3 p.-1 fig.We have constructed two plasmid vectors for cloning and expression of DNA fragments controlled by the lac promoter as a NotI-expression cassette. Whereas plasmid pSJ33 allows mobilization of the expression cassette into a wide variety of
Gram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmid
pSJP18Not facilitates cloning and expression in Escherichia coli when high gene dosage may be detrimental. In addition to their suitable cloning features (e.g. multiple
cloning site, lacZa fragment, compatible with ColE1-derived vectors), these plasmids
are particularly useful as auxiliary vectors for cloning of the expression cassettes at
the NotI site of mini-transposon elements [1, 2] and their eventual stable insertion
into the host chromosome.This work was funded by Contract BIO2-CT92-0084
of the BIOTECH program of the EU.Peer reviewe

Díaz, Eduardo

Jaenecke, Susanne

International Microbiology

Digital.CSIC

29A versatile expression plasmid should allow the expression ofcloned genes in as many different bacteria as possible, andtherefore should be endowed with a broad host-range promoterelement. The well characterized lac promoter (Plac) ofEscherichia coli has been shown to drive expression of clonedgenes in a wide variety of Proteobacteria. Such an expressioncan be constitutive or regulated depending on the absence orpresence, respectively, of the lacI q repressor gene. A numberof vectors for cloning and expression of heterologous genesunder the control of Plac are now available, and although mostof them are restricted to E. coli and other related enterobacterialspecies, some possess a wide-host-range among Gram-negativebacteria [6, 9, 12]. However, these vectors lack suitablerestriction sites when further subcloning of the Plac fusion isrequired, e.g. for its integration into the bacterial chromosomethrough transposon delivery systems. Therefore, the aim of thepresent study was to construct versatile Plac-based expressionvectors that allow easy excision of the cloned gene togetherwith Plac as an independent expression cassette.Plasmid pNot18, a pUC18 [13] derivative whose HaeIIlacZa fragment, i.e. the one containing Plac, the multiplecloning site (MCS) and the lacZa fragment, has been substitutedby an equivalent NotI lacZa fragment, was the source of thePlac-based expression cassette. We have inserted this cassetteas a 0.9-kb DNA fragment into the kanamycin(Km)-resistantbroad-host-range plasmid pVLT33 (Fig. 1), a RSF1010derivative and therefore able to replicate in a wide variety ofGram-negative bacteria and susceptible of efficient mobilizationby the RP4 conjugation system [3]. The resultant plasmid,pSJ33, has five unique restriction sites which facilitate thecloning of DNA fragments under the transcriptional control ofPlac (Fig. 1). Significant features of other broad-host-rangePlac-based expression plasmids [6, 9, 12] are also present inpSJ33: (i) positive selection of recombinant plasmids on 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (XGal)-containing Luria-Bertani indicator medium [10] can beaccomplished in appropriate host strains by inactivation of thea-complementation; (ii) sequencing of inserted DNA using theuniversal and/or reverse primers; (iii) plasmid pSJ33 iscompatible with replicons other than that of RSF1010 and theKm resistance gene is a widely useful marker. However, themost distinguishing feature of plasmid pSJ33 is that any genecloned into the MCS can be excised together with Plac as aNotI fragment, provided that there are no additional NotI sitesin the insert, and transferred afterwards as an expression cassetteinto the others vectors. Since NotI is a rare cutter, generationof Plac-based NotI-expression cassettes are the result in themajority of cases. Plasmid pSJ33 is particularly useful incombination with the previously described mini-Tn5 and mini-Tn10 transposon delivery vectors [1, 2], since the engineeredSusanne Jaenecke1Eduardo Díaz2Division of Microbiology, GBF-NationalResearch Centre for Biotechnology,Braunschweig, GermanyPresent addresses:1Department of Biomolecular Sciences,University of Manchester, Institute of Scienceand Technology (UMIST), Manchester, UK2Department of Molecular Microbiology, Center for Biological Research (CIB), NationalResearch Center (CSIC), Madrid, SpainReceived 28 October 1998Accepted 22 November 1998Correspondence to: Eduardo Díaz. Departamento de MicrobiologíaMolecular. Centro de Investigaciones Biológicas, CSIC. Velázquez, 144. 28006 Madrid. España. Tel.: +34-915611800. Fax: +34-915627518. E-mail: cibdf4f@fresno.csic.esRESEARCH ARTICLEINTERNATL MICROBIOL (1999) 2:29–31© Springer-Verlag Ibérica 1999Construction of plasmid vectorsbearing a NotI-expression cassettebased on the lac promoterSummary We have constructed two plasmid vectors for cloning and expression ofDNA fragments controlled by the lac promoter as a NotI-expression cassette. Whereasplasmid pSJ33 allows mobilization of the expression cassette into a wide variety ofGram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmidpSJP18Not facilitates cloning and expression in Escherichia coli when high genedosage may be detrimental. In addition to their suitable cloning features (e.g. multiplecloning site, lacZa fragment, compatible with ColE1-derived vectors), these plasmidsare particularly useful as auxiliary vectors for cloning of the expression cassettes atthe NotI site of mini-transposon elements [1, 2] and their eventual stable insertioninto the host chromosome.Key words lac Promoter · Broad-host-range vectors · Low-copy-number plasmids ·Mini-transposons · Gram-negative bacteriaNotI-expression cassettes can be directly inserted at the singleNotI site of the mini-transposons and subsequently stablyintegrated into the chromosome of a wide variety of Gram-negative bacteria. This combination also facilitates thecomparative study of gene expression in single copy ormulticopy using different microorganisms and the sameregulatory elements.We have used plasmid pSJ33 to replace its NotI lacZafragment by a 4.0-kb NotI cassette containing a complete lacZgene whose transcription is controlled by the Pr promoter-operator from the lambda bacteriophage [5]. When the resultingrecombinant plasmid was transformed into E. coli MV1190Dlac cells, all transformants showed a blue phenotype on mediacontaining the b-galactosidase indicator XGal. To check theperformance of this plasmid in a different Gram-negativebacteria, we performed tri-parental filter matings using E. coliMV1190 cells containing the recombinant plasmid as donor,E. coli HB101 (pRK600) providing the RP4-transfer functionsas helper and Pseudomonas putida KT2440 as recipient [2].Exconjugants were selected on M9 minimal medium [10]supplemented with 0.1% sodium citrate (for nutritionalcounterselection of donor and helper E. coli strains) and 50 µg/ml Km (to select for the presence of the recombinantplasmid), and they showed, as in the case of E. coli, a bluephenotype on XGal containing medium (data not shown).Levels of b-galactosidase activity were measured withpermeabilized P. putida KT2440 cells harboring the pSJ33derivative and were around 200 Miller units [8]. 30 INTERNATL MICROBIOL Vol. 2, 1999 Jaenecke, DíazSmoriVoriTEvrep/mobBsHpPtacKmRlacIqpVLT33(9.7 kb)ESmHBglacZaPlacoriVApRpNot18(2.7 kb)NNASsE S SmK B X Sa SpP HESSm KBXSaSp PH NNlacZapCK01(3.6 kb)oriVASc0.4 kb NotI Plac-lacZafragmentNotI 0.9 kb AflIII/SspIfragment8.4 kb HpaI/HindIIIfragmentMade blunt-ended with Klenow  fragmentT4-DNA ligaseT4-DNA ligaseMCSRCmMCSPlacpSJ33(9.3 kb)oriVlacZa Placrep/mobNBsKmREvNBgX B K S EoriTRCmlacZalacZaPlacPlacpSJP18Not(4.0 kb)NNAScESSm KBXSaSp PHoriVFig. 1 Construction of expression plasmids pSJ33 and pSJP18Not. Functional elements of the plasmids such as relevant restriction sites, promoters,selection markers (Ap, ampicillin resistance), MCS (at the top), origen for vegetative (oriV) and transfer (oriT) replication, and the location of the RSF1010replication and RP4-mediated mobilization functions (rep/mob) are shown. Abbreviations for restriction sites are: A, AflIII; B, BamHI; Bg, BglII; Bs,BstEII; E, EcoRI; Ev, EcoRV; H, HindIII; Hp, HpaI; K, KpnI; N, NotI; P, PstI; S, SacI; Sa, SalI; Sc, ScaI; Sm, SmaI; Sp, SphI; Ss, SspI; X, XbaI. It should benoted that within the NotI lacZa fragment of plasmid pSJP18Not, one of the two C at positions 464–465 in the nucleotide sequence of pUC18 ([13]; correspondingto nucleotides 12–13 of the 16-mer reverse sequencing primer) is missingTo facilitate the generation of Plac-based NotI-expressioncassettes in cases of deleterious gene dosage effects to the E. colihost cell, we inserted the 0.4-kb NotI lacZa fragment from plasmidpNot18 into the NotI digested low-copy-number pCK01 vector[4] (Fig. 1). The resultant plasmid, pSJP18Not, contains Plac,the MCS of pUC18 and the lacZa fragment bracketed by twoNotI sites. Cloning of DNA within the MCS can be easily detectedby a-complementation in appropriate host strains. Moreover, itschloramphenicol (Cm)-resistance selection marker and pSC101oriof replication [11] are compatible with those of the commonampicillin-resistant ColE1-replicon derived vectors of E. coli.Plasmid pSJP18Not was of great utility to clone the bacteriophagelambda repressor (cI gene). When we tried the cloning of thelambda repressor under control of a synthetic lac promoter,PA1/04/03, present in plasmid pUHE24-2 [7], the resulting plasmid,pSJ2 [5], showed mutations that abolished expression of the cIgene. Sequencing analysis of the pSJ2 mutant plasmids alwaysrevealed deletions in the promoter region (data not shown) thatmight be caused by a 18-nucleotide direct repeat present in thesynthetic lac promoter. Interestingly, cloning and expression ofthe cI gene in plasmid pSJP18Not under the control of the Placpromoter was successful and allowed us to transfer the cI-containing NotI cassette into a mini-Tn5 delivery vector for itschromosomal insertion [5]. In conclusion, the plasmids here described behave aseffective broad-host-range and low-copy-number compatibleexpression systems. They have been shown to be particularlyuseful as auxiliary vectors for the cloning of the expressioncassettes at the NotI site of mini-transposon elements [1, 2] andfor their eventual stable insertion into the host chromosome.Acknowledgments We are indebted to Prof. K. N. Timmis for his generoussupport and encouragement, and to Dr. V. de Lorenzo for providing plasmidspNot18 and pCK01. This work was funded by Contract BIO2-CT92-0084of the BIOTECH program of the EU. E. D. was the recipient of a postdoctoralBRIDGE-EU fellowship.References1. Alexeyev MF, Shokolenko IN (1995) Mini-Tn10 transposon derivativesfor insertion mutagenesis and gene delivery into the chromosome ofGram-negative bacteria. Gene 160:59–622. de Lorenzo V, Timmis KN (1994) Analysis and construction of stablephenotypes in Gram-negative bacteria with Tn5 and Tn10-derived mini-transposons. Methods Enzymol 235:386–4053. de Lorenzo V, Eltis L, Kessler B, Timmis KN (1993) Analysis ofPseudomonas gene products using lacI q/Ptrp-lac plasmids andtransposons that confer conditional phenotypes. Gene 123:17–244. Fernández S, de Lorenzo V, Pérez-Martín J (1995) Activation of thetranscriptional regulator XylR of Pseudomonas putida by release ofrepression between functional domains. Mol Microbiol 16:205–2135. Jaenecke S, de Lorenzo V, Timmis KN, Díaz E (1996) A stringentlycontrolled expression system for analysing lateral gene transfer betweenbacteria. Mol Microbiol 21:293–3006. Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene70:191–1977. Lanzer M, Bujard H (1988) Promoters largely determine the efficiencyof repression action. Proc Natl Acad Sci USA 85:8973–89778. Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor,NY: Cold Spring Harbor Laboratory Press9. Morales VM, Bäckman A, Bagdasarian M (1991) A series of wide-host-range low-copy-number vectors that allow direct screening forrecombinants. Gene 97:39–4710. Sambrook J, Fritsch E, Maniatis T (1989) Molecular Cloning: ALaboratory Manual (2nd edn.). Cold Spring Harbor, NY: Cold SpringHarbor Laboratory Press11. Takeshita S, Sato M, Toba M, Wakako M, Hashimoto-Gotoh T (1987)High-copy-number and low-copy-number plasmid vectors for lacZa-complementation and chloramphenicol- or kanamycin-resistance selection.Gene 61:63–7412. West SEH, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ (1994)Construction of improved Escherichia-Pseudomonas shuttle vectorsderived from pUC18/19 and sequence of the region required for theirreplication in Pseudomonas aeruginosa. Gene 128:81–8613. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 cloningvectors and host strains: nucleotide sequences of the M13np18 and pUC19vectors. Gene 33:103–11931Construction of plasmid vectors INTERNATL MICROBIOL Vol. 2, 1999

Construction of plasmid vectors bearing a NotI-expression cassette based on the lac promoter

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