A new mobilizable cosmid vector for use in Vibrio cholerae and other gram - bacteria

Gene, 153 (1995) 85-87 ©1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

85

GENE 08593

A new mobilizable cosmid vector for use in Vibrio cholerae and other G r a m - bacteria* (Recombinant DNA; conjugation; cloning vector; in vitro packaging; gene cloning)

T e r r y D. C o n n e l l a, A n d r e w J. M a r t o n e a a n d R a n d a l l K. H o l m e s b aDepartment of Microbiology, School of Medicine and Biomedical Science, University at Buffalo, State University of New York, Buffalo, N Y 14214, USA; and bDepartment of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. Tel. (I-305) 295-3400 Received by M. Bagdasarian: 10 August 1994; Revised/Accepted: 12 October/13 October 1994; Received at publishers: 7 November 1994

SUMMARY

A new mobilizable cosmid vector, pCOS5, was engineered for use in Vibrio cholerae (Vc). Plasmid pCOS5 is small in size (7286 bp), contains the oriT from plasmid RK2, and has several unique restriction sites. The complete nucleotide sequence of pCOS5 was deduced from the DNA fragments used in its construction. Biparental matings using Escherichia coli (Ec) SM10 and triparental matings using Ec DH5~[pRK2013] were used to measure the conjugation frequency of pCOS5 and pAJM1, a clc,ne containing a 40-kb insert of chromosomal DNA from Vc ligated into pCOS5. Transfer of pCOS5 or pAJM 1 to Vc o,zcurred at a frequency of between 1 0 - 2 10-3 transconjugants per recipient cell. The promiscuous nature of RP4/RK2 transfer functions makes pCOS5 a potentially useful vector for mobilizing large fragments of cloned DNA between diffierent Gram- bacteria that support replication of ColE1 plasmids or as a mobilizable suicide vector in Gram- bacteria where replication of ColE1 plasmids is not supported.

INTRODUCTION

Cosmid vectors have been instrumental as recombinant vehicles for introducing large DNA inserts into Ec and other Gram- bacteria (Collins and Bruning, 1978). Cosmid clones contain laJ~'ge DNA inserts, are packaged into bacteriophage L capsids, and are delivered into recipient cells by transfection. Transfection of the cosmid Correspondence to: Dr. T.D. Connell, Department of Microbiology, University at Buffalo, State University of New York, 3435 Main Street, Buffalo, NY 14214, USA. Tel. (1-716) 829-3364; Fax (1-716) 829-2158; e-mail: [email protected]~Llo.edu *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Short Communication. Abbreviations: A, absorbance ( 1 cm); Ap, ampicillin; bp, base pair(s); Cm, chloramphenicol; cos, cohesive sites from bacteriophage ~.; Ec, Escherichia coli; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); nt, nucleotide(s); oriT, origin c f conjugative transfer; oriV, origin of vegetative replication; R, resistance/resistant; Sin, streptomycin; Vc, Vibrio cholerae; 1-], denotes pla,;mid-carrier state. SSDI 0378-1119(94)00804-3

clones requires that the bacterium be susceptible to infection by bacteriophage L. The enteric pathogen Vc is not susceptible to infection by bacteriophage L. Expression of the cloned receptor for bacteriophage ~ in Vc made it possible to introduce cosmids, but the efficiency of transfection was very low (Morales et al., 1991). Transformation of Vc can be accomplished with high efficiency by electroporation for plasmids up to 10 kb but not for larger plasmids (Marcus et al., 1990). Therefore, the only practical method now for introducing large recombinant replicons into Vc is by conjugation. Conjugative plasmids for Vc have been derived from members of the lncQ and IncP (RK2/RP4) families (F0rste et al., 1986; Keen et al., 1988). Most of these plasmids are large and do not have fully characterized nt sequences. To facilitate genetic analysis of Vc, we have engineered pCOS5, a mobilizable cosmid vector consisting entirely of DNA fragments from plasmids with known nt sequences (Fig. 1). pCOS5 clones containing

86 .ClaI iHindlII

: i

E PvuI :.

.Sail i.BamHI

iiXbaI

~

.-Nc°l

oriT

Cm X

V'" SPahllI

pCOS5

~_.. NruI

oriV

COS " NruI SphI {EcoRVi Fig. 1. Restriction map of pCOS5. Arrows mark the location and orientation of the Ap R and Cm R genes. The regions encoding oriT, oriV and the bacteriophage ~, cohesive site (cos) are boxed. The plasmid has unique HindlII, ClaI, BamHI, XbaI, NcoI and PvuI sites. A unique site for EcoRV, shown in parentheses, was predicted from the sequences of the ancestral plasmids but could not be confirmed by digestion with EcoRV. The multiple sites for SphI, SalI and NruI have been included. The distance between tickmarks is equal to 1 kb. Methods: (a) Construction of pCOS5: The cosmid vector pCOS5 was engineered in three steps. First, a 270-bp BamHI-HindIII fragment encoding oriT (Waters et al., 1991) from plasmid pSS1910 (S. Stibitz, unpublished) was substituted for the 346-bp BamHI-HindIII fragment of pHC79, a non-mobilizable ColEl-based cosmid vector (Hohn and Collins, 1980). This intermediate plasmid containing the RK2 oriT was designated pHC79/oriT. Second, pHC79/oriT was subjected to complete digestion with BamHI and partial digestion with SphI, and a 6.2-kb BamHI-SphI fragment was isolated. Next, plasmid pUC18-CAT (which consists of a 1.0-kb Sau96I-Bsu36I fragment encoding the Cm R gene of pACYC184 (Chang and Cohen, 1978) ligated into the HinclI site of pUC18 (Yanisch-Perron et al., 1985; N. Carbonetti, unpublished)) was digested with BamHI+SphI, and the 1.05-kb fragment containing the Cm R marker was isolated. Finally, the 6.2-kb and 1.05-kb fragments were ligated together, and the structure of the final pCOS5 construct was confirmed by restriction mapping. (b) Nucleotide sequence of pCOS5: The nt sequence of pCOS5 was deduced from the known sequences of its constituent fragments (GenBank accession No. U13206).

DNA inserts up to approx. 50 kb can be packaged into bacteriophage ~, transfected into Ec, and subsequently mobilized into Vc by conjugation. EXPERIMENTAL AND DISCUSSION

(a) Constructing recombinant libraries using pCOS5 To test the usefulness of pCOS5 as a cosmid vector, two recombinant libraries of Vc chromosomal DNA were

constructed. The reactions were packaged into ~, capsids and the packaged mix transfected into Ec DH5~. To determine the average size of cosmid clones in the libraries, cosmid DNA was purified from a small number of random clones. The average insert size was 37 kb (data not shown). To test if the libraries were representative of the entire Vc chromosome, we screened 960 transfectants from one library for epsE (Sandqvist et al., 1993) and approx. 1000 transfectants from the other library for a gene identified in our laboratory that is associated with protease synthesis/secretion. A single clone encoding the epsE gene and six clones encoding the protease gene were isolated (data not shown).

(b) Conjugation frequency of pCOS5 Three experiments were designed to measure the ability of pCOS5 to be mobilized from Ec to Fc. To determine the frequency of direct conjugative transfer of the vector, pCOS5 was transformed into Ec SM10, a strain that expresses functions required for conjugative transfer of RK2 oriT-containing plasmids (Simon et al., 1983). Ec SM10[pCOS5] was then mated with Vc 569B str6, a spontaneous SmR mutant of strain 569B, and the frequency of conjugation was determined. To examine the potential of pCOS5 to be mobilized using triparental matings, the vector was transformed into Ec DH5ctmcr (Bethesda Research Laboratories, Gaithersburg, MD, USA), and Ec DH5ctmcr[pCOS5] was mated simultaneously with Ec DH5~[pRK2013] and Fc 569B str6. pRK2013 encodes mobilization functions that mediate transfer of RK2/RP4 plasmids and has been used previously to mobilize RK2 plasmids in triparental matings (Ditta et al., 1980). And finally, to determine the affect of cosmid size on conjugation frequency, pAJM1, a clone containing a 40-kb insert of chromosomal DNA from Vc 569B, was electroporated into Ec SM10, and Ec SM10[-pAJM1] was mated with Fc 569B str6 by direct conjugation. In all cases, pCOS5 mobilized at high frequencies into Vc 569B str6 (Table I). The conjugation frequency of pAJM1 was only slightly less than that observed for pCOS5. The data suggest that the 40-kb insert had little affect on the frequency of plasmid transfer. To determine the stability of pAJM1 in Vc, cosmid DNA was prepared from four independent transconjugants of the clone. No differences were detected in the restriction maps of the four isolates (data not shown). (c) Use of pCOS5 in Gram- bacteria other than Vc Although we have tested pCOS5 for conjugation only in Ec and Vc, the known promiscuity of RK2 (IncP) plasmids (FOrste et al., 1989) makes it likely that pCOS5 can also be used for transfer of large fragments of cloned DNA from Ec to a variety of other Gram- bacteria.

87 TABLE I Conjugative transfer of pCOS5 plasmids from Ec to Vc in biparental and triparental matings Mating combinations~ Donor strain Ec SM10[pCOS5] Ec SM10[pAJM1] ~ Ec DH5~mcr[pCOS5]

Helper strain

Recipient strain

Conjugation frequencyb

Ec DH5ot[pRK2013]

Vc 569B str6 Vc 569B str6 Vc 569B str6

7.4 × 10 -3 2.2 × 10 -3 2.3 × 10 -2

a Strains were grown to an A60o of 1.3-1.8 in LB broth containing appropriate antibiotics. Cells were collected by centrifugation, washed with LB broth to remove antibiotics, and =esuspended in LB broth to the original volume. Cell suspensions (20 ~tl)were combined, and matings were performed on BA-85 filters (Schleicher & Schuell, Keene, NH, USA) on LB agar plates. After overnight incubation at 37°C, bacteria were collected and resuspended in 1 ml LB broth. Serial dilutions of the cell suspensions were plated on selective media to determine conjugation frequencies. Total number of transconjugants b Conjugation frequency = Total number of recipients Plasmid pAJM1 contains a 40-kb insert of Vc 569B chromosomal DNA ligated into pCOS5 at the BamHI site.

Those bacteria that have the ability to replicate ColEI oriV-based plasmids will stably maintain pCOS5. For those species that do not support ColE1 oriV-based replication, we envision that pCOS5 can be used as a mobilizable suicide plasmid to introduce cloned DNA to heterologous recipients for subsequent homologous recombination with the chromosome (Nnalue, 1990).

(d) Conclusions (1 ~pCOS5 is the first cosmid vector available for use in ~: that combines: (i) the advantages of relatively small size; (ii) construction from ancestral plasmids of known nt sequence; (iii) efficient mobilization by conjugation and (iv) a moderately larige number of unique sites for cloning of foreign DNA fragments. (2) For those bacterial species that do not support replication of ColEI-based plasmids but are susceptible to conjugation by plasmids ti~at contain an RK2/RP4 oriT, pCOS5 may be a useful suicide plasmid for experiments where homologous recombination of the cloned DNA with the chromosome is desired.

ACK NOWLEDGEMENTS

We thank Drs. Scott Stibitz and Nicholas Carbonetti for the gifts of plasmids. This work was supported by research grant R01AI31940-03 from the National Institutes of Health, DHHS, by protocol R07301 from UHUHS and by funds awarded to T.D.C. from SUNY at Buffalo (Buffalo, NY, USA).

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