JOURNAL OF BACTERIOLOGY, Mar. 1977, p. 1653-1656 Copyright © 1977 American Society for Microbiology
Vol. 129, No. 3
Printed in U.S.A.
Properties of Derivatives of the Pseudomonas Plasmid pVS1 That Have Inherited Carbenicillin Resistance from RP1 VILMA A. STANISICH,I* P. M. BENNETT, AND M. H. RICHMOND
Department ofBacteriology, Bristrol University Medical School, University Walk, Bristol BS8 1 TD, England Received for publication 17 September 1976
A procedure is described for the isolation, in Pseudomonas aeruginosa PAO, of bacteria carrying derivatives of pVS1 that inherited the carbenicillin-resistance determinant from RP1 either alone or together with that for aeruginocin resistance. Such bacteria occur among the transconjugant progeny from both recombination-proficient or -deficient pVS1+ RP1+ donors, suggesting that the formation of these plasmids is due to the translocation of TnA from RP1 into pVS1. It is possible, therefore, that the aeruginocin-resistance determinant is part of TnA or is closely linked to it. Unexpectedly, none of these plasmids showed the 3 x 106- to 4 x 106-dalton increase in size predicted for TnA+ derivatives of pVS1. It is suggested that an interaction between TnA and the Tn501 translocation unit in pVS1 could account for this result.
The translocation units TnA (3 x 106 to 4 x 106 daltorts) and Tn501 (about 6 x 106 daltons) encode resistance to carbenicillin (Cbr) and mercuric ions (Hgr), respectively. TnA occurs on the P-group plasmid RP4 (4, 5), and Tn501 occurs on the nonconjugative plasmid, pVS1, from Pseudomonas aeruginosa PAT (11, 16). Transfer ofTn501 to RP1, a plasmid that is very similar to RP4 (7, 15), is reported in an accompanying paper (16). In this study we examined the phenotypic and molecular properties of derivatives of pVS1 that inherited the Cbr determinant from RP1. The compositions of the various media used have been described previously (14, 15). The bacterial strains were all derivatives of P. aeruginosa PAO (6) and were the pVS1+ sublines of PAO2001 (argH32 str-39 chl-2) and its recombination-deficient derivative PA02003 (rec-2) (2, 16), PA01670 (ade-136 leu-8 chl-3 rif-1) (2), and PA09504 (argB18 chl-2) (this paper), and an RP1+ derivative of PA09501 (ade-136 leu-8 chl-3 rif-1) (16). The P-group plasmid RP1(Cbr Nmr/Kmr Tcr Aerr Tra+) and the nonconjugative plasmid pVSl(Hgr Sur) have been described previously (15, 16). The donor-specific phage PR4 (12) was used to determine the transfer-phenotype of bacteria carrying derivatives of RP1, and F116L (9) was used for transduction experiments. The procedures for transduction and the isolation of transconjugants from matings carried out on the surface of agar medium were those described by StaniI Present address: Department of Microbiology, La Trobe University, Bundoora, 3083 Victoria, Australia.
sich and Holloway (14). The preparation of aeruginocin AP41 from P. aeruginosa PAF41 and the determination of bacterial response to this agent were as previously described (15). In a previous study (11) it has been shown that derivatives of pVS1 that inherited the Cbr determinant from R30 occur among the progeny of interspecific matings between P. aeruginosa PAT and P. aeruginosa PAO. Transconjugants that inherited these hybrid plasmids were unusual because they produced large colonies on the initial selective minimal medium which contained carbenicillin. This contrasted with the smaller colonies produced by the majority of the progeny that had inherited only the wildtype R30. Since R30 and RP1 are both members of the P-incompatibility group (Stanisich, unpublished data), we took advantage of this correlation between colonial morphology and plasmid type to isolate derivatives of pVS1 that had acquired the Cbr determinant from RP1. It was assumed that most, if not all, of these derivatives would arise from the translocation of TnA into pVS1. RP1 was therefore transferred to pVS1+ sublines of P. aeruginosa PAO2001 (rec+) and its recombination-deficient derivative PA02003 (rec-2), and several single colonies were purified once on the same selective medium. Broth cultures derived from these purified colonies were used as donors in subsequent mating experiments with PA01670 (ade-136 leu-8), and the transconjugants were selected on the appropriately supplemented minimal medium containing carbenicillin (1 mg/ml). Resistant progeny were recovered at a frequency of about 5 x 1653
1654
NOTES
J. BACTERIOL.
10-'/donor, and the few bacteria that were tested displayed all the characteristics normally associated with the wild-type RP1 (15). However, larger colonies occurred at a lower frequency (about 5 x 10-5/donor) and were superimposed on the background lawn of RP1+ bacteria. These were purified and tested for the various RP1- and pVS1-determined phenotypes. Although a diversity of phenotypes was obtained (see Table 1 for the results of two such experiments), these Cbr transconjugants could be subdivided into two main types that occurred at approximately equal frequencies and corresponded to the types previously reported by Stanisich (11). The first type was comprised of bacteria with the resistance phenotype Cbr Kmr Tcr Aerr and, hence, presumably carried derivatives of RP1. These bacteria were all transfer defective (Tra-), and about one-half displayed the additional resistance to mercuric ions (lines 1, 2, 5, and 6 of Table 1). In a separate study (15), bacteria with the latter phenotype have been shown to carry hybrid plasmids that arose by the translocation of Tn501 from pVS1 into RP1 (that is, RPl Hgr hybrids). Our concern was with the second type of transconjugants since these displayed both the mercuric-ion and sulfonamide-resistance phenotypes characteristic of pVS1, despite the fact that they had been selected initially because of their resistance to carbenicillin. It was likely that these bacteria carried derivatives of pVS1 that had inherited TnA, and this was supported
by their sensitivity to kanamycin and tetracycline and their resistance to the RPl-specific phage PR4. However, these transconjugants were found to be heterogeneous with respect to the RPl-determined phenotype of aeruginocin resistance. All the Cbr Hgr Sur transconjugants obtained from matings involving four independently isolated Rec- donors were resistant to aeruginocin AP41, whereas only about onethird of those from comparable matings with a Rec+ donor had this phenotype (see lines 3, 4, and 7 of Table 1). To confirm that in these various transconjugants the Cbr and Cbr Aerr determinants formed part of the same plasmid that carried the Hgr and Sur determinants (that is, carried possible pVS l Cbr and pVS l Cbr Aerr hybrid plasmids), transduction experiments were carried out using F116L as vector and PA09504 as recipient. Cbr and Hgr transductants were selected on nutrient medium containing carbenicillin (250 ,ug/ml) or mercuric chloride (12 ,ug/ ml), respectively, and were recovered at a frequency of about 1.5 x 10-7/plaque-forming units. In all instances (pVS1-1 to pVSl-9; Table 2) 100% co-inheritance of the respective unselected markers was found; namely, co-inheritance of Cbr, Hgr, Sur, and Aerr as appropriate. Therefore, it was concluded that these various transconjugants carried hybrid plasmids that were probably derivatives of pVS1. Furthermore, it was likely that at least those that had originated from matings involving a Rec- donor carried plasmids in which TnA had been translocated from RP1 into pVS1.
TABLE 1. Phenotype of Cbr transconjugants from matings of P. aeruginosa PAO involving pVS1 + RPI + donors of Cbr transconjugants Donor,Phenotype DJonor
No. of trans-
Km
Tc
Tra
Aer
Hg
Su
R R S S
R R S S
-
R R
-
R
S R R R
S S R R
-
R R R
cnuat
conjugants
Probable type of plasmid carid riedb
Rec+
S
11, 10, 11, 9,
7 7 11 10
Tra-RP1 Tra-RP1 Hg hybrid pVS1 Cb' hybrid pVS1-Cbr Aerr hybrid -
Rec-
S
S Tra-RP1 4, 3 S Tra-RP1 Hgr hybrid 8, 12 S R pVS1-Cbr Aerr hybrid 12, 13 a pVS1+ RP1+ derivatives of PA02001 (rec+) or PA02003 (rec-2) were mated with PA01670 (ade-136 leu8). Cbr transconjugants were selected on the appropriately supplemented minimal agar containing carbenicillin. Only those transconjugants that produced large colonies were tested for their inheritance of various RP1 or pVS1 determined phenotypes. The plasmid phenotype symbols follow those proposed by Novick et al. (10), and the genetic symbols are those proposed by Demerec et al. (3). Aer, Response to aeringinocin AP41; R/S, resistance/sensitivity to various antibacterial agents. b The transfer phenotype was determined by resistance (Tra-) or sensitivity (Tra+) of the strain to phage PR4. R R S
R R
R R
VOL. 129, 1977
NOTES
TABLE 2. Size ofpVS1 and its derivativesa AeruginoIncrease in Mol ci phenoPlasmidb (x 10) mol wt ( 01 type 18.5 pVSl S Rec+ derivatives pVSl-l pVS1-2 pVS1-3 pVS1-4 pVSl-5 Rec- derivatives pVS1-6 pVS1-7 pVS1-8
S S R S R
20.3 21.3 20.3 20.1 20.2
1.8 2.8 1.8 1.6 1.7
R R R R
23.1 20.8 20.6 20.0
5.4 2.3 2.1 1.5
pVSl-9 a DNA was precipitated from cleared lysates by polyethylene glycol 6000, and the plasmid DNA was separated from the contaminating chromosomal DNA by ultracentrifugation in cesium chlorideethidium bromide gradients (13, 16). The preparation of plasmid DNA for electron microscopy and the determination of molecular weight from contour length measurements followed the procedures described by Bennett and Richmond (1). b All pVSl RP1 hybrids conferred the phenotype HgCbr. Each isolate was from a separate mating experiment except for the phenotypically distinguishable pairs pVS1-2, pVS1-3 and pVS1-4, pVS1-5. e Molecular weight was determined relative to RP1 DNA (38 x 106 daltons) and was the average contour length of six to seven molecules. -
To confirm these conclusions, covalently closed circular deoxyribonucleic acid (DNA) was isolated from the Hgr Sur Cbr and Hgr Sur Cbr Aerr transconjugants and the sizes of their plasmid molecules were determined by contour length measurements. As would be expected for translocational derivatives of pVS1, all the plasmids had a size more closely similar to pVS1 (18.5 x 106 daltons [16]) than to RP1 (38 x 10; daltons [1]). However, although all were larger than pVS1 by a molecular weight of 1.5 x 106 to 2.8 x 106f, only the transconjugant carrying pVS1-2 had a size that approximated that predicted for a pVS1 derivative that had acquired the 3 x 10fi- to 4 x 106f-dalton fragment of TnA (4, 5). One other transconjugant that carried pVS1-6 was exceptional in that its plasmid exceeded the size of pVS1 by 5.4 x 106 daltons. As far as could be determined from the small number of transconjugants studied, no correlation was apparent between the size of the plasmid, its carriage of the Cbr or Cbr Aerr determinants, or its origin from either a Rec+ or Rec- donor parent.
1655
Several observations can be made from these various results. (i) It seems likely that the different colonial morphology (small versus large colonies) seen among the Cbr transconjugants selected on minimal medium is due to the presence or absence of the RP1 transfer system. Thus, the majority of transconjugants carried the wild-type (Tra+) RP1 and produced small colonies; whereas those that carried either Tramutants of RP1 (or of RP1 * Hgr hybrids) or derivatives of pVS1 produced large colonies. As there is no significant difference in the sizes of colonies produced by established RP1+ sublines of P. aeruginosa PAO and their R- parents, it appears that the production of small colonies is associated with the recent acquisition of RP1 and may simply be a phenomenon that is accentuated on minimal medium. (ii) Other studies have shown that RP1 and RP4 carry the TnA translocation unit (3 x 106 to 4 x 106 daltons) that encodes the bla gene (4, 5; Bennett, unpublished data). Our isolation of derivatives of pVS1 that have acquired the Cbr and Aerr determinants simultaneously suggests that these two loci are closely linked on the RP1 molecule and may both be carried on TnA. In support of this are the properties of a deletion mutant of RP1 that has apparently lost TnA (P. M. Bennett, Robinson, and M. H. Richmond, manuscript in preparation). This derivative is smaller than the parent plasmid by 3 x 106 daltons and confers resistance neither to carbenicillin nor to aeruginocin. (iii) The observation that all the pVS1 * RP1 hybrids from Rec- donors had the phenotype CbO Aerr compared with only about one-third from Rec+ donors, suggests that two different processes, translocation and recombination, may be involved in their formation. However, the heterogeneity found in the sizes of the different hybrids, irrespective of their origin, did not support this conclusion. It is known that although translocation is a Rec-independent process, it apparently occurs with equal fidelity against both a Rec+ or Rec- background, such that the translocational derivatives are homogeneous in size (1, 5, 16). Therefore, we suggest that the inheritance by pVS1 of both the Cbr and Aerr determinants probably represents the addition to this plasmid of TnA, but that events occur associated with this process, which result in a modification in the size of the final derivative. One factor that might conceivably influence the inheritance of TnA by pVS1 is the Tn501 translocation unit that already occurs on this plasmid. Studies on the reciprocal transfer event, namely the translocation of Tn501 into RP1, have shown that those integrations that
1656
NOTES
J. BACTERIOL.
the bla region yield plasmids of a smaller size than those following integrations elsewhere in the molecule (16). The particular RP1 * HgrCbs hybrids studied were also found to be aeruginocin sensitive, and thus may have arisen by a deletion induced by the integration ofTn501 within or near TnA. If such an interaction occurs, then it might reasonably be expected to be more common when TnA integrates into pVS1 than when Tn501 integrates into RP1, simply because TnA and Tn501 occupy about 8% and 33% of their respective parent plasmids and integration is known to have low site specificity.
occur near
Financial support for this work was from search Council Grant to M. H. Richmond.
a
Medical Re-
LITERATURE CITED 1. Bennett, P.M., and M. H. Richmond. 1976. Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Escherichia
coli. J. Bacteriol. 126:1-6. 2. Chandler, P. M., and V. Krishnapillai. 1974. Isolation and properties of recombination-deficient mutants of Pseudomonas aeruginosa. Mutat. Res. 23:15-23. 3. Demerec, M., E. A. Adelberg., A. J. Clark, and P. E. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-76. 4. Hedges, R. W., and A. E. Jacob. 1974. Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132:31-40. 5. Heffron, F., R. Sublett., R. W. Hedges., A. Jacob, and S. Falkow. 1975. Origin of the TEM beta-lactamase gene found on plasmids. J. Bacteriol. 122:250-256.
6. Holloway, B. W. 1969. Genetics of Pseudomonas. Bacteriol. Rev. 33:419-443. 7. Holloway, B. W., and M. H. Richmond. 1973. R factors used for genetic studies in strains of Pseudomonas aeruginosa and their origin. Genet. Res. 21:103-105. 8. Holloway, B. W., H. Rossiter, D. Burgess, and J. Dodge. 1974. Aeruginocin tolerant mutants of Pseudomonas aeruginosa. Genet. Res. 22:239-253. 9. Krishnapillai, V. 1971. A novel transducing phage. rts role in recognition of a possible new host-controlled modification system in Pseudomonas aeruginosa. Mol. Gen. Genet. 114:134-143. 10. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtiss III, and N. Datta. 1976. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40:168-189. 11. Stanisich, V. A. 1974. Interaction between an R factor and an element conferring resistance to mercuric ions in Pseudomonas aeruginosa. Mol. Gen. Genet. 128:201-212. 12. Stanisich, V. A. 1974. Properties and host range of male-specific bacteriophages ofPseudomonas aeruginosa. J. Gen. Microbiol. 84:332-342. 13. Stanisich, V. A., and P. M. Bennett. 1976. The properties of hybrids formed between the P-group plasmid RP1 and various plasmids from Pseudomonas aeruginosa. Molec. Gen. Genet. 149:217-223. 14. Stanisich, V. A., and B. W. Holloway. 1972. A mutant sex factor of Pseudomonas aeruginosa. Genet. Res. 19:91-108. 15. Stanisich, V. A., and J. M. Ortiz. 1976. Similarities between plasmids of the P-incompatibility group derived from different bacterial genera. J. Gen. Microbiol. 94:281-289. 16. Stanisich, V. A., P. M. Bennett, and M. H. Richmond. 1976. Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a nonconjugative plasmid in Pseudomonas aeruginosa. J. Bacteriol. 129:1227-1233.
Vol. 129, No. 3
Printed in U.S.A.
Properties of Derivatives of the Pseudomonas Plasmid pVS1 That Have Inherited Carbenicillin Resistance from RP1 VILMA A. STANISICH,I* P. M. BENNETT, AND M. H. RICHMOND
Department ofBacteriology, Bristrol University Medical School, University Walk, Bristol BS8 1 TD, England Received for publication 17 September 1976
A procedure is described for the isolation, in Pseudomonas aeruginosa PAO, of bacteria carrying derivatives of pVS1 that inherited the carbenicillin-resistance determinant from RP1 either alone or together with that for aeruginocin resistance. Such bacteria occur among the transconjugant progeny from both recombination-proficient or -deficient pVS1+ RP1+ donors, suggesting that the formation of these plasmids is due to the translocation of TnA from RP1 into pVS1. It is possible, therefore, that the aeruginocin-resistance determinant is part of TnA or is closely linked to it. Unexpectedly, none of these plasmids showed the 3 x 106- to 4 x 106-dalton increase in size predicted for TnA+ derivatives of pVS1. It is suggested that an interaction between TnA and the Tn501 translocation unit in pVS1 could account for this result.
The translocation units TnA (3 x 106 to 4 x 106 daltorts) and Tn501 (about 6 x 106 daltons) encode resistance to carbenicillin (Cbr) and mercuric ions (Hgr), respectively. TnA occurs on the P-group plasmid RP4 (4, 5), and Tn501 occurs on the nonconjugative plasmid, pVS1, from Pseudomonas aeruginosa PAT (11, 16). Transfer ofTn501 to RP1, a plasmid that is very similar to RP4 (7, 15), is reported in an accompanying paper (16). In this study we examined the phenotypic and molecular properties of derivatives of pVS1 that inherited the Cbr determinant from RP1. The compositions of the various media used have been described previously (14, 15). The bacterial strains were all derivatives of P. aeruginosa PAO (6) and were the pVS1+ sublines of PAO2001 (argH32 str-39 chl-2) and its recombination-deficient derivative PA02003 (rec-2) (2, 16), PA01670 (ade-136 leu-8 chl-3 rif-1) (2), and PA09504 (argB18 chl-2) (this paper), and an RP1+ derivative of PA09501 (ade-136 leu-8 chl-3 rif-1) (16). The P-group plasmid RP1(Cbr Nmr/Kmr Tcr Aerr Tra+) and the nonconjugative plasmid pVSl(Hgr Sur) have been described previously (15, 16). The donor-specific phage PR4 (12) was used to determine the transfer-phenotype of bacteria carrying derivatives of RP1, and F116L (9) was used for transduction experiments. The procedures for transduction and the isolation of transconjugants from matings carried out on the surface of agar medium were those described by StaniI Present address: Department of Microbiology, La Trobe University, Bundoora, 3083 Victoria, Australia.
sich and Holloway (14). The preparation of aeruginocin AP41 from P. aeruginosa PAF41 and the determination of bacterial response to this agent were as previously described (15). In a previous study (11) it has been shown that derivatives of pVS1 that inherited the Cbr determinant from R30 occur among the progeny of interspecific matings between P. aeruginosa PAT and P. aeruginosa PAO. Transconjugants that inherited these hybrid plasmids were unusual because they produced large colonies on the initial selective minimal medium which contained carbenicillin. This contrasted with the smaller colonies produced by the majority of the progeny that had inherited only the wildtype R30. Since R30 and RP1 are both members of the P-incompatibility group (Stanisich, unpublished data), we took advantage of this correlation between colonial morphology and plasmid type to isolate derivatives of pVS1 that had acquired the Cbr determinant from RP1. It was assumed that most, if not all, of these derivatives would arise from the translocation of TnA into pVS1. RP1 was therefore transferred to pVS1+ sublines of P. aeruginosa PAO2001 (rec+) and its recombination-deficient derivative PA02003 (rec-2), and several single colonies were purified once on the same selective medium. Broth cultures derived from these purified colonies were used as donors in subsequent mating experiments with PA01670 (ade-136 leu-8), and the transconjugants were selected on the appropriately supplemented minimal medium containing carbenicillin (1 mg/ml). Resistant progeny were recovered at a frequency of about 5 x 1653
1654
NOTES
J. BACTERIOL.
10-'/donor, and the few bacteria that were tested displayed all the characteristics normally associated with the wild-type RP1 (15). However, larger colonies occurred at a lower frequency (about 5 x 10-5/donor) and were superimposed on the background lawn of RP1+ bacteria. These were purified and tested for the various RP1- and pVS1-determined phenotypes. Although a diversity of phenotypes was obtained (see Table 1 for the results of two such experiments), these Cbr transconjugants could be subdivided into two main types that occurred at approximately equal frequencies and corresponded to the types previously reported by Stanisich (11). The first type was comprised of bacteria with the resistance phenotype Cbr Kmr Tcr Aerr and, hence, presumably carried derivatives of RP1. These bacteria were all transfer defective (Tra-), and about one-half displayed the additional resistance to mercuric ions (lines 1, 2, 5, and 6 of Table 1). In a separate study (15), bacteria with the latter phenotype have been shown to carry hybrid plasmids that arose by the translocation of Tn501 from pVS1 into RP1 (that is, RPl Hgr hybrids). Our concern was with the second type of transconjugants since these displayed both the mercuric-ion and sulfonamide-resistance phenotypes characteristic of pVS1, despite the fact that they had been selected initially because of their resistance to carbenicillin. It was likely that these bacteria carried derivatives of pVS1 that had inherited TnA, and this was supported
by their sensitivity to kanamycin and tetracycline and their resistance to the RPl-specific phage PR4. However, these transconjugants were found to be heterogeneous with respect to the RPl-determined phenotype of aeruginocin resistance. All the Cbr Hgr Sur transconjugants obtained from matings involving four independently isolated Rec- donors were resistant to aeruginocin AP41, whereas only about onethird of those from comparable matings with a Rec+ donor had this phenotype (see lines 3, 4, and 7 of Table 1). To confirm that in these various transconjugants the Cbr and Cbr Aerr determinants formed part of the same plasmid that carried the Hgr and Sur determinants (that is, carried possible pVS l Cbr and pVS l Cbr Aerr hybrid plasmids), transduction experiments were carried out using F116L as vector and PA09504 as recipient. Cbr and Hgr transductants were selected on nutrient medium containing carbenicillin (250 ,ug/ml) or mercuric chloride (12 ,ug/ ml), respectively, and were recovered at a frequency of about 1.5 x 10-7/plaque-forming units. In all instances (pVS1-1 to pVSl-9; Table 2) 100% co-inheritance of the respective unselected markers was found; namely, co-inheritance of Cbr, Hgr, Sur, and Aerr as appropriate. Therefore, it was concluded that these various transconjugants carried hybrid plasmids that were probably derivatives of pVS1. Furthermore, it was likely that at least those that had originated from matings involving a Rec- donor carried plasmids in which TnA had been translocated from RP1 into pVS1.
TABLE 1. Phenotype of Cbr transconjugants from matings of P. aeruginosa PAO involving pVS1 + RPI + donors of Cbr transconjugants Donor,Phenotype DJonor
No. of trans-
Km
Tc
Tra
Aer
Hg
Su
R R S S
R R S S
-
R R
-
R
S R R R
S S R R
-
R R R
cnuat
conjugants
Probable type of plasmid carid riedb
Rec+
S
11, 10, 11, 9,
7 7 11 10
Tra-RP1 Tra-RP1 Hg hybrid pVS1 Cb' hybrid pVS1-Cbr Aerr hybrid -
Rec-
S
S Tra-RP1 4, 3 S Tra-RP1 Hgr hybrid 8, 12 S R pVS1-Cbr Aerr hybrid 12, 13 a pVS1+ RP1+ derivatives of PA02001 (rec+) or PA02003 (rec-2) were mated with PA01670 (ade-136 leu8). Cbr transconjugants were selected on the appropriately supplemented minimal agar containing carbenicillin. Only those transconjugants that produced large colonies were tested for their inheritance of various RP1 or pVS1 determined phenotypes. The plasmid phenotype symbols follow those proposed by Novick et al. (10), and the genetic symbols are those proposed by Demerec et al. (3). Aer, Response to aeringinocin AP41; R/S, resistance/sensitivity to various antibacterial agents. b The transfer phenotype was determined by resistance (Tra-) or sensitivity (Tra+) of the strain to phage PR4. R R S
R R
R R
VOL. 129, 1977
NOTES
TABLE 2. Size ofpVS1 and its derivativesa AeruginoIncrease in Mol ci phenoPlasmidb (x 10) mol wt ( 01 type 18.5 pVSl S Rec+ derivatives pVSl-l pVS1-2 pVS1-3 pVS1-4 pVSl-5 Rec- derivatives pVS1-6 pVS1-7 pVS1-8
S S R S R
20.3 21.3 20.3 20.1 20.2
1.8 2.8 1.8 1.6 1.7
R R R R
23.1 20.8 20.6 20.0
5.4 2.3 2.1 1.5
pVSl-9 a DNA was precipitated from cleared lysates by polyethylene glycol 6000, and the plasmid DNA was separated from the contaminating chromosomal DNA by ultracentrifugation in cesium chlorideethidium bromide gradients (13, 16). The preparation of plasmid DNA for electron microscopy and the determination of molecular weight from contour length measurements followed the procedures described by Bennett and Richmond (1). b All pVSl RP1 hybrids conferred the phenotype HgCbr. Each isolate was from a separate mating experiment except for the phenotypically distinguishable pairs pVS1-2, pVS1-3 and pVS1-4, pVS1-5. e Molecular weight was determined relative to RP1 DNA (38 x 106 daltons) and was the average contour length of six to seven molecules. -
To confirm these conclusions, covalently closed circular deoxyribonucleic acid (DNA) was isolated from the Hgr Sur Cbr and Hgr Sur Cbr Aerr transconjugants and the sizes of their plasmid molecules were determined by contour length measurements. As would be expected for translocational derivatives of pVS1, all the plasmids had a size more closely similar to pVS1 (18.5 x 106 daltons [16]) than to RP1 (38 x 10; daltons [1]). However, although all were larger than pVS1 by a molecular weight of 1.5 x 106 to 2.8 x 106f, only the transconjugant carrying pVS1-2 had a size that approximated that predicted for a pVS1 derivative that had acquired the 3 x 10fi- to 4 x 106f-dalton fragment of TnA (4, 5). One other transconjugant that carried pVS1-6 was exceptional in that its plasmid exceeded the size of pVS1 by 5.4 x 106 daltons. As far as could be determined from the small number of transconjugants studied, no correlation was apparent between the size of the plasmid, its carriage of the Cbr or Cbr Aerr determinants, or its origin from either a Rec+ or Rec- donor parent.
1655
Several observations can be made from these various results. (i) It seems likely that the different colonial morphology (small versus large colonies) seen among the Cbr transconjugants selected on minimal medium is due to the presence or absence of the RP1 transfer system. Thus, the majority of transconjugants carried the wild-type (Tra+) RP1 and produced small colonies; whereas those that carried either Tramutants of RP1 (or of RP1 * Hgr hybrids) or derivatives of pVS1 produced large colonies. As there is no significant difference in the sizes of colonies produced by established RP1+ sublines of P. aeruginosa PAO and their R- parents, it appears that the production of small colonies is associated with the recent acquisition of RP1 and may simply be a phenomenon that is accentuated on minimal medium. (ii) Other studies have shown that RP1 and RP4 carry the TnA translocation unit (3 x 106 to 4 x 106 daltons) that encodes the bla gene (4, 5; Bennett, unpublished data). Our isolation of derivatives of pVS1 that have acquired the Cbr and Aerr determinants simultaneously suggests that these two loci are closely linked on the RP1 molecule and may both be carried on TnA. In support of this are the properties of a deletion mutant of RP1 that has apparently lost TnA (P. M. Bennett, Robinson, and M. H. Richmond, manuscript in preparation). This derivative is smaller than the parent plasmid by 3 x 106 daltons and confers resistance neither to carbenicillin nor to aeruginocin. (iii) The observation that all the pVS1 * RP1 hybrids from Rec- donors had the phenotype CbO Aerr compared with only about one-third from Rec+ donors, suggests that two different processes, translocation and recombination, may be involved in their formation. However, the heterogeneity found in the sizes of the different hybrids, irrespective of their origin, did not support this conclusion. It is known that although translocation is a Rec-independent process, it apparently occurs with equal fidelity against both a Rec+ or Rec- background, such that the translocational derivatives are homogeneous in size (1, 5, 16). Therefore, we suggest that the inheritance by pVS1 of both the Cbr and Aerr determinants probably represents the addition to this plasmid of TnA, but that events occur associated with this process, which result in a modification in the size of the final derivative. One factor that might conceivably influence the inheritance of TnA by pVS1 is the Tn501 translocation unit that already occurs on this plasmid. Studies on the reciprocal transfer event, namely the translocation of Tn501 into RP1, have shown that those integrations that
1656
NOTES
J. BACTERIOL.
the bla region yield plasmids of a smaller size than those following integrations elsewhere in the molecule (16). The particular RP1 * HgrCbs hybrids studied were also found to be aeruginocin sensitive, and thus may have arisen by a deletion induced by the integration ofTn501 within or near TnA. If such an interaction occurs, then it might reasonably be expected to be more common when TnA integrates into pVS1 than when Tn501 integrates into RP1, simply because TnA and Tn501 occupy about 8% and 33% of their respective parent plasmids and integration is known to have low site specificity.
occur near
Financial support for this work was from search Council Grant to M. H. Richmond.
a
Medical Re-
LITERATURE CITED 1. Bennett, P.M., and M. H. Richmond. 1976. Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Escherichia
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