (10 Fg/ml) or Sp (100 tJg/ml) and transferred to HT agar containing no antibiotic. Spores were diluted, plated as single colonies on HT agar, and then repfica-plated to HT agar containing Th, Sp, or no antibiotic (total). na, not applicable (i.e., pTSS5 does not contain the stm/sl¢ resistance gene); nd, not done.

attL tsr sfm/spcodRor/T intalfR Pstl Ligation Transformation

TABLE I

I

J

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Fig. 4. Recovery and analysis of pPM925-cloned fragments from the $. Iivldans chromosome. CA)Random BamHl fragments of S. iividans chromosomal DNA were cloned into the BamHl site of pPM927 using E. coliJMlO! as host. These plasmids were transferred by conjugation to

(e) Recovery of cloned fragments from the Streptomyces li~idans chromosome We were able to recover cloned DNA from the S. lividans chromosome conveniently in E. coil Genomic DNA was isolated from each strain containing one of the four pPM927-derived recombinant plasmids (described above) integrated at attB. Digestion of these DNAs with Pstl, an $. llvldans (Mazodier et al., 1989) and integrated into the chromosome. Transconjupnts were purified on HT medium ~Hickey and Tresner, 1952) containing Sp (I00/Ag/ml)and Nl (200/Ag/ml)and grown in YEME medium (Hopwood etal., 1985). Genomic DNA was extracted (Hopwood et ai., 1985) and cleaved with Pstl. This liberated a fragment between the two underlined Pstl sites (cloned fragments were not cleaved with Pstl). Since these fragments contained the pBR322 replicon, they could be self-ligated and used to transform E. co//. (B) Plasmid DNAs isolated from E. colitransformants were compared to their nonintegrated parent alter digestion with BamHl. Lanes: ~,,phage ~ DNA cleaved with Pstl; A, pPM927; B, D, F, H, four pPM927-derived plasmids containing different $.llvldans chromosomal inserts (pPM927-4, pPM927-$, pPM927-10); C, E, 6, l, the corresponding series of clones recovered as described above from the 8. lividans chromosome; J, pOS229 (pOS229 contained a 7.5-kb fragment of the $. livldans chromosome which included the attB region cloned into pUCI2 (Boecard et al., 1988). Five common fragments as well as the clone-specific S. lividans insert were observed. Three of the common fragments comigrated with S. lividans chromosomal BamHl fragments (cloned in pOS229) found in a region of the $. li~dans chromosome immediately adjacent to attO, The two other fragments common to pPM927 and its derivatives contained vector and chromosomal DNA including attL,

58 enzyme which did not cut within the cloned fragments, was expected to generate fragments of chromosomal DNA including attL (Boccard et al., 1988), vector D N A from attL to tsr, tsr, the BamHI cloning site, the insert, ptipA, ter, and the pBR322 origin of repfication (Fig. 4A). Total DNAs were isolated from four clones described above, self-ligated and used to transform E. coll. Digestion of plasmids demonstrated that integration in these four cases had occurred at attB in the same orientation and that cloned fragments were intact (Fig. 4B). In one exceptional case (among the five analyzed; data not shown), the recovered DNA did not contain the attB region. Restriction mapping indicated that integration had occurred by homologous recombination between cloned DNA and its chromosomal counterpart. In summary, recovery of cloned D N A demonstrated that most recombinant plasmids had inserted at the attB site in the S. lividans chromosome and that integrated cloned DNA had not undergone any major rearrangements in this process. (f) Conclusions (1) We have constructed vectors which allow the stable site-specific integration of cloned fragments into the chromosome of S. lividans and which are designed for the analysis and the expression of genes in Streptomyces. pTS55, which contained six unique restriction sites available for cloning (BamHl, Sstl, Kpnl, Xbal, Xhol, HindIII) could be introduced to Streptomyces by protoplast transformation, pPM927, a derivative of pTS55, could be transferred from E. coil to Streptomyces by conjugation and the tipA promoter could be used to control the expression of genes cloned in its unique BamHl site. (2) Transfer by conjugation and stable integration of $. lividans genomic fragments verified the reliability of the system. Th-induced expression of heterologous genes was demonstrated using the Tn5 kan and hie genes. (3) Cloned fragments could be recovered from the chromosome of S. lividans by cleavage of genomic DNA isolated from transformants or transconjugants, intramolecular ligation, and transformation of E. coil for convenient analysis.

ACKNOWLEDGEMENTS

We thank M.-J. Virolle for communicating results before publication, C. Stuttard, D. Holmes, M. Bibb, and P. Kaiser for their editorial comments as well as J.-L. Pernodet, J. Davies, and A. Sabatier for their continued interest in this work. Support was provided by the Minist~re de la Recherche et de la Technologic (CHVP Program), a research contract No. BAP-0268-F-CEE2 (CD) of the Commission of the European Communities, and by RhOne-

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Gu6rinean, M.: Functional analysis of the Streptomyces ambofaciens element pSAM2. Plasmid (1990) in press. Thompson, CJ., Ward, J.M. and Hopwood, D.A.: DNA cloning in Streptomyces: resistan~ genes from antibiotic-producing species. Nature 286 (1980) 525-527. Thompson, CJ., Ward, J.M. and Hopwood, D.A.: Cloning of antibiotic and nutritional genes in streptomycetes. J. Bacteriol. 151 (1982) 668-677. Trieu-Cuot, P., Carlier, C., Martin, P. and Courvalin, P.: Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol. Lett. 48 (1987) 289-294. Tsai, J.F.-Y. and Chen, C.W.: Isolation and characterization of Sueptomyces li~lans mutants deficient in intraplasmid recombination. Mol. Gen. Genet. 208 (1987) 211-218. Ward, J.M., Janssen, G.R., Kieser, T., Bibb, MJ., Butmer, MJ. and Bibb, M.J.: Construction and characterization of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from T ~ as indicator. Mol. Gen. Genet. 203 (1986) 468-478. Zuber, P., Healy, J.M. and Losick, R.: Effects ofplasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subglis. J. Bacteriol. 169 (1987) 461-469.