JOURNAL

OF

Vol. 138, No. 3

BACTERIOLOGY, June 1979, p. 1036-1037

0021-9193/79/06-1036/02$02.00/0

R Plasmids R91 and R9la from Pseudomonas aeruginosa Share Only the Gene for Carbenicillin Resistance DAVID MORRIS AND PAUL BRODA* Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland Received for publication 7 March 1979

Plasmid R9la of Pseudomonas aeruginosa strain 9169 is homologous with RP1. Plasmid R91 carried by the same strain is related in the Tnl region but is otherwise unrelated to R91a. Pseudomonas aeruginosa strain 9169, isolated at the Birmingham Accident Hospital in 1969 by Lowbury et al. (10), was reported to contain a plasmid expressing resistances to carbenicillin, kanamycin, and tetracycline in Escherichia coli, but resistance only to carbenicillin in Pseudomonas strains (2). Further physical and genetic studies have now revealed that strain 9169 contains two plasmids which differ with respect to their molecular sizes and restriction endonuclease recognition sites (9). One is an incompatibility group (Inc) P-1 plasmid, R9la, which determines resistance to carbenicillii, kanamycin, and tetracycline. The size of plasmid R9la, 39 megadaltons, as determined by electron microscopy, is similar to that of the prototype Inc P-1 plasmid RP1; like RP1, it has only a single EcoRI recognition site. However, plasmid R9la is unusual among Inc P-1 plasmids in that its transfer functions are expressed with barely detectable frequency. The other plasmid present in strain 9169, R91, confers resistance to carbenicillin only and has been included in a new Pseudomonas Inc group, P-10. It has a smaller molecular size than R9la (35 megadaltons compared with 39 megadaltons) and has at least nine EcoRI recognition sites. A more highly transmissible derivative of R91, R91-5, allows multiplication of phages PRD1, PR3, and PR4. Although these phages also lyse RP1-carrying strains, they also bind to pili specified by plasmids of other Inc groups (1). In contrast, phages PRR1 and Pf3, which attach specifically to P-1 pili, do not lyse a strain carrying R91 (9). We were interested in studying the interrelationships among RP1, R91, and R9la. This was done by hybridization of radioactively labeled probes of each of these DNA species to endonuclease-cleaved fragmentation patterns of the other plasmids bound to nitrocellulose filters by Southern transfer (7, 11). Plasmid R9la, in an E. coli host strain, J53, and plasmid R91, in P. aeruginosa strain PU21 (9), were provided by G. A. Jacoby. Plasmid DNA was prepared from

1-liter cultures grown to log phase in L broth by the technique of Guerry et al. (5). After removal of the bulk of the chromosomal DNA by saltsodium dodecyl sulfate precipitation, plasmid DNA was concentrated by polyethylene glycol precipitation (8). Final purification was by ethidium bromide-cesium chloride equilibrium gradient centrifugation at 38,000 rpm for 40 h in a type Ti 50 rotor on a Beckman model L2-50 ultracentrifuge. Plasmid RP1 DNA was kindly supplied by J. M. Watson. Plasmid DNA was digested by restriction endonucleases PstI and EcoRI (MRE, Porton Down, Wilts.). Digestion with PstI was carried out for 1 h at 37°C in 6 mM Tris-hydrochloride (pH 7.4)-6 mM NaCl-0.5 mM dithiothreitol, after which the NaCl concentration was increased to 100 mM and EcoRI restriction was performed for another hour' Electrophoresis of plasmid DNA in 1% agarose gels was essentially as described by Duggleby et al. (3), except horizontal gels were used. Transfer of DNA fragments to nitrocellulose filters (11), radioactive labeling of plasmid DNA by "nick translocation," and hybridization of the plasmid probe to nitrocellulose-bound single-stranded DNA have been described previously (7, 11). Double digests of both RP1 and R9la with restriction endonucleases PstI and EcoRI gave rise to seven fragments which could be resolved by electrophoresis on 1% agarose gels. The restriction patterns obtained for the two plasmid DNA species were identical. On the other hand, the R91 DNA gave a different fragmentation pattern. At least 15 bands were resolved, some of which, as deduced from densitometric traces of gel photographic negatives, consisted of two fragment species having the same mobilities. Only two bands in the R91 digest had mobilities identical to equivalent bands in the RP1 and R9la fragmentation patterns. The total of all of the fragments in the R91 digest gave a value of about 33 megadaltons as the size of the whole plasmid. This value compares with the size of 35

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VOL. 138, 1979

megadaltons (9) cited above. When radioactively labeled RP1 DNA was hybridized with the endonuclease-derived fragments of RP1, R91, and R9la DNAs, it was observed (Fig. 1) that it hybridized strongly to each of the RP1 and R9la fragments. When R9la DNA was used as the probe, it also hybridized strongly to all of the RP1 and R9la fragments. Therefore, RP1 and R9la are also indistinguishable at this level of analysis. In contrast, only four bands of the R91 pattern hybridized to the RP1 probe. The two bands which hybridized most strongly corresponded in mobility to RP1 bands E and G. Also, only four bands in the RP1 and R9la patterns hybridized to the R91 probe, and again only two bands showed strong homology. These corresponded to bands E and G in the RP1 pattern; relatively less hybridization occurred with bands A and D, indicating less homology. Reference to the restriction map of plasmid RP1 (4) shows that fragments E and G, together with small portions of fragments A (adjacent to G) and D (adjacent to E), correspond to the transposable element Tnl. Therefore, our results lead us to conclude that the homology between R91 and RP1 that is detectable by our methods is limited to this transposon. Both R91 and R9la are known to determine a TEM-2-type beta-lactamase (9), an enzyme known to be carried by Tnl (6). Our results therefore confu-m the conclusion of Jacoby et al. (9) that R91 originated by transposition of Tnl to another plasmid in the Birmingham P. aeruginosa strains, where intensive use of carbenicillin led to the emergence of the carbenicillin resistance strain 9169. We thank G. Jacoby for strains and discussions and the Medical Research Council for support.

LITERATURE CITED 1. Bradley, D. E. 1977. Pili and associated bacteriophages of Pseudomonas aeruginosa, p. 127-133. In D. Schlessinger (ed.), Microbiology-1977. American Society for

Microbiology, Washington, D.C. 2. Chandler, P. M., and V. Krishnapillai. 1974. Phenotypic properties of R factors of Pseudomonas aeruginosa: R factors transferable only in Pseudomonas aeruginosa. Genet. Res. 23:596-603. 3. Duggleby, C. J., S. A. Bayley, M. J. Worsey, P. A.

4. 5. 6. 7.

Williams, and P. Broda. 1977. Molecular sizes and relationships of TOL plasmids in Pseudomonas. J. Bacteriol. 130:1274-1280. Grinsted, J., P. M. Bennett, and M. H. Richmond. 1977. A restriction enzyme map of R-plasmid RP1. Plasmid 1:34-37. Guerry, P., D. J. LeBlanc, and S. Falkow. 1973. General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116:1064-1066. Hedges, R. W., and A. E. Jacob. 1974. Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132:34-40. Heinaru, A. L., C. J. Duggleby, and P. Broda. 1978.

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0.4 A B FIG. 1. Extent of hybridization between RPI and R91 DNA probes against the digests of RPI, R91, and R9l a DNAs. The fragments released by digestion of RPI, R91, and R9la DNAs with a mixture of endonucleases EcoRI and PstI were resolved by electrophoresis in 1% agarose gels, denatured, transferred to nitrocellulose filters, and hybridized with (A) 32P-labeled RPI DNA and (B) 32P-labeled R91 DNA. (A) is overexposed so as to reveal homology of this probe with the smaller fragments. The scale indicates the sizes of the DNA fragments in megadaltons. The letters identify the RPI fragments in descending order of size. Molecular relationships of degradative plasniids determined by in situ hybridisation of their endonucleasegenerated fragments. Mol. Gen. Genet. 160:347-351. 8. Humphreys, G. O., G. A. Willshaw, and E. S. Anderson. 1975. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 383:457-463. 9. Jacoby, G. A., R. Weiss, T. R. Korfhagen, V. Krishnapillai, A. E. Jacob, and R. W. Hedges. 1978. An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression. J. Bacteriol. 136: 1159-1164. 10. Lowbury, E. J. L., A. Kidson, H. A. Lilly, G. A. J. Ayliffe, and R. J. Jones. 1969. Sensitivity of Pseudomonas aeruginosa to antibiotics: emergence of strains highly resistant to carbenicillin. Lancet ii:448452. 11. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

R plasmids R91 and R91a from Pseudomonas aeruginosa share only the gene for carbenicillin resistance.

JOURNAL OF Vol. 138, No. 3 BACTERIOLOGY, June 1979, p. 1036-1037 0021-9193/79/06-1036/02$02.00/0 R Plasmids R91 and R9la from Pseudomonas aerugin...
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