Conversion of pBR322-Based Plasmids into Broad-Host-Range ...

JOURNAL OF BACTERIOLOGY, Nov. 1994, p. 6566-6571

Vol. 176, No. 21

0021-9193/94/$04.00+0

Copyright X) 1994, American Society for Microbiology

Conversion of pBR322-Based Plasmids into Broad-Host-Range Vectors by Using the Tn3 Transposition Mechanism MENNO KOKl* MONIQUE REKIK&' BERNARD WITHOLT,2 AND SHIGEAKI HARAYAMA1'3 Departement de Biochimie Medicale, Universitg de Geneve, Geneva,' and Institut ftir Biotechnologie, Eidgenossische Technische Hochschule, Zurich,2 Switzerland and Marine Biotechnology Institute, Kamaishi City, Japan3 Received 20 May 1994/Accepted 29 August 1994

We constructed a series of transposon vectors which allow efficient in vitro gene manipulation and subsequent introduction of cloned DNA into a variety of gram-negative bacteria. Transfer of the cloned fragment from these multicopy plasmids into self-transmissible broad-host-range vectors is achieved in vivo, using the Tn3 transposition mechanism. Transposition into a variety of broad-host-range plasmids proceeds efficiently, and the resulting recombinant plasmids can be readily transferred and maintained in a variety of gram-negative bacteria. The utility of the transposable vectors was demonstrated by the introduction and expression of the lacIPOZY sequences of Escherichia coli into Pseudomonas putida strains, allowing them to utilize lactose as a sole source of carbon and energy. Ever since its first appearance in the literature in the 1970s (3), plasmid pBR322 has been used in numerous gene cloning experiments and its structure has been modified to yield a wide variety of expression- and promoter-probing plasmids, cosmids, and other specific-purpose vectors (1). The majority of these pBR322 derivatives have inherited the same 1.6-kb fragment from the progenitor of pBR322, plasmid pMB8. This fragment contains the P-lactamase gene, one of two 38-bp inverted repeats of transposon Tn2 (identical to the Tn3 inverted repeats), and the origin of replication. Because of their small sizes and high copy numbers, pBR322-based plasmids are easy to handle. Unfortunately, the application of these plasmids is limited to a small group of members of the family Enterobacteriaceae. During the last decade, many other gram-negative bacteria have become important targets for basic and applied molecular research. As these bacterial species do not support the replication of typical Escherichia coli plasmids, new vector systems, based on broad-host-range plasmids such as RK2 and RSF1010, have been developed. These plasmids can be transferred very efficiently from E. coli to a wide range of gramnegative bacteria by mobilization. However, as broad-hostrange plasmids are usually large in size, gene cloning, subcloning, and subsequent introduction of the cloned DNA fragment into the desired bacterial host may be a laborious process. A solution to this problem is to combine the advantages of high-copy-number pBR322 vectors with the convenience of conjugative plasmids. To this end, we have converted multicopy pBR322-type plasmids into transposable elements by the introduction of a second copy of the 38-bp Tn3 inverted repeat, satisfying the requirements in cis for transposition (12, 21). The transposable elements thus created can be stably integrated into broad-host-range plasmids and transferred into bacterial hosts of choice. We describe this approach in this paper and illustrate the use of these transposable vectors with two examples. First, the alkST genes, encoding the positive regulator and one enzyme component of the Pseudomonas putida (P. oleovorans) alkane pathway (7, 20), were cloned in

transposable vectors and transferred to and expressed in P. putida. Second, the vector system was used for the introduction of a reconstituted E. coli lac operon into P. putida, allowing it to utilize lactose (milk sugar) as a source of carbon and energy. MATERIALS AND METHODS Bacterial strains and plasmids. Bacterial strains and plasmids are listed in Table 1. Plasmid pDVN-1, which carries an inverted repeat of Tn3 flanked by restriction sites, was kindly supplied by M. Fennewald; plasmids pSC259, which encodes Tn3 transposase, R751, pSa, and R1162 were supplied by J. Shapiro; pJRD184 and pJRD215 were supplied by J. Davison; and plasmid pCHR71 was supplied by C. Sasakawa. Microbiological and recombinant DNA methods. Bacterial media, growth conditions, and procedures for transformation of E. coli, replica plating, and conjugations on solid media have been described before (8, 20). Glucose and lactose (Sigma) were each used at a final concentration of 40 mM in solid and liquid mineral media. Antibiotics (Sigma) were used at the following concentrations per liter: ampicillin, 70 mg; kanamycin, 25 mg for E. coli and P. putida; streptomycin, 200 mg; tetracycline, 10 mg; trimethoprim, 200 mg; and piperacillin, 150 mg. X-Gal (5-bromo-4-chloro-3-indolyl-galactoside) was used at 30 mg/liter. Restriction enzymes, exonuclease III, T4 DNA ligase, and the Klenow fragment of DNA polymerase I were supplied by New England Biolabs, GIBCO BRL, Boehringer Mannheim, and United States Biochemical and were used according to the recommendations of the suppliers. Nucleotide sequencing was done with the Pharmacia T7 sequencing kit, using 3'S-dATP supplied by Amersham. Transposition assay. The frequency of transposition into plasmid R751 was determined as follows. First, E. coli MS967 (containing R751 and pSC259, encoding tnpA) was transformed by an appropriate transposon vector. Second, at least four independent transformant colonies were suspended in 0.2 ml of Luria-Bertani (LB) broth, mixed with greater than a 10-fold excess of the recipient (E. coli MC1061, unless stated otherwise), and incubated for 8 h on LB agar plates at 370C. Third, cells were taken from the plate and resuspended in 0.5 ml of LB broth, and serial dilutions were plated on LB agar plates containing streptomycin and trimethoprim. Finally, ex-

* Corresponding author. Mailing address: Departement de Biochimnie Medicale, Universit6 de Geneve, 1 Rue Michel Servet, 1211 Geneve 4, Switzerland. Phone: 41-22-70 25508. Fax: 41-22-347 3334.

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TABLE 1. List of strains and plasmids Relevant characteristic(s)

Strain or plasmid

Strains E. coli MC1061 DH5a D1210

MS967 SF800

Source or reference

F- araD139 A(ara-leu)7696 galE15 galK16 A(1acIPOZYA)X74 rpsL hsdR2 mcrA mcrBl endAl hsdR17 supE44 thi-1 recAl gyrA relA1 A(1acZYA-argF)U169 deoR [4t80d1acA(lacZ)M15] F- A(gpt-proA)62 leu ara-14 supE44 galK2 lacd lacYl A(mcrC-mrr) rpsL20 xyl-5 mtl-i recA13 pSC259 (tnpA+) R751 thyA polA

Laboratory collection Laboratory collection

Prototroph, biotype A Prototroph, biotype A

Laboratory collection Laboratory collection

Apr, source of the Tn3 inverted repeat Derivative of pSC101 (Kmr tnpA) IncW R338 rep (Ts) Tmpr Tra+ IncPl Tmpr Tra+ Apr Tcr IncP4 Kmr Smr

New et al. (19) Owen et al. (20) Sasakawa and Yoshikawa (23) J. A. Shapiro Heusterspreute et al. (14) Davison et al. (4) M. Casadaban Bethesda Research Laboratories (10) Promega 8 8 This study

P. Green Owen et al. (20) Laboratory collection

P. putida

GPo12 KT2440 Plasmids pDVN-1

pSC259 pCHR71 R751 pJRD184 pJRD215 pMC903 pSPORT1

pGEM7(+) pGEc29 pGEc47 pGMK822 pSPORTn3 pGMK956 pGMK1004 pGMK1004Cm

pACYC177::lacZY lacIq lacPOZa Apr

lacPOZa Apr pLAFRI::alkBFGHJKL pLAFRI::alkBFGHJKL alkST Insertion of one Tn3 inverted repeat into lacZca of pGEM7(+) Tn3 inverted repeat inserted upstream of lacI in pSPORT1 pSPORTn3::lacIPOZY Apr xylM inserted into pGMK822 pGMK1004 with a chloramphenicol resistance cassette inserted outside the transposable element sequence

conjugant colonies (>300) were replica plated on LB agar plates containing trimethoprim, streptomycin, and a third antibiotic corresponding to the marker of the transposable fragment. The transposition efficiency is defined as the frequency of transfer of the transposon marker divided by the frequency of transfer of the trimethoprim marker of the conjugative plasmid R751. Cointegrate resolution. Host RecA-dependent resolution of cointegrate structures was measured using plasmid pGMK 1004Cm as a transposon donor. This plasmid is a derivative of pGMK822; it encodes nontransposable chloramphenicol resistance and transposable ampicillin resistance. Transposition was performed as indicated in the previous section, using R751 as a target plasmid. The transfer of cointegrates was detected by cotransfer of the chloramphenicol and ampicillin markers, while transfer of resolved plasmids was detected by the transfer of the ampicillin marker alone. Vector constructions. Plasmid pDVN-1 carries a synthetic DNA fragment consisting of one inverted repeat of transposon Tn3 flanked by EcoRI and BamHI sites (19). This fragment was purified and inserted into pGEM7(+) digested with EcoRI and BamHI. Plasmid pGEM7(+) already carries one copy of the Tn3 inverted repeat, downstream of the ,-lactamase gene. Insertion of a second copy of the inverted repeat into the multiple cloning site would render part of the plasmid, including the ampicillin resistance (Apr) gene, transposable. As all DH5a transformants obtained from this ligation were found to express a functional lacZa peptide, pGEM7(+) recombinants into which the second copy of the inverted repeat had been

This study This study This study This study

inserted could not be identified directly. We therefore screened the plasmid population for derivatives capable of acting as a (transposon) donor in a transposition experiment. To this end, E. coli MS967 was transformed with the plasmid preparation obtained from DH5oa. MS967 transformants capable of integrating the Apr fragment into plasmid R751 by transposition would be able to efficiently transfer this marker by conjugation to E. coli MC1061. In contrast, MS967 transformants carrying an Apr plasmid with only one Tn3 inverted repeat [such as unmodified pGEM7(+)] would not be able to transfer the Apr marker efficiently. The MS967 transformants were replica plated on a lawn of MC1061, incubated at 37°C for 6 h, and subsequently replica plated on LB agar containing streptomycin and ampicillin. Three transposition-proficient pGEM7(+) derivatives were identified and purified from the original MS967. The structures of these recombinant plasmids were confirmed by restriction enzyme mapping and nucleotide sequencing. One recombinant was designated pGMK822. We subsequently removed the BamHI and SmaI sites from pGMK822 by BamHI digestion and limited exonuclease III treatment followed by ligation and a second BamHI digestion prior to the transformation of DH5ct. In one of the resulting plasmids, pGMK823, four nucleotides were found to be deleted (CCCC), thus eliminating the BamHI and SmaI sites. In order to facilitate the use of transposon vectors in gram-negative species other than E. coli, we subsequently replaced the ampicillin resistance of pGMK822 by a kanamycin resistance marker in a two-step procedure. First, an XbaIXhoI fragment from plasmid pJRD215, encoding the lambda

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KOK ET AL.

I ,

Spe SnaBi

tV'~~~~~~~~~~~~~~~~~su j N~

Xbal

Sacil NOJIl

fI AAeI

pGMK836

Bcn Drial

9

Spel SnaBi

spn Xbal Avrill

\\0os

Stuil

AfllI Sacll

Noil

Sf1l Spel BcIl

Ndel Nsil

~~~~Ndel

FIG. 1. Construction of general purpose transposon vectors. The plasmids pGMK833, pGMK836, and pGMK839 carry two inverted repeats of transposon Tn3 (filled arrowhead), flanking two antibiotic resistance genes and multiple cloning sites. Upon transposition, all the DNA flanked by these inverted repeats is copied into the recipient replicon. The 4,902-bp plasmid pGMK833 encodes the kanamycin resistance gene, the lambda cos site, and part of the multiple cloning site of plasmid pJRD215, linked to the fi origin of single-stranded DNA replication, the ampicillin resistance gene, and the origin of replication of plasmid pGEM7(+). pGMK836 (4,686 bp) was derived from pGMK833 by DraI deletion, followed by the insertion of an EcoRI-Xh1oI fragment from pGMK7O5. In pGMK839 (4,970 bp), the same fragment encoding tetracycline resistance was inserted into pGMK823.

Sacil

Apal Smal Hpal Mlul Ncol BsaBI Psil

BsiBI Kpnl Sacl

C site and a kanamycin resistance marker, was introduced into pGMK822, to yield pGMK833 (Fig. 1), and second, the 1-lactamase sequence was removed from pGMK833 by Dral deletion, resulting in pGMK834. A second selectable marker was taken from plasmid pGMK705. Plasmid pGMK705 is identical to plasmid pJRD184, except for an insertion of a SfiI-NotI linker sequence into the unique BglII site. The tetracycline resistance gene and the adjacent restriction site bank were introduced into pGMK823 and pGMK834 as an EcoRI-XhoI fragment, resulting in plasmids pGMK836 and pGMK839, respectively. pSPORTn3 was derived from pSPORT1 by a two-step procedure. First, a SmaI fragment of pGMK836, containing the tetracycline resistance gene and one Tn3 inverted repeat, was inserted into pSPORT1, which had been partially digested with BssHII and filled with Klenow polymerase. The ligation mixture was used to transform E. coli MC1061 to ampicillin and tetracycline resistance. pSPORT1 recombinants, in which cos

the tetracycline resistance cassette had been inserted into the right BssHII site and in the desired orientation, were identified by restriction mapping. One recombinant was denoted pSPORTc and used for further experiments. The tetracycline marker was removed from pSPORTc by ClaI-NcoI double digestion, Klenow treatment, and ligation, resulting in pSPORTn3 (Fig. 2). The plasmid maps (Fig. 1 and 2) were constructed using the available nucleotide sequence information and were verified by restriction enzyme digestion and occasionally by additional nucleotide sequencing. Other plasmid constructions. Two plasmids were constructed which carry the P. putida alkST region. pGMK916 was constructed by insertion of a 10-kb ClaI fragment, derived from pGEc47, into the unique ClaI site of pGMK836. Plasmid pGMK851 is a derivative of transposon vector pGMK834 into which a 4.9-kb SalI fragment (internal to the 10-kb ClaI fragment) was inserted using the XhzoI site downstream of the kanamycin resistance gene. The lactose vector pGMK956 was constructed by insertion of a BamHI lacZY fragment of plasmid pMC9O3 into BamHI-digested pSPORTn3. In the resulting plasmid, a iacIPO::'trpBA lacZY fragment and the ,B-lactamase gene are contained within the Tn3 inverted repeats. Plasmid stability. The propagation of plasmids, in the absence of selective pressure, was monitored by growing serial batch cultures of F. coli and P. putida recombinants on 5 ml of LB broth. After growth to saturation, 100- to 500-fold dilutions were made in fresh medium, and growth was continued. Samples were appropriately diluted in 10 mM MgCl2 and plated on LB agar plates, to yield 300 to 500 isolated colonies

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TABLE 2. Transposition of Tn3 derivatives

Agel

fad /s

RsrII

~

° $

BspEII EcoPJ

Sail

pSPORTn3

XbaI

/ Hired_\I SnaBI /t BamH

.

Transposon donor'

Size of transposon (kb)

pSPORT1 pGMK823 pSPORTn3 pGMK839 pSPORTc pGMK956 pGMK916

2.11 3.30 3.88 4.95 6.48 14.1

%

transposition

frequency"