Gene, 97 (1991) 109-112

109

Elsevier

GENE

03828

Escherichia-Pseudomonas (Recombinant

DNA;

shuttle vectors derived from pUC18/19

broad-host-range

expression

vectors;

multiple

cloning

site; DNA

sequencing)

H.P. Schweizer Department of Microbiology and Infectious Diseases, Universityof Calgary Health Sciences Center, Calgary, Alberta T2N 4NI (Canada) Received by M. Bagdasarian: Revised: 30 July 1990 Accepted: 25 August 1990

31 May 1990

SUMMARY

Two new broad-host-range plasmid vectors, pUCP18 and pUCP19, which are stably maintained in Escherichia coli and Pseudomonas aeruginosa have been constructed. The plasmids are based on the E. coli pUC18 and pUC19 vectors and possess all their features: (i) convenient direct screening of recombinants; (ii) versatile multiple cloning site; (iii) use as sequencing and expression vectors; (iv) small size; and (v) intermediate to high copy number.

The pUC plasmids (Vieira and Messing, 1982; 1987; Messing, 1983; Norrander et al., 1983; Yanisch-Perron et al., 1985) provide a series of valuable pMB 1 (or ColEl)based cloning vectors. These vectors express the N-terminal fragment of the E. cdi IacZ gene product (@Gal) and display a-complementation in appropriate host strains. Since a MCS is engineered early within the lacZ portion, recombinant plasmids generated by insertion of foreign DNA within the MCS can be identified by histochemical screening. Direct screening for r~combin~ts and multiple

Correspondenceto: Dr. H.P. Schweizer, Infectious

Diseases,

University

Department

of Calgary

Health

of Microbiology Sciences

Center,

and 3330

Hospital Dr. N.W., Calgary, Alberta T2N 4N1 (Canada) Tel. (403)220-8302; Fax (403)283-4740. Abbreviations: Ap, ampicillin; bp, base pair(s); pGa1, P-galactosidase; Cb, carbenicillin; IPTG, isopropyl-/?‘-D-galactopyranoside; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); MCS, multiple cloning site; nt, nucleotide(s); ori, origin of DNA replication; P., Pseudomonas; PAGE, polyacryi~ide-gel electrophoresis; R, resistance; strand(ed); u, unit(s) (~mol/min); wt, wild type; XGaI, chloro-3-indolyl-P-D-galactopyranoside;

[ 1, designates

ss, single 5-bromo-4-

plasmid-carrier

state.

0378-I 119/91/$03.50 0 1991 Elsevier Science Publishers B.V. (Biomedical Division)

other advantages, small size, high copy number, and versatile MCS, have made the pUC vectors popular as cloning and sequencing vectors. One disadvantage is their narrow host-range due to the ColEl replicon. Recently, Chen et al. (1987) and Frank et al. (1989) reported that insertion of the entire 1%kb pROl600-derived portion of PRO1614 (Olson et al., 1982) as a PstI fragment into the pUC vectors yields plasmids which are stably maintained in both E. coli and P, aeruginosa. However, since the stabilizing fragment was inserted into the MCS two of the most desirable features of the pUC vectors, direct screening of recombinants and versatile MCS, were lost. Here I report the construction and properties of two vectors, pUCP18 and pUCP19, which contain the stabilizing fragment inserted into a non-essential region of the pUC18/19 vectors, thus preserving all their essential features.

EXPERIMENTAL

AND DISCUSSION

(a) construction of plasmid pUCPl9 As the site of insertion of the stabilizing fragment into the pUC vectors the single NarI site at nt 235 was chosen.

110 Although this site is located within iacZa (82 nt from the termination codon recognized in wt lacZAM15 strains or 131 nt from the termination codon recognized in supE ZacZAM15 strains) (Yanisch-Perron et al., 1985), it has been found that the C-terminal part of LacZa can be highly variable due to fusion of the IacZa gene fragment and the sequences located 3’ of it (for a discussion see Martinez et al., 1988). For construction of pUCP19, pUC19 was digested with NarI and the ends were rendered flush by treatment with T4 DNA polymerase (Sambrook et al., 1989). Similarly, pUC181.8 containing the 1.8-kb PstI stabilizing fragment of pR0 1614 inserted at the PstI site in the MCSofpUCl&@rank eta4., l9%9) WastreatedwithPI and T4 DNA polymerase and the 1.8-kb fragment was isolated from low-melting agarose. The fragment was then ligated to the ca. 2.69 kb pUC19 fragment and the DNA was used to transform strain DHSaF’ {E. coli K-12 ($8O~acZAMl5) A(ZacZYA-argF)U169 recA1 e&A 1 hsdR 17 (r;mk+ ) supE44 thi-1 gyrA relA1; Liss, 1987). Plasmids were isolated from colonies which were blue on LB XGal indicator plates by an alkaline lysis method (Sambrook et al., 1989) and a plasmid (pUCP19) of the expected size (approx. 4.5 kb) was retained for further experimentation. (b) Construction of pUCP18 For construction of pUCPl8, an approx. 2750-bp PvuI fragment was isolated from pUCP19 and ligated to the 1790-bp PvuI fragment from pUC18. This orients the MCS opposite to that in pUCP19. The correct orientation of the MCS was determined by digesting pUCP18 and pUCP19 DNA with EcoRI and PvuII followed by PAGE using similar digests of pUCl8 and pUC19 DNA as reference (data not shown). (c) Applications and~properties of the new plasmids (1) Escherichia coli In E. coli, tlie new vectors possess all-the features of the pUC vector series: (i) Successful cloning into the MCS can be monitored by direct screening on lactose-indicator medium [XGal medium (Vieira and Messing, 1982) or MacConkey medium (Miller, 1972)]. In host strain DHScrF’ (ZacI) both vectors form blue colonies on LB XGal indicator medium with and without IPTG, and red colonies on MacConkey lactose indicator medium. In host strain XL1 E. coli K-12 ([F’ proAB + lacIQ lacZAM15 zzf: : TnlO] recA 1 endA 1 gyrA96 thil hsdR 17 (rk rnz ) supE44 reIA 1 Alac) (Bullock et al., 1987) both vectors form colorless to very light blue colonies without IPTG and blue colonies with IPTG. On MacConkey lactose indicator medium they produced red colonies. In contrast to the pUCP vectors, the pUC vectors form white colonies on

Fig. 1. Physical map ofplasmid (SF) from pUC181.8

pUCP18.

is indicated

The 1.8-kb stabilizing

by the shadowed

of the ApR gene (Ap), ColEl origin of replication (Plac) contains

are shown.

The open arrow

the MCS in the opposite

the MCS, all but AccI and HincII

indicates

orientation.

portion.

fragment

The location

(ORI) and lac promoter laczcr. Plasmid Of the restriction

pUcPl? sites of

are unique. A single AccI site and two

HincII sites also occur within the SF, along with single sites for HpaI and NarI. Restriction site locations within the SF have only been determined by restriction

analysis

and are numbered

relative to coordinate

0 of the

pUC18 sequence. Among the restriction enzymes which do not cut these vectors, some of the widely used are: BglII, EcoRV and 2301.

MacConkey lactose medium in both strain backgrounds. As discussed previously (Martinez et al., 1988) this finding offers an added advantage, since screening of recombinants in the pUCP vectors can be performed on MacConkey lactose medium. In both lacl + and lad’? strains, screening on this medium avoids the need of inducing the lac promoter with the fortuitous inducer IPTG, a situation which can result in selective loss or even inviability of certain recombinants. In recombinants, insertions with ZacZcr will abolish a-complementation and there will be no active PGal. Hence, on MacConkey lactose medium no inducer allolactose will be formed and no induction will occur; (ii) genes inserted in the proper orientation into then MCS can be expressed from the Zac promoter; (iii) DNA fragments inserted into the MCS may be sequenced directly using the universal primer followingconstru~ of ExoIII/Sl nuclease deletions (Henikoff, 1984). For ssDNA sequencing, fragments can be readily subcloned into the corresponding pUC118 and pUC119 (Vieira and Messing, 1987) or M13mp18 and M13mp19 (YanischPerron et al., 1985) vectors via appropriate flanking polylinker restriction sites; (iv) the vectors can be isolated with high yields from small cultures and transform very efficiently. (2) Pseudomonas aeruginosa To ascertain replication and maintenance of the newly constructed plasmids in P. aeruginosa, they were transformed into strain PA01 (Cuskey and Phibbs, 1985) by the method of Olson et al. (1982). Selection was on VBMM

111 medium [VB medium with 0.3% trisodium citrate (Vogel and Bonner, 1956)] containing 500 pg Cb/ml. DNA was isolated from the CbR transformants by an alkaline lysis method (Sambrook et al., 1989) and phenol-extracted before ethanol precipitation. The DNA was analyzed in restriction digests and used for transformation of DHSaF’. Although the amount of DNA obtained from P. aeruginosa PA01 was significantly less (five- to tenfold, as estimated by agarose gel electrophoresis; data not shown) than the amounts obtained from E. coli cells following the same procedure, the DNA was unaltered in size and transformed very efficiently. The relative copy number of pUCP 19 in P. aeruginosa in comparison to E. coli was estimated by determining P-lactamase activities (Cohenford et al., 1988) in logarithmically growing cells ofDHSaF’[pUCP19] and PAOl[pUCP19]. The /?-lactamase activity recovered from toluenized E. coli DHScrF’ cells (average 0.448 u/ml) was between four- and eightfold higher than the activity recovered from toluenized P. aeruginosa PA01 cells (average 0.077 u/ml). Assuming that the new vectors are present in E. coli at the same copy number as the pUC vectors (at least 100 copies per cell) and assuming that the p-lactamase is expressed with the same efficiency in both bacteria, the new vectors are present in P. aeruginosa at between ten and 25 copies per cell. This finding is in agreement with that of Frank et al. (1989) who reported that pUC18 derivatives containing the 1.8-kb PstI-stabilizing fragment cloned in the MCS were present at 13-14 copies per cell. To test the functionality of the plasmids in P. aeruginosa, several gel-purified BarnHI-, EcoRI- and BumHI + EcoRIgenerated DNA fragments encompassing the chromosomal region containing the glpR gene, encoding a putative activator gene for glycerol catabolism (Cuskey and Phibbs, 1985), were subcloned into pUCP19. As the host strain, DHSaF’ was used and ApR transformants were selected on LB XGal indicator medium containing 100 pg Ap/ml. Recombinants were obtained with all fragments and comprised between 2 and 5% of all ApR transformants when subcloning BamHI or EcoRI fragments and more than 98 y0 when subcloning BarnHI-EcoRI fragments into similarly cleaved, nondephosphorylated vector. Cloning of a l.O-kb EcoRI fragment produced light blue colonies. They probably arose from formation of an active LacZcr in-frame fusion protein either due to the absence of stop codons early in the cloned DNA or due to restart of an active LacZa. When the recombinant plasmids were transformed into a P. aeruginosa glpR mutant (Cuskey and Phibbs, 1985), only clones containing the expected 3.4-kb BarnHI-EcoRI fragment gave rise to glycerol-positive colonies (H.P.S., details to be published elsewhere). The results prove the biological usefulness of pUCP19. Although the new vectors cannot be mobilized from

E. coli to P. aeruginosa by conjugation,

they can easily be introduced into P. aeruginosa by transformation (Olsen et al., 1982) or electroporation (Smith and Iglewski, 1989).

(d) Conclusions Besides being a valuable

addition

to the currently

rather

poor arsenal of genetic tools available for cloning and gene manipulation in P. aeruginosa, the improved vectors described here should facilitate the cloning, sequencing and expression of genes from various Grambacteria. Olsen et al. (1982) have shown that pBR322 containing the stabilizing fragment replicated not only in E. coli and P. aeruginosa but also in P. putida, P. jluorescens and Klebsiellapneumoniae. In addition, it is conceivable that the new vectors will be useful in developing a LacZa complementation system for direct screening of recombinants in P. aeruginosa modeled after a similar system recently described for Bacillus subtilis (Haima et al., 1990).

ACKNOWLEDGEMENTS

I thank A. Schryvers for generous support and D. Storey for the gift of pUC 18 1.8 DNA. This research was supported by a grant from the Canadian Cystic Fibrosis Foundation administered by D.E. Woods and by grants from the Medical Research Council of Canada and the University of Calgary. H.P.S. is a MRC Medical Scholar.

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