Vol. 131, No. 3
JOURNAL OF BACTERIOLOGY, Sept. 1977, p. 765-769 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Inhibition and Facilitation of Transfer Among Pseudomonas aeruginosa R Plasmids HITOSHI SAGAI, SHIZUKO IYOBE,* AND SUSUMU MITSUHASHI Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan Received for publication 22 September 1976
Examining 12 plasmids in Pseudomonas aeruginosa, we found two types of interaction in their transfer (inhibition and facilitation), using donor cells carrying two compatible plasmids. (i) Ten plasmids representing incompatibility groups P-1, P-2, P-5, P-6, and P-7 were all transmissible at a high frequency, 10-2 to 10-1, except for one with a lower frequency of about 10-3. The transfer of P-5
plasmids was inhibited by P-2 plasmids reciprocally or unilaterally, and the unilateral transfer inhibition was observed in other combinations between plasmids belonging to groups P-1, P-2, P-6, and P-7. It was characteristic of Pseudomonas plasmids that most plasmids with high transferability inhibited the transfer of other coexisting plasmids without distinct inhibition of their own transfer. (ii) Two plasmids, Rmsl49 of P-8 group and Rlb679, which was not classified, were transmissible at an exceptionally low frequency of 10-7 to 10-6, but their transfer was facilitated by plasmids with high transferability. We have isolated R plasmids from clinical plex (Gm; Shionogi), carbenicillin (Cb; Fujisawa isolates of Pseudomonas aeruginosa strains Pharmaceutical Co.), rifampin (Rp; Daiichi Seiand mercuric chloride (Hg; Wako Jyunyaku) and classified them into eight groups by incom- yaku),used. patibility properties, using the intraspecies were Media. Nutrient broth and nutrient agar were conjugation system in P. aeruginosa (19). Most used. Nutrient broth consisted of 10 g of beef extract, of the Pseudomonas R plasmids were transmis- 10 g of peptone, and 3 g of NaCl in 1,000 ml of sible at a high frequency, 10-1 to 10-2, whereas distilled water. Nutrient agar consisted of nutrient a few of them were transmitted at a low fre- broth supplemented with 1.5% agar. A-glucose agar quency, 10-7 to 10-i (19, 20). We found that the consisted of medium A (5) without sodium citrate transfer frequency of the former type of R plas- but containing 0.008% bromothymol blue, 1.5% mids was decreased when two compatible R agar, and 0.1% glucose. For the assay of Su resistance or the selection of Su-resistant transconjuplasmids coexisted in the same donor. On the gants, semisynthetic medium was used, which conother hand, the transfer of the latter type of R sisted of 1,000 ml of medium A, 2 g of Casamino plasmids was facilitated by another R plasmid Acids, 10 mg of tryptophan, 2 mg of nicotinic acid, 10 coexisting in the donor. This paper deals with mg of thiamine, 2 g of glucose, and 15 g of agar. inhibition or facilitation of Pseudomonas R Mating experiments. Two milliliters of recipient plasmid transfer from a donor carrying two and 0.5 ml of donor cultures were mixed at the early stationary phase of growth. The mixture was gently compatible R plasmids. shaken at 37°C for 2 h, and then a portion of the appropriately diluted culture was spread on selecMATERIALS AND METHODS tive plates. The transfer frequency was expressed by Bacterial strains. Two auxotrophic and isogenic the number of transconjugants per input donor cell. strains of P. aeruginosa, ML4262 (trp his ilv met rif; A selection plate for the transcorjugants was nutririf = rifampin resistance) and ML4600 (trp his) (20), ent agar containing Rp (200 ,ug/ml) and one of the were used as the host strains for plasmids. selective drugs when ML4262 was used as the recipiR plasmids. R plasmids and their resistance ent. A-glucose agar supplemented with tryptophan, markers are shown in Table 1. RP4 (4), FP2 (8), and Rlb679 (2) were obtained, respectively, from N. Datta (Royal Postgraduate Medical School, London, England), M. Kageyama (Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan), and B. W. Holloway (Monash University, Victoria, Australia). Drugs and abbreviations. Tetracycline (Tc; Lederle Japan), chloramphenicol (Cm; Sankyo), streptomycin (Sm; Kyowa Hakko), sulfanilamide (Su; Dainippon Pharmaceutical Co.), gentamicin C com-
histidine, and a drug was also used as a selection plate when ML4600 was used as the recipient. The concentrations of drugs used for selection were (micrograms per milliliter): Tc, 50; Cm, 75; Sm, 12.5; Su, 800; Gm, 0.8; Cb, 200; and Hg, 10.
RESULTS Inhibition of transfer between R plasmids. The transfer frequency of most R plasmids in P. 765
766
J. BACTBrzoL.
SAGAI, IYOBE, AND MITSUHASHI
aeruginosa ranged from 10-2 to 10-1 (Table 1). quency of 7 x 10-5, which was about 10-fold However, their transfer frequency from the do- lower than the level of Rmsl61 itself. We examined further the transfer inhibition between P-2 nor carrying two compatible plasmids was decreased when compared with the frequency plasmids, and P-5 plasmid R s176. Since from the donor carrying only one of the plas- Rmsl76 mediated only Sm resistance, we used mids (Table 2). The transfer frequency of P-5 P-2 plasmid mutants, Rmsl61-1 and Rmsl64-1, plasmid Rmsl63 was decreased from 100-fold to that had lost Sm resistance (19). Rmsl61-1 inmore than 2,000-fold by coexisting P-2 plas- hibited the transfer of Rmsl76 to a frequency of 10-4 (450-fold), which was the same as the mids. The degree of transfer inhibition of P-2 plas- transfer level of Rms161-1, and Rmsl64-1 inmids Rmsl47, Rmsl59, and Rsu38 by coexisting hibited the Rms176 transfer to a frequency of Rmsl63 ranged from 10- to 200-fold. By con- 10- (18,000-fold). The transfer frequencies of trast, the transfer of two other P-2 plasmids, both Rmsl61-1 and Rmsl64-1 did not change Rmsl61 and Rmsl64, was not inhibited by the with the presence of Rmsl76 in the donor. presence of the P-5 plasmid Rmsl63. Rmsl61 These results indicated that there were two inhibited the P-5 plasmid transfer to a fre- types of inhibition in the transfer between P-2 TABLz 1. R plaumide used Imcompatibility group
Plasmid
Drug resi
Transfer frequencyr
e patrn
Tc Km Cb P-1 RP4 P-2 Tc Sm Su Km Gm Hg Rmsl47 P-2 Tc Cm Sm Hg Rmsl59 P-2 Cm Sm Su Km Hg Rmsl6l P-2 Cm Km Hg Rmsl61-1 P-2 Rmsl64 Sm Su Gm Hg Rmsl64-1 P-2 Su Gm Hg P-2 Rsu38 Tc Sm Su Hg P-5 Tc Cm Su Rmsl63 Rmsl76 P-5 Sm FP2 P-6 Hg P-7 Sm Rmsl48 P-8 Rmsl49 Sm Su Gm Cb NDC Rlb679 Sm Su * Abbreviations for drug resistance are listed in Materials and Methods. * Donor, ML4262; recipient, ML4600. c ND, Not determined. TABLz 2. Transfer inhibition of R pkzsmids
Trnfer frquency of each plasmid
fom donor carrying both Rl and R2 plasmids
Plsmids in donor'
Rms147(Sm) Rms159(8m) Rsu38(Sm)
Rmsl63(Cm) Rmsl63(Su) Rmsl63(Cm)
RI 2 x 10-2 6 x 10-3 1 x 10-3
Rmsl6l(Hg) Rmsl64(Gm)
Rmsl63(Tc) Rmsl63(Tc) Rmsl76(Sm) Rmsl76(Sm)
7 x 2x 1 x 1x
Rl
Rmsl6l-1(Hg) Rmsl64-1(Hg)
8 2 2 1 3 2 2 2 2 9 4 3 6 2
R2
10-4 10-1 10-4 10-'
R2
2,000 280 100 450 18,000
1 x 10-' Rmsl64-1(Gm) 2 x 10-1 1.5 20,000 FP2(Hg) 7 x 10-' 6 x 10-5 4.3 660 1 x 10-' RP4(Cb) 3 x 10-' 1 80 2 x 10-' RP4(Cb) 5 x 10-' 0.6 40 Donor, ML4262; recipient, ML4600. bC Drug in parentheses was used for selection of the R plasmid. Transfer inhibition is expremed by the ratio ofthe transfer fiequency of R plasmid from the donor strain carrying a single plasmid to that from the donor strain carrying two plasmids. A high value indicates a high level of inhibition.
Rmsl48(Sm) Rms148(Sm) alRmsl48(Sm) Rmsl59(Sm)
VOL. 131, 1977
R PLASMID TRANSFER IN P. AERUGINOSA
and P-5 group plasmids. One was a reciprocal inhibition in which each plasmid inhibited the transfer of another plasmid, and the other was a unilateral inhibition in which only one of the two plasmids inhibited the transfer of another plasmid. The degree of transfer inhibition did not differ when ML4600 was used as a donor instead of ML4262. The frequency of the Rmsl63 transfer that had been obtained by transfer from the donor carrying two plasmids, Rmsl63 and Rmsl64, was the same as that of the original Rmsl63, indicating that the Rmsl63 was not modified in its transferability, and its transfer was similarly inhibited by the introduction of Rmsl64 to the same donor cells. These facts indicated that the transfer inhibition occurred irrespective of the host strains only when two plasmids coexisted in the same donor cells. We also found inhibition of transfer among other incompatibility group plasmids. P-7 plasmid Rmsl48 unidirectionally inhibited the transfer of P-2 (Rmsl64-1), P-6 (FP2), and P-1 (RP4) plasmids (Table 2). A P-2 plasmid, Rmsl59, unidirectionally inhibited the transfer of a P-1 plasmid, RP4. Mode of entry exclusion. The two P-5 plasmids, Rmsl63 and Rmsl76, showed entry exclusion with each other, and these exclusions were not affected by coexisting P-2 plasmids, Rmsl61-1 and Rms164-1, in the recipient (Table 3). These facts indicated that the entry exclusion specified by P-5 plasmids was not affected by the coexistence of P-2 plasmids, although the transfer of P-5 plasmids was inhibited by coexisting P-2 plasmids. Facilitation of R plasmid transfer by coexisting R plasmids. Two R plasmids, Rlb679 and Rms149, were transferred at the very low frequencies of 10-6 and 1O-7, respectively (Table 1), and their transfer was facilitated by another coexisting plasmid (Table 4). The mode of transfer facilitation was, however, different between Rlb679 and Rms149. The Rlb679 transfer was highly facilitated by coexisting RP4 and Rmsl63 plasmids but not significantly facilitated by coexisting Rmsl64-1 and FP2. The facilitated level of Rlb679 by RP4 (or Rms163) was the same as the transfer level of RP4 (or Rms163), and about 30% of the transconjugants accepted only resistance markers on Rlb679 and the remaining 70% accepted resistance markers on both plasmids, Rlb679 and RP4 (or Rmsl63). Among transconjugants selected by the resistance markers on RP4 (or Rms163), about 20% (or 2%) accepted only resistance markers on RP4 (or Rmsl63), and the remaining carried both Rlb679 and RP4 (or Rmsl63) plasmids. On the other hand, the transfer frequency of
767
TABLE 3. Entry exclusion of group P-5 plasmids Donor
Recipient
Entry exclusion a
ML4600(Rmsl63)
ML4262 1 ML4262(Rms176) 75,000 ML4262(Rms176, 60,000 Rmsl61-1) ML4262(Rms176, 100,000 Rmsl64-1) ML4262(Rmsl76) ML4600 1 ML4600(Rms163) 6,250 ML4600(Rmsl63, 6,500 Rmsl64-1) a Entry exclusion is expressed by the ratio of the transfer frequency of the R plasmid to the R- recipient to that to the R+ recipient.
Rmsl49 was increased 100-fold by any of the used plasmids coexisting in the same donor, and the facilitated level of the Rmsl49 transfer was about 100-fold lower than the transfer level of coexisting plasmids. All transconjugants accepted resistance markers on both plasmids (Rmsl49 and another coexisting plasmid) from the donor when selected for resistance markers on Rmsl49, and when the selection was done for the markers on the plasmid coexisting with Rmsl49 in the donor, only the selected plasmid was found in the transconjugants. We tried to transfer again from each one of the transconjugants that accepted both plasmids from each donor in Table 4 and found that Rlb679 (or Rmsl49) and coexisting plasmids were transferred by the same mode as seen in the first transfer experiments. The resistance markers were transferred as a unit at the original low frequency from the transconjugant that accepted only resistance markers on Rlb679 or from the strain carrying only resistance markers on Rmsl49, which was obtained from a stock culture of transconjugants harboring markers on both Rmsl49 and RP4. Furthermore, these resistance markers were not excluded by superinfection of RP4, indicating that Rlb679 and Rmsl49, with their original properties, were isolated after the facilitated transfer by RP4. These facts suggested that a stable recombinant formation was not concerned with the facilitated transfer of Rlb679 and Rmsl49. Exclusion of the facilitated transfer. Since RP4 and Rmsl63 show a specific entry exclusion (7, 19), it was expected that the facilitated transfer of Rlb679(or Rmsl49) from the donor carrying both Rlb679 (or Rmsl49) and RP4 (or Rmsl63) would be excluded by RP4 (or Rmsl63) existing in the recipient. The Rlb679 (or Rmsl49) transfer from the donor carrying both
J. BAcTzitIOL. SAGAI, IYOBE, AND MITSUHASHIB
768
TABLz 4. Facilitation of the transfer of Rlb679 or Rmsl49 by coxisting R plasmidsba
Transconjugants
Transfer frequency of:
Drug used for selection
Donor carrying
of: Donor c
marryein
on both
( when (%)plasmide
selected for':
RI
R2
Rl
R2
Rlb679 Rlb679 R1b679 Rlb679 R1b679
RP4 Rmsl63 Rmsl64-1 FP2
Sm Sm Sm Sm Sm
Cb Tc Gm Hg
Sm Rmsl49 Gm Rmsl49 Tc Sm RP4 Rmsl49 Tc Sm Rmsl63 Rmsl49 Gm Tc Rmsl59 Rmsl49 FP2 Hg Sm Rmsl49 a Donor, ML4600; recipient, ML4262. b One hundred transconjugants were tested.
TABz 5. Exclusion of facilitated transfer in Rlb679 and Rms149a Plasmids in Plasmid in Selection Transfer fredonor
Rlb679, RP4
Rlb679, Rmsl63
Rmsl49, RP4 Rmsl49, Rmsl63
recipient
for..
quency 1 x 10-2 2 x 10-5 2 x 10-2
RP4 Rmsl63
Rlb679
RP4 Rmsl63
Rlb679
4 x 10-2 2 x 10-2 3 x 10-5
RP4 Rmsl63
Rmsl49
1 x 10-4 2 x 10-6 7 x 10-4
3 x 10-4
1 x 10-4 RP4 Rmsl49 2 x 10-6 Rmsl63 a Donor, ML4600; recipient, ML4262. ° Sm was used as a selective drug for transconjugants that accepted Rlb679 or Rmsl49.
Rlb679 (or Rmsl49) and RP4 (or Rmsl63) was decreased about 10-3-fold by the presence of RP4 (or Rmsl63) in the recipient (Table 5). In contrast, the facilitated transfer of Rlb679 (or Rmsl49) by RP4 was not inhibited by Rmsl63 in the recipient, and that by Rms163 was also not inhibited by RP4. These facts indicate that the facilitation of the transfer in Rlb679 and Rmsl49 by RP4 (or Rmsl63) is dependent on the transfer system determined by the coexisting RP4 (or Rmsl63). DISCUSSION Two types of interaction in conjugal transfer were found between plasmids coexisting in the
R2
RI
10-7 10-2 10-2 10-7 10-6
2 1 3 5 2
x x x x x
5 3 5 2 4 2
x 10-6 x 10-6 x 10-4 x 10-4 x 10-4 x 10-4
Rl
R2
2 x 10-2 1 x 10-2 4 x 10-l 1 x 10-2
65 69
78 98
10-2 10-2 10-1 10-2
100 100 100 100
0 0 0 0
4 1 3 2
x x x x
same donor cells of P. aeruginosa. Transfer inhibition occurred reciprocally or unilaterally in various combinations of compatible plasmids representing five incompatibility groups. Reciprocal transfer inhibition was observed between P-2 and P-5 plasmids, and the degree of inhibition was 10- to 200-fold in one direction and 2,000-fold or more in another direction. This type of transfer inhibition in Pseudomonas plasmids appeared more striking when compared with that reported in Escherichia coli, in which RP1 and R6K plasmids inhibited transfer reciprocally by 10- to 30-fold (16). Unilateral transfer inhibition between plasmids was first reported in E. coli, e.g., transfer (or fertility) inhibition of the F plasmid by various kinds of fi+ R plasmids (22), and later observed among R plasmids belonging to the P, N, W, or X incompatibility group (16, 17). In Pseudomonas, the fertility of the sex plasmids FP2 and FP5 or transfer of RP1 plasmid was also inhibited unilaterally by some plasmids (11, 21). Unilateral inhibition in our case was seen between all plasmids with a high transfer frequency of 10-2 to 10-1, except for the combination between Rmsl61 (or its derivative Rmsl611) and P-5 plasmids Rmsl63 and Rmsl76. It seems characteristic in Pseudomonas that most plasmids can transfer at a high frequency (19) and strongly inhibit the transfer of another plasmid coexisting in the same cells, without a remarkable decrease in their own transfer. Rmsl61 and its mutant Rmsl61-1 were transferable at the low frequency of 10-4 to 10-3 and inhibited the transfer of coexisting P-5 plasmids to a level similar to that of their transfer frequency. This type of transfer inhibition in P.
VOL. 131, 1977
R PLASMID TRANSFER IN P. AERUGINOSA
aeruginosa is similar to the transfer inhibition in E. coli between F and fi+ R plasmids. The entry exclusion specified by P-5 plasmids was not inhibited by P-2 plasmids, although their transfer was inhibited. A similar type of inhibition was reported in E. coli; that is, fi+ R plasmids R455 and R485 (6) inhibited the transfer of F plasmid without inhibition of entry exclusion. Their inhibition mechanism was considered to be due to an inhibitor which acts directly on the gene(s) or gene product(s) of the transfer loci. Transfer facilitation was observed in Rlb679 and Rms149, which were characteristic in their low transferability. Both Rlb679 and Rmsl49 were transferred without detectable recombinant formation with coexisting other plasmids and could use the transfer system of coexisting plasmids, because their facilitated transfer was blocked when the entry of the coexisting other plasmid was excluded by its residence in recipients. In Enterobacteriaceae, nonconjugative plasmids such as ColEl or Sm (or ampicillin) determinants were mobilized by conjugative plasmids such as F or A, respectively (1, 12). They were mobilized at a high frequency comparable to that of the coexisting conjugative plasmid, and transconjugants carrying only nonconjugative plasmids could be isolated. This may be explained by the association of plasmids; i.e., a plasmid aggregate (3) is formed between nonconjugative and conjugative plasmids in donor cells. There are many reports suggesting association and dissociation between plasmid deoxyribonucleic acid: (i) joint transduction of staphylococcal plasmids without formation of a stable recombinant (9); (ii) joint transformation by mixed deoxyribonucleic acids (15); and (iii) an example of dissociation and association of a complex replicon (18). A similar mechanism might exist in P. aeruginosa plasmids, although the mechanisms of low transferability in Rlb679 and Rms149 were not elucidated. In surveys of clinical isolates for plasmids conferring drug resistance, nonconjugative plasmids or conjugative plasmids with a low transmission frequency were often detected by transfer facilitation due to coexisting plasmids (13, 20). LITERATURE CITED 1. Anderson, E. S., and M. J. Lewis. 1965. Characterization of a transfer factor associated with drug resistance in Salmonella typhimurium. Nature (London)
208:843-849. 2. Bryan, L. E., S. D. Semeka, M. H. Van Den Elzen, J. E. Kinner, and R. E. S. Whitehouse. 1973. Characteristics of R931 and other Pseudomonas aeruginosa R factors. Antimicrob. Agents Chemother. 3:625-637. 3. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405.
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4. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:12441249. 5. Davis, R. D., and E. S. Mingioli. 1960. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-29. 6. Gasson, M. J., and N. S. Willetts. 1975. Five control systems preventing transfer of Escherichia coli K-12 sex factor F. J. Bacteriol. 122:518-525. 7. Hedges, R. W., and A. E. Jacob. 1974. Properties of an R factor from Bordetella bronchiseptica. J. Gen. Microbiol. 84:199-204. 8. Holloway, B. W. 1969. Genetics ofPseudomonas. Bacteriol. Rev. 33:419-443. 9. Inoue, M., T. Okubo, H. Oshima, and S. Mitauhashi. 1975. Staphylococcal plasmids carrying tetracycline and chloramphenicol resistance, p. 153-164. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. 10. Iyobe, S., K. Hasuda, and S. Mitauhashi. 1974. Demonstration of R factors from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 5:547-552. 11. Jacoby, G. A. 1975. R plasmids determining gentamicin or tobramycin resistance in Pseudomonas aeruginosa, p. 287-295. In S. Mitsuhashi, L. Rosival, and V. Krtm6ry (ed.), Drug-inactivating enzymes and antibiotic resistance. Avicenum, Czechoslovak Medical
12.
13.
14.
15.
Press, Prague, and Springer-Verlag, Berlin, Heidelberg, and New York. Kahn, P., and D. R. Helinaki. 1964. Relationship between colicinogenic factors E, and V and an F factor in Escherichia coli. J. Bacteriol. 88:1573-1579. Korfhagen, T. R., J. A. Ferrel, C. L. Menefee, and J. C. Loper. 1976. Resistance plasmids of Pseudomonas aeruginosa: change from conjugative to nonconjugative in a hospital population. Antimicrob. Agents Chemother. 9:810-816. Kawakami, Y., F. Mikoshiba, S. Nagasaki, H. Matsumoto, and T. Tazaki. 1972. Prevalence of Pseudomonas aeruginosa strains possessing R factor in a hospital. J. Antibiot. 25:607-09. Kretschmer, P. J., A. C. Y. Chang, and S. N. Cohen. 1975. Indirect selection of bacterial plasmids lacking identifiable phenotypic properties. J. Bacteriol.
124:225-231. 16. Olsen, R. H., and P. L. Shipley. 1975. PR1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids. J. Bacteriol. 123:28-35. 17. Pinney, R. J., and J. T. Smith. 1974. Fertility inhibition of an N group R factor by a group X R factor, R6K. J. Gen. Microbiol. 82:415-418. 18. Rownd, R. H., N. Goto, E. R. Appelbaum, and D. Perlman. 1975. Dissociation and transition of R plasmids in Proteus mirabilis, p. 3-25. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. 19. Sagai, H., K. Hasuda, S. Iyobe, L. E. Bryan, B. W. Holloway, and S. Mitsuhashi. 1976. Classification of R plasmids by incompatibility in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 10:573578. 20. Sagai, H., V. Kremkry, K. Hasuda, S. Iyobe, H. Knothe, and S. Mitauhashi. 1975. R factor mediated resistance to aminoglycoside antibiotics in Pseudomonas aerugino8a. Jpn. J. Microbiol. 19:427-432. 21. Stanisich, V. A. 1974. The properties and host range of male-specific bacteriophages of Pseudomonas aeruginosa. J. Gen. Microbiol. 84:332-342. 22. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. 1964. Episome-mediated transfer of drug resistance in Enterobacteriaceae. Vfl. Two types of naturally occurring R factors. J. Bacteriol. 88:716726.
JOURNAL OF BACTERIOLOGY, Sept. 1977, p. 765-769 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Inhibition and Facilitation of Transfer Among Pseudomonas aeruginosa R Plasmids HITOSHI SAGAI, SHIZUKO IYOBE,* AND SUSUMU MITSUHASHI Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan Received for publication 22 September 1976
Examining 12 plasmids in Pseudomonas aeruginosa, we found two types of interaction in their transfer (inhibition and facilitation), using donor cells carrying two compatible plasmids. (i) Ten plasmids representing incompatibility groups P-1, P-2, P-5, P-6, and P-7 were all transmissible at a high frequency, 10-2 to 10-1, except for one with a lower frequency of about 10-3. The transfer of P-5
plasmids was inhibited by P-2 plasmids reciprocally or unilaterally, and the unilateral transfer inhibition was observed in other combinations between plasmids belonging to groups P-1, P-2, P-6, and P-7. It was characteristic of Pseudomonas plasmids that most plasmids with high transferability inhibited the transfer of other coexisting plasmids without distinct inhibition of their own transfer. (ii) Two plasmids, Rmsl49 of P-8 group and Rlb679, which was not classified, were transmissible at an exceptionally low frequency of 10-7 to 10-6, but their transfer was facilitated by plasmids with high transferability. We have isolated R plasmids from clinical plex (Gm; Shionogi), carbenicillin (Cb; Fujisawa isolates of Pseudomonas aeruginosa strains Pharmaceutical Co.), rifampin (Rp; Daiichi Seiand mercuric chloride (Hg; Wako Jyunyaku) and classified them into eight groups by incom- yaku),used. patibility properties, using the intraspecies were Media. Nutrient broth and nutrient agar were conjugation system in P. aeruginosa (19). Most used. Nutrient broth consisted of 10 g of beef extract, of the Pseudomonas R plasmids were transmis- 10 g of peptone, and 3 g of NaCl in 1,000 ml of sible at a high frequency, 10-1 to 10-2, whereas distilled water. Nutrient agar consisted of nutrient a few of them were transmitted at a low fre- broth supplemented with 1.5% agar. A-glucose agar quency, 10-7 to 10-i (19, 20). We found that the consisted of medium A (5) without sodium citrate transfer frequency of the former type of R plas- but containing 0.008% bromothymol blue, 1.5% mids was decreased when two compatible R agar, and 0.1% glucose. For the assay of Su resistance or the selection of Su-resistant transconjuplasmids coexisted in the same donor. On the gants, semisynthetic medium was used, which conother hand, the transfer of the latter type of R sisted of 1,000 ml of medium A, 2 g of Casamino plasmids was facilitated by another R plasmid Acids, 10 mg of tryptophan, 2 mg of nicotinic acid, 10 coexisting in the donor. This paper deals with mg of thiamine, 2 g of glucose, and 15 g of agar. inhibition or facilitation of Pseudomonas R Mating experiments. Two milliliters of recipient plasmid transfer from a donor carrying two and 0.5 ml of donor cultures were mixed at the early stationary phase of growth. The mixture was gently compatible R plasmids. shaken at 37°C for 2 h, and then a portion of the appropriately diluted culture was spread on selecMATERIALS AND METHODS tive plates. The transfer frequency was expressed by Bacterial strains. Two auxotrophic and isogenic the number of transconjugants per input donor cell. strains of P. aeruginosa, ML4262 (trp his ilv met rif; A selection plate for the transcorjugants was nutririf = rifampin resistance) and ML4600 (trp his) (20), ent agar containing Rp (200 ,ug/ml) and one of the were used as the host strains for plasmids. selective drugs when ML4262 was used as the recipiR plasmids. R plasmids and their resistance ent. A-glucose agar supplemented with tryptophan, markers are shown in Table 1. RP4 (4), FP2 (8), and Rlb679 (2) were obtained, respectively, from N. Datta (Royal Postgraduate Medical School, London, England), M. Kageyama (Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan), and B. W. Holloway (Monash University, Victoria, Australia). Drugs and abbreviations. Tetracycline (Tc; Lederle Japan), chloramphenicol (Cm; Sankyo), streptomycin (Sm; Kyowa Hakko), sulfanilamide (Su; Dainippon Pharmaceutical Co.), gentamicin C com-
histidine, and a drug was also used as a selection plate when ML4600 was used as the recipient. The concentrations of drugs used for selection were (micrograms per milliliter): Tc, 50; Cm, 75; Sm, 12.5; Su, 800; Gm, 0.8; Cb, 200; and Hg, 10.
RESULTS Inhibition of transfer between R plasmids. The transfer frequency of most R plasmids in P. 765
766
J. BACTBrzoL.
SAGAI, IYOBE, AND MITSUHASHI
aeruginosa ranged from 10-2 to 10-1 (Table 1). quency of 7 x 10-5, which was about 10-fold However, their transfer frequency from the do- lower than the level of Rmsl61 itself. We examined further the transfer inhibition between P-2 nor carrying two compatible plasmids was decreased when compared with the frequency plasmids, and P-5 plasmid R s176. Since from the donor carrying only one of the plas- Rmsl76 mediated only Sm resistance, we used mids (Table 2). The transfer frequency of P-5 P-2 plasmid mutants, Rmsl61-1 and Rmsl64-1, plasmid Rmsl63 was decreased from 100-fold to that had lost Sm resistance (19). Rmsl61-1 inmore than 2,000-fold by coexisting P-2 plas- hibited the transfer of Rmsl76 to a frequency of 10-4 (450-fold), which was the same as the mids. The degree of transfer inhibition of P-2 plas- transfer level of Rms161-1, and Rmsl64-1 inmids Rmsl47, Rmsl59, and Rsu38 by coexisting hibited the Rms176 transfer to a frequency of Rmsl63 ranged from 10- to 200-fold. By con- 10- (18,000-fold). The transfer frequencies of trast, the transfer of two other P-2 plasmids, both Rmsl61-1 and Rmsl64-1 did not change Rmsl61 and Rmsl64, was not inhibited by the with the presence of Rmsl76 in the donor. presence of the P-5 plasmid Rmsl63. Rmsl61 These results indicated that there were two inhibited the P-5 plasmid transfer to a fre- types of inhibition in the transfer between P-2 TABLz 1. R plaumide used Imcompatibility group
Plasmid
Drug resi
Transfer frequencyr
e patrn
Tc Km Cb P-1 RP4 P-2 Tc Sm Su Km Gm Hg Rmsl47 P-2 Tc Cm Sm Hg Rmsl59 P-2 Cm Sm Su Km Hg Rmsl6l P-2 Cm Km Hg Rmsl61-1 P-2 Rmsl64 Sm Su Gm Hg Rmsl64-1 P-2 Su Gm Hg P-2 Rsu38 Tc Sm Su Hg P-5 Tc Cm Su Rmsl63 Rmsl76 P-5 Sm FP2 P-6 Hg P-7 Sm Rmsl48 P-8 Rmsl49 Sm Su Gm Cb NDC Rlb679 Sm Su * Abbreviations for drug resistance are listed in Materials and Methods. * Donor, ML4262; recipient, ML4600. c ND, Not determined. TABLz 2. Transfer inhibition of R pkzsmids
Trnfer frquency of each plasmid
fom donor carrying both Rl and R2 plasmids
Plsmids in donor'
Rms147(Sm) Rms159(8m) Rsu38(Sm)
Rmsl63(Cm) Rmsl63(Su) Rmsl63(Cm)
RI 2 x 10-2 6 x 10-3 1 x 10-3
Rmsl6l(Hg) Rmsl64(Gm)
Rmsl63(Tc) Rmsl63(Tc) Rmsl76(Sm) Rmsl76(Sm)
7 x 2x 1 x 1x
Rl
Rmsl6l-1(Hg) Rmsl64-1(Hg)
8 2 2 1 3 2 2 2 2 9 4 3 6 2
R2
10-4 10-1 10-4 10-'
R2
2,000 280 100 450 18,000
1 x 10-' Rmsl64-1(Gm) 2 x 10-1 1.5 20,000 FP2(Hg) 7 x 10-' 6 x 10-5 4.3 660 1 x 10-' RP4(Cb) 3 x 10-' 1 80 2 x 10-' RP4(Cb) 5 x 10-' 0.6 40 Donor, ML4262; recipient, ML4600. bC Drug in parentheses was used for selection of the R plasmid. Transfer inhibition is expremed by the ratio ofthe transfer fiequency of R plasmid from the donor strain carrying a single plasmid to that from the donor strain carrying two plasmids. A high value indicates a high level of inhibition.
Rmsl48(Sm) Rms148(Sm) alRmsl48(Sm) Rmsl59(Sm)
VOL. 131, 1977
R PLASMID TRANSFER IN P. AERUGINOSA
and P-5 group plasmids. One was a reciprocal inhibition in which each plasmid inhibited the transfer of another plasmid, and the other was a unilateral inhibition in which only one of the two plasmids inhibited the transfer of another plasmid. The degree of transfer inhibition did not differ when ML4600 was used as a donor instead of ML4262. The frequency of the Rmsl63 transfer that had been obtained by transfer from the donor carrying two plasmids, Rmsl63 and Rmsl64, was the same as that of the original Rmsl63, indicating that the Rmsl63 was not modified in its transferability, and its transfer was similarly inhibited by the introduction of Rmsl64 to the same donor cells. These facts indicated that the transfer inhibition occurred irrespective of the host strains only when two plasmids coexisted in the same donor cells. We also found inhibition of transfer among other incompatibility group plasmids. P-7 plasmid Rmsl48 unidirectionally inhibited the transfer of P-2 (Rmsl64-1), P-6 (FP2), and P-1 (RP4) plasmids (Table 2). A P-2 plasmid, Rmsl59, unidirectionally inhibited the transfer of a P-1 plasmid, RP4. Mode of entry exclusion. The two P-5 plasmids, Rmsl63 and Rmsl76, showed entry exclusion with each other, and these exclusions were not affected by coexisting P-2 plasmids, Rmsl61-1 and Rms164-1, in the recipient (Table 3). These facts indicated that the entry exclusion specified by P-5 plasmids was not affected by the coexistence of P-2 plasmids, although the transfer of P-5 plasmids was inhibited by coexisting P-2 plasmids. Facilitation of R plasmid transfer by coexisting R plasmids. Two R plasmids, Rlb679 and Rms149, were transferred at the very low frequencies of 10-6 and 1O-7, respectively (Table 1), and their transfer was facilitated by another coexisting plasmid (Table 4). The mode of transfer facilitation was, however, different between Rlb679 and Rms149. The Rlb679 transfer was highly facilitated by coexisting RP4 and Rmsl63 plasmids but not significantly facilitated by coexisting Rmsl64-1 and FP2. The facilitated level of Rlb679 by RP4 (or Rms163) was the same as the transfer level of RP4 (or Rms163), and about 30% of the transconjugants accepted only resistance markers on Rlb679 and the remaining 70% accepted resistance markers on both plasmids, Rlb679 and RP4 (or Rmsl63). Among transconjugants selected by the resistance markers on RP4 (or Rms163), about 20% (or 2%) accepted only resistance markers on RP4 (or Rmsl63), and the remaining carried both Rlb679 and RP4 (or Rmsl63) plasmids. On the other hand, the transfer frequency of
767
TABLE 3. Entry exclusion of group P-5 plasmids Donor
Recipient
Entry exclusion a
ML4600(Rmsl63)
ML4262 1 ML4262(Rms176) 75,000 ML4262(Rms176, 60,000 Rmsl61-1) ML4262(Rms176, 100,000 Rmsl64-1) ML4262(Rmsl76) ML4600 1 ML4600(Rms163) 6,250 ML4600(Rmsl63, 6,500 Rmsl64-1) a Entry exclusion is expressed by the ratio of the transfer frequency of the R plasmid to the R- recipient to that to the R+ recipient.
Rmsl49 was increased 100-fold by any of the used plasmids coexisting in the same donor, and the facilitated level of the Rmsl49 transfer was about 100-fold lower than the transfer level of coexisting plasmids. All transconjugants accepted resistance markers on both plasmids (Rmsl49 and another coexisting plasmid) from the donor when selected for resistance markers on Rmsl49, and when the selection was done for the markers on the plasmid coexisting with Rmsl49 in the donor, only the selected plasmid was found in the transconjugants. We tried to transfer again from each one of the transconjugants that accepted both plasmids from each donor in Table 4 and found that Rlb679 (or Rmsl49) and coexisting plasmids were transferred by the same mode as seen in the first transfer experiments. The resistance markers were transferred as a unit at the original low frequency from the transconjugant that accepted only resistance markers on Rlb679 or from the strain carrying only resistance markers on Rmsl49, which was obtained from a stock culture of transconjugants harboring markers on both Rmsl49 and RP4. Furthermore, these resistance markers were not excluded by superinfection of RP4, indicating that Rlb679 and Rmsl49, with their original properties, were isolated after the facilitated transfer by RP4. These facts suggested that a stable recombinant formation was not concerned with the facilitated transfer of Rlb679 and Rmsl49. Exclusion of the facilitated transfer. Since RP4 and Rmsl63 show a specific entry exclusion (7, 19), it was expected that the facilitated transfer of Rlb679(or Rmsl49) from the donor carrying both Rlb679 (or Rmsl49) and RP4 (or Rmsl63) would be excluded by RP4 (or Rmsl63) existing in the recipient. The Rlb679 (or Rmsl49) transfer from the donor carrying both
J. BAcTzitIOL. SAGAI, IYOBE, AND MITSUHASHIB
768
TABLz 4. Facilitation of the transfer of Rlb679 or Rmsl49 by coxisting R plasmidsba
Transconjugants
Transfer frequency of:
Drug used for selection
Donor carrying
of: Donor c
marryein
on both
( when (%)plasmide
selected for':
RI
R2
Rl
R2
Rlb679 Rlb679 R1b679 Rlb679 R1b679
RP4 Rmsl63 Rmsl64-1 FP2
Sm Sm Sm Sm Sm
Cb Tc Gm Hg
Sm Rmsl49 Gm Rmsl49 Tc Sm RP4 Rmsl49 Tc Sm Rmsl63 Rmsl49 Gm Tc Rmsl59 Rmsl49 FP2 Hg Sm Rmsl49 a Donor, ML4600; recipient, ML4262. b One hundred transconjugants were tested.
TABz 5. Exclusion of facilitated transfer in Rlb679 and Rms149a Plasmids in Plasmid in Selection Transfer fredonor
Rlb679, RP4
Rlb679, Rmsl63
Rmsl49, RP4 Rmsl49, Rmsl63
recipient
for..
quency 1 x 10-2 2 x 10-5 2 x 10-2
RP4 Rmsl63
Rlb679
RP4 Rmsl63
Rlb679
4 x 10-2 2 x 10-2 3 x 10-5
RP4 Rmsl63
Rmsl49
1 x 10-4 2 x 10-6 7 x 10-4
3 x 10-4
1 x 10-4 RP4 Rmsl49 2 x 10-6 Rmsl63 a Donor, ML4600; recipient, ML4262. ° Sm was used as a selective drug for transconjugants that accepted Rlb679 or Rmsl49.
Rlb679 (or Rmsl49) and RP4 (or Rmsl63) was decreased about 10-3-fold by the presence of RP4 (or Rmsl63) in the recipient (Table 5). In contrast, the facilitated transfer of Rlb679 (or Rmsl49) by RP4 was not inhibited by Rmsl63 in the recipient, and that by Rms163 was also not inhibited by RP4. These facts indicate that the facilitation of the transfer in Rlb679 and Rmsl49 by RP4 (or Rmsl63) is dependent on the transfer system determined by the coexisting RP4 (or Rmsl63). DISCUSSION Two types of interaction in conjugal transfer were found between plasmids coexisting in the
R2
RI
10-7 10-2 10-2 10-7 10-6
2 1 3 5 2
x x x x x
5 3 5 2 4 2
x 10-6 x 10-6 x 10-4 x 10-4 x 10-4 x 10-4
Rl
R2
2 x 10-2 1 x 10-2 4 x 10-l 1 x 10-2
65 69
78 98
10-2 10-2 10-1 10-2
100 100 100 100
0 0 0 0
4 1 3 2
x x x x
same donor cells of P. aeruginosa. Transfer inhibition occurred reciprocally or unilaterally in various combinations of compatible plasmids representing five incompatibility groups. Reciprocal transfer inhibition was observed between P-2 and P-5 plasmids, and the degree of inhibition was 10- to 200-fold in one direction and 2,000-fold or more in another direction. This type of transfer inhibition in Pseudomonas plasmids appeared more striking when compared with that reported in Escherichia coli, in which RP1 and R6K plasmids inhibited transfer reciprocally by 10- to 30-fold (16). Unilateral transfer inhibition between plasmids was first reported in E. coli, e.g., transfer (or fertility) inhibition of the F plasmid by various kinds of fi+ R plasmids (22), and later observed among R plasmids belonging to the P, N, W, or X incompatibility group (16, 17). In Pseudomonas, the fertility of the sex plasmids FP2 and FP5 or transfer of RP1 plasmid was also inhibited unilaterally by some plasmids (11, 21). Unilateral inhibition in our case was seen between all plasmids with a high transfer frequency of 10-2 to 10-1, except for the combination between Rmsl61 (or its derivative Rmsl611) and P-5 plasmids Rmsl63 and Rmsl76. It seems characteristic in Pseudomonas that most plasmids can transfer at a high frequency (19) and strongly inhibit the transfer of another plasmid coexisting in the same cells, without a remarkable decrease in their own transfer. Rmsl61 and its mutant Rmsl61-1 were transferable at the low frequency of 10-4 to 10-3 and inhibited the transfer of coexisting P-5 plasmids to a level similar to that of their transfer frequency. This type of transfer inhibition in P.
VOL. 131, 1977
R PLASMID TRANSFER IN P. AERUGINOSA
aeruginosa is similar to the transfer inhibition in E. coli between F and fi+ R plasmids. The entry exclusion specified by P-5 plasmids was not inhibited by P-2 plasmids, although their transfer was inhibited. A similar type of inhibition was reported in E. coli; that is, fi+ R plasmids R455 and R485 (6) inhibited the transfer of F plasmid without inhibition of entry exclusion. Their inhibition mechanism was considered to be due to an inhibitor which acts directly on the gene(s) or gene product(s) of the transfer loci. Transfer facilitation was observed in Rlb679 and Rms149, which were characteristic in their low transferability. Both Rlb679 and Rmsl49 were transferred without detectable recombinant formation with coexisting other plasmids and could use the transfer system of coexisting plasmids, because their facilitated transfer was blocked when the entry of the coexisting other plasmid was excluded by its residence in recipients. In Enterobacteriaceae, nonconjugative plasmids such as ColEl or Sm (or ampicillin) determinants were mobilized by conjugative plasmids such as F or A, respectively (1, 12). They were mobilized at a high frequency comparable to that of the coexisting conjugative plasmid, and transconjugants carrying only nonconjugative plasmids could be isolated. This may be explained by the association of plasmids; i.e., a plasmid aggregate (3) is formed between nonconjugative and conjugative plasmids in donor cells. There are many reports suggesting association and dissociation between plasmid deoxyribonucleic acid: (i) joint transduction of staphylococcal plasmids without formation of a stable recombinant (9); (ii) joint transformation by mixed deoxyribonucleic acids (15); and (iii) an example of dissociation and association of a complex replicon (18). A similar mechanism might exist in P. aeruginosa plasmids, although the mechanisms of low transferability in Rlb679 and Rms149 were not elucidated. In surveys of clinical isolates for plasmids conferring drug resistance, nonconjugative plasmids or conjugative plasmids with a low transmission frequency were often detected by transfer facilitation due to coexisting plasmids (13, 20). LITERATURE CITED 1. Anderson, E. S., and M. J. Lewis. 1965. Characterization of a transfer factor associated with drug resistance in Salmonella typhimurium. Nature (London)
208:843-849. 2. Bryan, L. E., S. D. Semeka, M. H. Van Den Elzen, J. E. Kinner, and R. E. S. Whitehouse. 1973. Characteristics of R931 and other Pseudomonas aeruginosa R factors. Antimicrob. Agents Chemother. 3:625-637. 3. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405.
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4. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:12441249. 5. Davis, R. D., and E. S. Mingioli. 1960. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-29. 6. Gasson, M. J., and N. S. Willetts. 1975. Five control systems preventing transfer of Escherichia coli K-12 sex factor F. J. Bacteriol. 122:518-525. 7. Hedges, R. W., and A. E. Jacob. 1974. Properties of an R factor from Bordetella bronchiseptica. J. Gen. Microbiol. 84:199-204. 8. Holloway, B. W. 1969. Genetics ofPseudomonas. Bacteriol. Rev. 33:419-443. 9. Inoue, M., T. Okubo, H. Oshima, and S. Mitauhashi. 1975. Staphylococcal plasmids carrying tetracycline and chloramphenicol resistance, p. 153-164. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. 10. Iyobe, S., K. Hasuda, and S. Mitauhashi. 1974. Demonstration of R factors from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 5:547-552. 11. Jacoby, G. A. 1975. R plasmids determining gentamicin or tobramycin resistance in Pseudomonas aeruginosa, p. 287-295. In S. Mitsuhashi, L. Rosival, and V. Krtm6ry (ed.), Drug-inactivating enzymes and antibiotic resistance. Avicenum, Czechoslovak Medical
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15.
Press, Prague, and Springer-Verlag, Berlin, Heidelberg, and New York. Kahn, P., and D. R. Helinaki. 1964. Relationship between colicinogenic factors E, and V and an F factor in Escherichia coli. J. Bacteriol. 88:1573-1579. Korfhagen, T. R., J. A. Ferrel, C. L. Menefee, and J. C. Loper. 1976. Resistance plasmids of Pseudomonas aeruginosa: change from conjugative to nonconjugative in a hospital population. Antimicrob. Agents Chemother. 9:810-816. Kawakami, Y., F. Mikoshiba, S. Nagasaki, H. Matsumoto, and T. Tazaki. 1972. Prevalence of Pseudomonas aeruginosa strains possessing R factor in a hospital. J. Antibiot. 25:607-09. Kretschmer, P. J., A. C. Y. Chang, and S. N. Cohen. 1975. Indirect selection of bacterial plasmids lacking identifiable phenotypic properties. J. Bacteriol.
124:225-231. 16. Olsen, R. H., and P. L. Shipley. 1975. PR1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids. J. Bacteriol. 123:28-35. 17. Pinney, R. J., and J. T. Smith. 1974. Fertility inhibition of an N group R factor by a group X R factor, R6K. J. Gen. Microbiol. 82:415-418. 18. Rownd, R. H., N. Goto, E. R. Appelbaum, and D. Perlman. 1975. Dissociation and transition of R plasmids in Proteus mirabilis, p. 3-25. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. 19. Sagai, H., K. Hasuda, S. Iyobe, L. E. Bryan, B. W. Holloway, and S. Mitsuhashi. 1976. Classification of R plasmids by incompatibility in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 10:573578. 20. Sagai, H., V. Kremkry, K. Hasuda, S. Iyobe, H. Knothe, and S. Mitauhashi. 1975. R factor mediated resistance to aminoglycoside antibiotics in Pseudomonas aerugino8a. Jpn. J. Microbiol. 19:427-432. 21. Stanisich, V. A. 1974. The properties and host range of male-specific bacteriophages of Pseudomonas aeruginosa. J. Gen. Microbiol. 84:332-342. 22. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. 1964. Episome-mediated transfer of drug resistance in Enterobacteriaceae. Vfl. Two types of naturally occurring R factors. J. Bacteriol. 88:716726.
