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parahaemolyticus. ATIN R. DATTA,l* JAMES B. KAPER,2 AND ANTHONY M. MACQUILLAN1 ..... Kaper, J. B., S. L. Moseley, and S. Falkow. 1981. Molecular ... 113:237-251. 16. Struhl, K., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979.
JOURNAL

OF

BACTERIOLOGY, Nov. 1984,

p.

808-811

Vol. 160, No. 2

0021-9193/84/110808-04$02.00/0 Copyright © 1984, American Society for Microbiology

Shuttle Cloning Vectors for the Marine Bacterium Vibrio parahaemolyticus ATIN R. DATTA,l* JAMES B. KAPER,2 AND ANTHONY M. MACQUILLAN1 Department of Microbiology, University of Maryland, College Park, Maryland 20742,1 and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 212022 Received 5 April 1984/Accepted 20 August 1984

Two cosmid cloning vectors containing k cos sequences and a 42-base-pair multipurpose cloning sequence were constructed. pAD22 also contains a 1.4-kiobase TRP-ARS fragment from Saccharomyces cerevisiae. These cosmids transformed Escherichia coli and S. cerevisiae cells and could be mobilized into Vibrio parahaemolyticus strains with a conjugative plasmid, pRK2013. The cosmid pAD22 was genetically and structurally stable during passage through V. parahaemolyticus and E. coli strains.

Vibrio parahaemolyticus is a moderately halophilic marine bacterium that is of interest to both marine and medical microbiologists. It is a nutritionally versatile organism which produces a variety of extracellular enzymes capable of degrading substrates such as gelatin, chitin, starch, casein, lecithin, and erythrocytes. The salt requirements and salt tolerance of this organism, recently reviewed by Joseph et al. (9), appear to vary depending on the growth medium but are not physiologically well understood. V. parahaemolyticus can also cause acute gastroenteritis. Diarrheal disease caused by this species is a foodbome infection related chiefly to the ingestion of contaminated seafoods. A distinction has been made between those strains of V. parahaemolyticus isolated from human disease and those strains widely distributed in the marine and estuarine environment. This distinction is based on the ability of some strains to produce a heat-stable hemolysin termed the Kanagawa phenomenon (KP) hemolysin (14). Nearly all (99%) environmental isolates of V. parahaemolyticus are KP-, whereas more than 96% of clinical isolates produce the hemolysin and are termed KP+ (9). Despite this correlation, the role of the KP hemolysin in disease is uncertain. Although much research has been directed towards the study of the physiology and pathogenicity of V. parahaemolyticus genetic studies are still in their infancy. Neither transformation, transduction, nor chromosomal mobilization have been described for this species. Although cryptic plasmids have been found in KP+ strains, their function is not at all understood (7). Some means of genetic transfer would greatly aid the study of several problems of concern and interest in V. parahaemolyticus, such as the mechanism and physiology of its halotolerance and pathogenicity. To facilitate the genetic study of V. parahaemolyticus and the cloning of Vibrio genes in Escherichia coli and their subsequent reintroduction back into Vibrio spp., we constructed two cosmid shuttle vectors (Fig. 1). These vectors, pCVD301 and pAD22, were constructed from the broad host range plasmid pRK290 (6) as follows. To transfer the 1.4kilobase (kb) BglII fragment containing the A cos sequence of pREG153 derived from pHC79 (4), we digested both pREG153 and pRK290 with BglII and ligated them with phage T4 DNA ligase. E. coli HB101 cells were transformed with this ligated DNA, and the tetracycline-resistant (Tet')

colonies were subjected to colony hybridization (10) with a [32P]dATP-labeled (15), 1.4-kb BglII fragment of pREG153 as the probe. After autoradiography, one clone was selected, and the plasmid DNA (pCVD300) was shown to contain the 1.4-kb cos fragment by BglII digestion and by its ability to serve as a substrate in a A in vitro packaging reaction (2). Additional restriction enzyme sites were introduced into pCVD300 by inserting the 42-base-pair sequence from the replicative form of the mp7 (Bethesda Research Laboratories) strain of phage M13 (12). After transformation of HB101 cells, plasmid DNA from several Tetr colonies was analyzed by agarose gel electrophoresis. Plasmid pCVD301 was found to be linearized by BamHI and PstI and also yielded a 4-kb SaiI fragment, revealing the presence of the 42-base-pair sequence. Finally, the 1.4-kb EcoRI fragment from the E. coli-Saccharomyces cerevisiae shuttle vector YRp7, which contains the ARS1 replicon and the TRPJ gene of S. cerevisiae (16), was introduced into an EcoRI site of pCVD301. For this, JA300 was transformed with the ligated DNA, and several Tetr Trp+ colonies were screened for the presence of plasmid DNA which yielded a 1.4-kb fragment after EcoRI digestion. The presence of a 4-kb SalI fragment in one of these plasmids (pAD22), similar to pCVD301, placed the TRP-ARS fragment in the right-hand EcoRI site in the 42-base-pair sequence. Each of the vectors contains a A cos fragment so that it can be used for packaging large fragments of DNA (20 to 30 kb) and for infecting susceptible E. coli cells to make gene libraries. The introduction of a 42base-pair multipurpose cloning site provided unique restriction sites for cloning. One of the vectors, pAD22, also contains a 1.4-kb TRP-ARS fragment from S. cerevisiae (16) so that it can also replicate in yeasts. The strains and plasmids used in this study are shown in Table 1. All the bacterial strains were routinely grown at 37°C in LB medium (1% tryptone [Difco Laboratories], 1% NaCl, 0.5% yeast extract) or on LB agar plates (LB gelled with 1.5% agar [Difco]). Thiosulfate-citrate-bile salts-sucrose agar medium (Difco) on which E. coli strains do not grow was used for streak purification of V. parahaemolyticus strains. Bacterial strains containing plasmids were always grown in the presence of tetracycline (10 ,ug/ml) or kanamycin (25 ,ug/ml) as required. Plasmid DNA was extracted by the method of Bimboim and Doly (1). The basic synthetic solid medium used was M9 salt solution (13) containing CaCl2 (50 mM) and MgSO4 (1 mM) and solidified with 1.5% agar (Difco). This basal medium was supplement-

* Corresponding author. 808

VOL. 160, 1984

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Sm

Bg E

rix

Hx E

E

pCVD 301

kt 5.8 kb 3 Eco RI DIGESTION and N ISOLATIOI OF rrp-Ars FRAG.

E

pAD 22 23 kb Bg E

E

FIG. 1. Construction of cosmid vectors. Arrows inside the circles indicate locations of the genes. Tcr, Tetracycline resistance; Apr, ampicillin resistance; rlx, relaxation complex site; RF, replicative form; Frag., fragment. Bars indicate the restriction enzyme cleavage sites. Sm, SmaI; S, SaII; Bg, BglII; E, EcoRI; P, PstI; B, BamiHI; H, HindlIl; A, Aval; C, Clal; X, XhoI.

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J. BACTERIOL.

NOTES

TABLE 1. Strains and plasmids used in this study Relevant genotype or phenotype

Source

E. coli K-12 JA300 AD29 AD21 AD31 AD30 AD32 AD33 HB101

recA trpC117 Str r-K mK JA300(pRK2013) JA300(pAD22) JA300(pCVD301) JA300(pRK2013)(pAD22) JA300(pRK2013)(pCVD301) JA300 Rifr leuB6 proA2 r-B mB- Strr

D. Morris This work This work This work This work This work This work D. Morris

V. parahaemolyticusa AD12 AD15

Prototroph KP- Rif' Prototroph KP+ Rifr

This work This work

S. cerevisiae RH218

Mata trpl gal2 mal SUC2

D. Morris

Strain or plasmid

CUP' Plasmid pRK290 pRK2013 YRp-7 pCVD301 pAD22 pREG153

Tetr non conjugative Kanr conjugative Tetr Ampr non conjugative Tetr non conjugative Tetr non conjugative Carr

S. Falkow S. Falkow D. Morris This work This work R. Gill

a V. parahaemolyticus strains S162-71 KP- and 3D38 (KP+), obtained from M. J. Voll, were used to obtain the Rifr strains AD12 and AD15, respectively. AD12 and AD15 were isolated by selecting for spontaneous Rifr mutants on LB agar plates supplemented with 100 p.g of rifampicin per ml and were then purified by two streakings.

ed with 0.4% glucose and other appropriate nutrients required for growth. Yeast strains were grown in either YEPD medium (1% yeast extract, 2% peptone, 2% dextrose) or YNBD medium (0.67% yeast nitrogen base without amino acids and with 2% dextrose) supplemented with appropriate nutrients required for growth at 31°C. Required amino acids were added at 20 ,xg/ml. It has been shown previously that pRK290 can be mobilized, with the help of the conjugative plasmid pRK2013, into E. coli and other gram-negative bacteria at a high frequency (6). Transfer frequencies of the new cosmid vectors into KP+ and KP- V. parahaemolyticus strains were determined from filter matings with JA300 derivatives (Table 2) as follows. For matings, cultures were grown overnight at 37°C, donors under stationary conditions and recipients with shaking. Cultures were then diluted 25- to 100-fold in fresh media and incubated at 37°C under similar conditions. When the cell concentrations were ca. 2 x 108 to 5 x 108 cells per ml, 0.5-ml amounts of donor cultures were dispensed into Eppendorf tubes and centrifuged for 10 s. The supernatants were discarded, and the pellets were very gently suspended in 1-ml amounts of recipient cultures. These mixtures were then filtered through 0.45-,um filters (Millipore Corp.). The filters were then incubated at 37°C on LB agar plates for 3 h, after which the cells were suspended in 1 ml of 0.85% NaCl. Appropriate dilutions in saline were then mixed with topping agar (0.65% agar [Difco]) and plated onto selective media. Plates were incubated at 37°C for ca. 40 h before colonies were counted. When JA300 strains carrying only pCVD301 or pAD22 were used as donors, no Tetr colonies were selected, indicating that these plasmids are not self transmissible (data not shown). However, pRK2013 was very efficiently transferred into E. coli and into each of the V. parahaemolyticus strains, indicating little restriction of pRK2013 in the recipients. High-frequency transfer of the

cosmid vectors was achieved in binary plasmid systems in which donor strains carried both pRK2013 and pCVD301 plasmids or both pRK2013 and pAD22 plasmids. The frequencies of transfer, expressed as the number of Kanr Rif' (for pRK2013) and Tetr Rif' (for pCVD301 and pAD22) colonies per donor cell, were quite comparable in E. coli and in the KP+ V. parahaemolyticus strains (Table 2). In addition to these binary plasmid mating systems, the cosmid vectors were also mobilized into E. coli and V. parahaemolyticus strains in triparental matings. In these matings, two different donors were used instead of a single donor as in the binary plasmid system. Table 2 shows the transfer frequencies of pCVD301 and pAD22 into E. coli and V. parahaemolyticus with this triparental mating system and demonstrates that they are comparable to those obtained with the binary plasmid system. The transfer frequencies of pCVD301 and pAD22 were found to be somewhat lower in AD12 than in AD15 or AD33. The reason for this difference was not apparent. It is possible that AD12 was more sensitive to the selective antibiotics. Alternatively, we have found that AD12 grows more slowly than AD15 in LB, and lower numbers of transconjugants could be a reflection of this slower growth rate. However, the frequency of transfer was sufficiently high to warrant the use of this system for gene transfer experiments in AD12, the KP- V. parahaemolyticus strain that we used. Although high frequencies of transconjugants resulted from E. coli-E. coli and E. coli-V. parahaemolyticus matings with both the binary and the triparental mating systems, the latter system has the advantage that the two plasmids, conjugative and nonconjugative, are kept apart, in separate cells, until the time of mating. This feature greatly reduces the chance of homologous recombination between these plasmids (6) and, thereby, the loss of any cloned DNA fragment. The triparental mating system is also more useful for transferring different cloned DNA fragments from large TABLE 2. Conjugal transfer frequencies in the pRK2013pCVD301 and pRK2013-pAD22 binary plasmid systems for E. coli and V. parahaemolyticus strainsa Donor

Recipient

No. of indicated transconjugants per donor cell Kanr Rifr

Tetr Rifr

AD29(pPK2013)

AD12 AD15 AD33

1.29 x 10-2 1.37 x 10-1 4.61 x 10-1

AD30 (pRK2013)(pAD22)

AD12

5.5 x 10-2

3.4 x 10-4

AD15 AD33

5.4 x 10-1 7.1 x 10-1

1.19 x 106.9 x 10-1

AD12

1.12 x 10-2

2.3 x 10-2

AD15 AD33

2.30 x 10-1 6.80 x 10-1

8.0 x 10-2 5.7 x 10-1

AD29(pRK2013) + AD21(pAD22)

AD12 AD15 AD33

7.6 x 10-2 3.5 x 10-1 5.2 x 10-1

7.8 x 10-4 7.7 x 10-2 3.8 x 10-1

AD29 (pRK2013) + AD31(pCVD301)

AD12 AD15 AD33

3.5 x 10-3 2.8 x 10-1 2.3 x 10-1

7.2 x 10-2 2.8 x 10-1 1.4 x 10-1

AD32 (pRK2013)(pCVD301)

a All the recipients were Rif'. pRK2013 is Tetr.

Kanr; pCVD301 and pAD22 are

NOTES

VOL. 160, 1984

numbers of cells in that the helper plasmid, pRK2013, need not be incorporated separately into all the prospective donors. Therefore, the search for specific V. parahaemolyticus genes, by genetic complementation of known E. coli mutants, is greatly facilitated. After mhating and initial selection, one streaking of the transconjugant clone on a selective antibiotic medium completely eliminates the possibility of both plasmids being harbored in the same cell, as pRK2013 and the cosmids are in the same incompatibility group. The stability of the plasmids in V. parahaemolyticus was investigated first by exploiting the presence of the S. cerevisiae TRP1 gene in pAD22. This gene can complement the E. coli trpC117 mutation. Five Tetr Rif' transconjugants from each of the binary and triparental crosses were purified (a total of 20 colonies, 10 from AD12 and 10 from AD15) by restreaking on LB-rifampin-tetracycline plates. Plasmid DNA was isolated from each and used to transform JA300. About 100 Tetr colonies from each of these transformations were selected and tested for the Trp marker. All of them were Trp+, indicating that in the vast majority of cases pAD22 can be transferred into and stably maintained in V. parahaemolyticus strains, even without selection for the trp+ gene. Second, plasmid DNA isolated from these Trp+ transformants and original pAD22 DNA were digested with endonuclease HaeIII, and the bands obtained on agarose gels were compared (data not shown). Identical banding patterns were obtained, indicating that pAD22 did tiot undergo any gross structural alteration in V. parahaemolyticus. pAD22 had been constructed to extend the usefulness of this gene transfer system (binary and triparental). Therefore, we compared the frequencies of transformation of E. coli and S. cerevisiae by pAD22 with those by YRp7 (Table 3). Despite their different sizes, the two plasmids transformed at similar high frequencies but also exhibited a similar instability in S. cerevisiae on nonselective mediutn. These data suggest, overall, that pAD22 could enhance the prospect of examining cloned eucaryotic (yeast) genes in halotolerant as well as non-halotolerant procaryotes. However, because of the relative ease of their mobilization into TABLE 3. Transformation of E. coli K-12 and S. cerevisiae by YRp-7 and pAD22

oftrpr

No. of Trp' Loss of Trp' transformants phenotype after tasfrasphnyeatr per recipient eight generations

cellc

(%)d

100 100

2.1 x 10-3 2.3 x 10-3

95.7 %.4

Tetrf f % 0 ~+ No. o Ttr Plasmid transformants per among Tet tanfomats cfl recipient tranSformantsb cell0 reipen

YRp-7 pAD22

1.6 x 10-6 1.9 x 10-6

a JA300 was transformed by the method of Cohen et al. (3) with minor modifications. The number of Tetr transformants per recipient cell were calculated by dividing the total number of viable cells by the total number of Tetr transformants obtained with 0.5 pg of YRp-7 DNA or an equimolar amount of pAD22 DNA. b A total of 100 single colonies from each type was transferred onto LBtetracycline plates and incubated at 37°C for 48 h. These colonies were replica plated onto minimal glucose plates with and without tryptophan. S. cerevisiae RH218 was transformed by the method of Hinnen et al. (8). The total number of transformants obtained with 2.5 F.g of YRp-7 DNA or an equilnolar amount of pAD22 DNA was divided by the total number of regenerating cells to get the number of Trp+ transformants per recipient cell. d Transformant stability was determined after growth of Trp+ transformants on non selective YEPD medium.

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V. parahaemolyticus, these vectors will greatly aid its genetic exploitation both as a host and as a source of cloned genes. V. parahaemolyticus offers certain advantages as a host for cloned genes because of its very short generation time (as short as 8 to 9 mim) (11) and its ability to excrete a variety of proteins (9). For these reasons, this species could serve as a prototype of marine bacteria and aid the development of marine biotechnology, an area of increasing interest recently reviewed by Colwell (5). Finally, because of its broad host range, pAD22 should also be useful for the study of yeast genes in a wide variety of gram-negative bacteria. We thank S. W. Joseph for his interest in this work. Funding was provided by Office of Naval Research contract N00014-82-K-0636, P0001. LITERATURE CITED 1. Birmboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Blattner, F. R., A. E. Blechl, K. Denniston-Thompson, H. E. Faber, J. E. Richards, J. L. Slightom, P. W. Tucker, and 0. Smithies. 1978. Cloning human fetal y-globulin and mouse atype globin DNA: preparation and screening of shotgun collections. Science 202:1279-1284. 3. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114. 4. Collins, J. 1979. Escherichia coli plasmids packageable in vitro in bacteriophage particles. Methods Enzymol. 68:309-326. 5. Colwell, R. R. 1983. Biotechnology in the marine sciences. Science 222:19-24. 6. Ditta, G., S. Stanfied, D. Corbin, and D. R. Helimski. 1980. Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. U.S.A. 77:7347-7351. 7. Guerry, P., and R. R. Colweli. 1977. Isolation of cryptic plasmid deoxyribonucleic acid from Kanagawa-positive strains of Vibrio parahaemolyticus. Infect. Immun. 16:328-334. 8. Hinnen, A., J. B. Hicks, and G. R. Fihik. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. U.S.A. 75:1929-1933. 9. Joseph, S. W., R. R. Colwell, and J. B. Kaper. 1983. Vibrio parahaemolyticus and related halophilic vibrios. Crit. Rev. Microbiol. 10:77-124. 10. Kaper, J. B., S. L. Moseley, and S. Falkow. 1981. Molecular characterization of environmental and nontoxigenic strains of Vibrio cholerae. Infect. Immun. 32:661-667. 11. Katoh, H. 1%5. Studies on the growth rate of various food bacteria. I. On the generationi time of Vibrio parahaemolyticus Fujino. Jpn. J. Bacteriol. 20:94-99. 12; Messing, J., R. Crea, and P. H. Seeburg. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309-321. 13. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14, Miyamoto, Y., T. Kato, Y. Obara, S. Akiyamai k. Takizawa, and S. Yamai. 1969. In vitro hemolytic characteristic of Vibrio parahaemolyticus: its close correlation with human pathogenicity. J. Bacteriol. 100:1147-1149. 15. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237-251. 16. Struhl, K., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979. High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 76:1035-1039.