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Restriction-modification systems determined by Pseudomonas plasmids
PLASMID
8,
141-147 (1982)
Restriction-Modification Systems Determined by Pseudomonas Plasmids G. A. JACOBY AND L. SUTTON Massachusetts General Hospital, Boston, Massachusetts 021I4 Received March 10, 1982 Four additional Pseudomonas R plasmids determining the PaeR7 restriction-modification system have been detected. All are transfer deficient and appear to belong to the same incompatibility group. The Pseudomonas fertility plasmid FP 110 determines a different restrictionmodification system and also inhibits the propagation of phage B39 by a separate mechanism. Pseudomonas R plasmid pMG73 has a third distinct restriction-modification specificity. PaeFPl 10 and PaeR73 are proposed as designations for these new plasmid-determined systems for restriction and modification.
Although a variety of plasmids found in Pseudomonas aeruginosa interfere with the propagation of DNA bacteriophages (reviewed by Jacoby, 1979), to date only a single plasmid-determined restriction-modification system, PaeR7, has been described (Jacoby and Sutton, 1979). We have detected four additional R plasmids that determine PaeR7 and have characterized two more restriction-modification systems determined by Pseudomonas plasmids. One of these is determined by the FP1 10 fertility plasmid described by Royle and Holloway ( 1980). MATERIALS
AND METHODS
Bacterial strains, plasmids, and phages. The following R- Fp- P. aeruginosa strains were used: PA038 (leu-38) (Haas and Holloway, 1976); PA038 Rip, a spontaneous rifampin-resistant mutant; PA0303 (argBI8 chl-2) (Haas and Holloway, 1976); and PU2 1 (ilv ieu str rif) (Jacoby, 1974). Plasmid FPI 10 was obtained from D. Bradley in PA02 (ser-3) (Royle et al., 1981). RP4::Tn7 was transferred to PU21 by conjugation from E. coli W3 1lOT- (RP4::TnCl) (Barth et al., 1976). RPl-1 (Ingram et al., 1972) and pMG7 (Falkiner et al., 1977) have been described previously. Multiresistant 141
clinical P. aeruginosa isolates in which pMG32, pMG33, pMG5 1, and pMG67 were detected were supplied by G. Miller (Schering Corporation, Bloomfield, N. J.). D. Goldmann (Children’s Hospital Medical Center, Boston) provided the clinical isolate that yielded pMG73. The sources of phages B3, B39, C5, D3, E79, F116, GlOl, M6, PBl, and PR4 have been described (Jacoby, 1974). Media. Phage was propagated using ZC agar overlays on Z agar plates and harvested and diluted in Z broth (Weppelman and Brinton, 1970). Lysates were sterilized with chloroform except for chloroform-susceptible C5, F116, and PR4 which were passed through 0.45-pm pore size membrane filters (Millipore Corp.). Phage titrations were performed using the same media. For liquid cultures P. aeruginosa strains were grown in nutrient broth (Difco) containing 4 mg of potassium nitrate (NNB)/ml (Jacoby, 1974). Selection plates contained minimal medium A (Davis and Mingioli, 1950), supplemental growth factors as required, 2% agar (Difco), and 0.5% glucose. Supplements were added at the following concentrations (in rg/ml): L-arginine, 100; L-leucine, 80; L-isoleucine, 70; L-set-me,100, and L-valine, 117. Antibiotics were added at the following concentrations (in rg/ml): car0 147-619X/82/050 I4 l-07$02.00/0 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
142
JACOBY AND SUTTON TABLE 1 PROPERTIESOFFLASMIDSDETERMININGTHEPU~R~
SYSTEM
Plasmid
Origin
Properties”
Molecular weight (X106)
pMG7 pMG32 pMG33 pMG5 1 pMG67
Dublin, Ireland Chicago, Illinois Chicago, Illinois Rochester, New York Newark, New Jersey
Gm Km Su Tm Hg PueR7 TraGm Km Sm Su Tm PueR7 TmCb Gm Km Sm Su Tm PaeR7 TmHg PaeR7 TraGm Km Sm Su PaeR7 Tm-
28 23 26 64 28
’ Resistance abbreviations: carbenicillin (Cb), gentamicin (Gm), kanamycin (Km), streptomycin (Sm), sulfonamide (Su), tobramycin (Tm), and mercuric chloride (Hg). Tm- indicates transfer deficient.
benicillin, 500; gentamicin, 20; kanamycin, 500; rifampin, 100; streptomycin, 100; sulfadiazine, 5000; tobramycin, 10; and trimethoprim, 2000. For selecting or scoring Hg2+ resistance 0.1 mM HgC12was added to appropriately supplemented minimal plates. Mating. Matings were performed in NNB medium by mixing equal volumes of exponentially growing cultures of the donor at 10’ cells/ml and the recipient at 5 X lo8 cells/ml at 37°C usually for 2 h (Jacoby, 1974). The frequency of transfer was calculated with respect to the number of donor cells at the end of mating. Transformation. Plasmid DNA was isolated by cesium chloride-ethidium bromide gradient centrifugation (Clewell and Helinski, 1969; Jacoby et al., 1978). Transformation was performed as described by Lederberg and Cohen (1974) with MgCl, substituted for CaC12(Mercer and Loutit, 1979) in some experiments. Gel electrophoresis. Agarose gel electrophoresis was performed as described by Meyers et al. ( 1976) or Crosa and Falkow ( 1981). Plasmid molecular weight was estimated from the mobility in the same gel of standard plasmids of known size. Plasmid detection and characterization. Plasmids pMG32, pMG33, and pMG73 were detected in overnight crosses between multiresistant P. aeruginosa clinical isolates and PA038 Rif by plating on antibiotic-containing media with rifampin for counterselection. Plasmids pMG51 and pMG67 were
detected by transformation of PA038 with DNA purified from plasmid-containing multiresistant P. aeruginosa isolates selecting for resistance to Hg2+ or gentamicin. PA038 R+ derivatives were scored for unselected resistances by spotting on antibiotic or He contaming plates, tested for lysogeny by spotting a supematant of overnight growth on a lawn of PA038, crossedwith PA0303 to establish transfer proficiency, spotted with phage lysatesto screen for inhibition of phage prop agation, and their plasmid content determined by agarosegel electrophoresis. Construction of FPllO transposon derivatives. PA02 (FPllO) (RP4::Tn7) was constructed by mating PA02 (FP 110) with PU2 1 (RP4::Tn7). A phage-resistant derivative in which RP4::Tn7 became Tm- was selected with the donor-specific phage PR4 on Z plates containing 500 &ml carbenicillin to circumvent phage resistance from plasmid loss. This strain transferred resistance to carbenicillin or streptomycin and trimethoprim at a frequency of about lo-’ to PA038. Transconjugants resistant to carbenicillin were susceptible to streptomycin and trimethoprim and vice versa indicating that RP4::Tn7 had not been mobilized. RESULTS
Properties of PaeR7 Plasmids Four additional plasmids producing the PaeR7 restriction-modification system have been identified. Their properties are listed in
Pseudomonas RESTRICTION-MODIFICATION
Table 1 together with those of the original PaeR7 plasmid, pMG7, for comparison. Plasmids pMG32 and pMG33 came from strains isolated at the same hospital in Chicago and differed only in resistance to carbenicillin. The other PaeR7 plasmids had diverse geographical origins and less uniformity of resistance markers and molecular weights. All were transfer deficient (Tra-). Plasmids pMG5 1 and pMG67 were obtained by transformation of strain PA038 to mercuric chloride or gentamicin resistance, respectively, using DNA isolated from plasmid-containing P. aeruginosa clinical isolates. Plasmids pMG32 and pMG33 were detected in overnight crossesbetween clinical isolates and PA038 Rif’ that at a frequency of about 1O-’ yielded gentamicin- and rifampin-resistant transconjugants. Tra- plasmids are not uncommonly obtained in such crosses (Iyobe et al., 1974). The mechanism of plasmid transfer is not known, but the PA038 R+ derivatives were not lysogenized and TABLE 2 EFFICIENCYOF PLATINGON PueR7 STRAINS’ StEiiIl Phage B3.RB3.R+.RD3.RD3.R+.RFl16.RFll6.R+.RGlOl.RGlOl.R+.R-
PA038 (pMG32)
PA038 (PMG~ 1)
PA038 (pMG67)
2x 2x 2x 2x 1x 7x 3x 6X
5x 3x 4x 1x 7x 1x 1x 2x
3x 4x 5x 3x 7x 7x 3x 1x
lo-* 10-Z 1o-4 10-2 lo-’ 10-S lo-’ IO-*
1o-4 1o-3 10-5 10-j 10-6 10-S 10-5 lo-’
1o-2 lo-* 1o-4 10-2 10-s 10-7 Io-8 10-S
a The indicated phage was grown on PA038 (designated phage.R-) and titered on PA038 and PA038(R+) to determine the effect of plasmid-caniage on plating efficiency. Phage from a plaque on PA038(R+) was regrown on PA038(R+) and titered on PA038 and PA038(R+) where the efficiency of plating was identical (data not shown). Finally phage from a plaque on PA038 from this titration was regrown on PA038 (designated phage.R+.R-) and again titered on PA038 and PAO38(R+). Propagation of phages B39, C5, E79, M6, and PBl was not effected.
143
SYSTEMS TABLE 3
EFFKIENCYOF PLATINGON FP 110 DERIVATIVES’ Strain
Phage B3.FP B3.pMG70.FPB39.FP B39.pMG70.FP D3.FP D3.pMG70.FP GlOl.FIGlOl.pMG70.FP M6.FP PBlFI-
PA0303 (pMG70) 4x 3x 3x 8x 3x 4x 8x 7x <7 x 12 x
lo-’ 1o-6 lo-’ 1O-3 10-6 10-7 1o-5 1o-5 10-S 10-a
PA0303 (pMG7 1) 1.0 3 x lo+ 1.0 1.0 1.0 1.0
(1Plasmid pMG70 is an FPl lO::Tn7 derivative that retains the broad phage inhibiting properties of FPl 10. Plasmid pMG7 1is an FPI 10::Tn7derivative that effects only phage B39 propagation. The same convention for describing the host for phage growth shown in Table 2 has been followed. Propagation of phages C5, E79, or F116 was not effected.
demonstrated only a single plasmid band on agarosegel electrophoresis. When pMG7 was transformed into PA038(pMG32) or PA038(pMG67) by selecting for mercury resistance, streptomycin resistance determined by the resident plasmid was lost. When pMG7 was transformed ,into PA038(pMG51) by selecting for gentamicin resistance, the 64 X 1O6band of plasmid pMG51 was no longer present on agarose gel electrophoresis. These results suggest that in addition to determining the PaeR7 restriction-modification system, these plasmids also belong to the same incompatibility group.
The pattern of phage inhibition produced by plasmids pMG32, pMG5 1, and pMG67 is illustrated in Table 2. Like pMG7 (Jacoby and Sutton, 1979) these plasmids inhibited the propagation of phagesB3, D3, F116, and G 101. Phage from rare plaques on the R+ host could subsequently grow on an R+ or R- strain with equal efficiency, but once propagated on an R- strain the phage showed the samelow efficiency of propagation on the
144
JACOBY
AND SUTTON
tested) or FP 1lO::Tn7 (four of 40 tested) derivatives had this property, but the majority, EFFICIENCY OF B39 PLATING ON FP 110 DERIVATIVES’ like FP 110, effectedthe propagation of phages B3, D3, GlOl, M6, and PBl as well (Table Strain 3). For convenience an FPl lO::Tn7 derivaPA0303 PA0303 tive with broad phage inhibition properties Phage (pMG70) (PMG’J 1) will be termed pMG70 and a derivative effecting only B39 propagation will be desigB39.FP 3 x 1o-8 5 x 10-6 nated pMG7 1. For phages M6 and PB 1 inB39.pMG70 1.0 1.0 hibition was complete, but for the other B39.pMG7 1 8 x 1om3 1.0 B39.pMG70.FP 4 x 10-3 1.0 phages effected by pMG70 plaques were obB39.pMG7 l.FI5 x 10-3 1.0 served at low ( 1O-5 to lo-‘) frequency. Phagesfrom these plaques plated with equal a See footnote to Table 2 for the convention used to efficiency on pMG70 or FPl lo- hosts, and describe the hosts for phage growth. phages repropagated on an FPl lo- strain R+ host as the original stock thus indicating showed the same low efficiency of propagathat restriction and modification was re- tion as the original stock for phages B3, D3, sponsible for phage inhibition. Strain and G 101 indicating that a restriction-modPA038(pMG33) gave a similar pattern of ification system was involved. Phage B39, phage inhibition. In addition phage modified however, plated with a 104-fold greater effiby growth on PA038 containing each of the ciency after passage through pMG70 and five plasmids shown in Table 1 plated with FPl lo- hosts. Royle and Holloway (1980) showed that high efficiency on PA038 containing any of FP 110 as the resident plasmid is incompatthe other plasmids demonstrating that their ible with R plasmid RPl-1 (also known as restriction-modification systems were idenRI 8-l) or R56Be, an observation that we tical. have confirmed. These plasmids are known to inhibit the propagation of phage B39 Identification of a SecondPseudomonas (Krishnapillai, 1974; Michel-Briand et al., Restriction-ModiJication System 1977) by a process different from restriction Plasmid FP 110 is a fertility plasmid iso- since when rare B39 phage that have overlated by Royle and Holloway (1980) from a come plasmid interference are retested after clinical strain of P. aeruginosa.We observed TABLE 5 that PA02 (FPl 10) inhibited the propagation of phages B3, B39, D3, GlOl, M6, and EFFICIENCY OF PLATING ON PA038 (pMG73y PB 1. FP 110 carries no antibiotic resistances. To facilitate genetic manipulations Strain PA038 (pMG73) Phage FPI 1O::Tnl and F’PI lO::Tn7derivatives were constructed by outcrossing from a donor 1 x lo-’ B3.Rcontaining FPl 10 and a Tra- derivative of 2 x lo-’ B3.R+.RRP4 carrying Tnl and Tn7 selecting for car4 x lo-’ B39.Rbenicillin-resistant (Tnl) or streptomycin1 x lo-’ B39.Rf.Rand trimethoprim-resistant (Tn7) transcon9 x lo-6 D3.R4 x lo-3 D3.Rf.Rjugants. By agarose gel electrophoresis these 6 x lo-’ GlOl.Rderivatives were larger than FP 110 by the 1 x lo-2 GlOl.R+.Rexpected sizes of Tnl or Tn7. Royle and Holloway (1980) reported that a See footnote to Table 2 for the convention used to FP 110 inhibits the propagation of phage B39. describe the hosts for phage growth. Propagation of About 10% of the FPllO::Tnl (two of 19 phages C3, E79, Fl16, M6, and PBl was not effected. TABLE 4
Pseudomonas BESTRICTION-MODIFICATION
145
SYSTEMS
TABLE 6 EF’F’EC~ OFDMG~~ ON FPl lO::Tn7 TRANSFER
Crosses PU21 (FPl 10: :Tnl) X PU21 (FPI 1O::Tnl) X PA0303 (FPl 10: :Tnl) PA0303 (FPI 1O::Tnl)
PA0303 PA0303 (pMG32) (pMG32) X PA038 (pMG32) X PA038 (pMG32)
Frequency of carbenicillin-resistant transconjugants 1x 2x 1x 9x
10-l 10-7 1o-3 lo-4
Property of transconjugants”
0 Gm’/20 Cb’ 20 Gm’/20 Cb’
e The frequency of carbenicillin-resistant (Cb’) transconjugants also resistant to gentamicin (Grn? is indicated.
growth on an R- host, they plate with normal efficiency on a RP 1-1 host as though hostrange phage mutants had been selected. Phagesfrom plaques of B39 on a host carrying pMG7 1, the FP llO::Tn 7 derivative resistant only to this phage, had this same property. They plated with high efficiency on a pMG71 host whether grown on that strain or an FPl lo- host (Table 4). Such phage were, however, restricted 10e3-fold by a pMG70 strain. Phages from plaques of B39 that survived growth on a pMG70 host plated with high efficiency on a pMG7 I host whether or not they had been propagated on an FPl 10 derivative indicating that they also were host-range mutants but with a 1OV3-fold reduction in efficiency after growth on an FPl lo- strain indicating that their susceptibility to the FP 110 restriction system had not been altered. The 1O-’ to 1O-*-fold reduction in B39 plating seen with a pMG70 host is thus the combined effect of restriction and a requirement for phage host-range mutation. Furthermore, since B39 grown on pMG71 is still restricted by pMG70, modification has been lost as well as restriction in this FPl lO::Tn7 derivative. Characterization of a Third Pseudomonas Plasmid Restriction-Modification System Plasmid pMG73 was detected in an overnight cross between a multiresistant P. aeruginosa clinical isolate from Boston and PA038 Rif r that at a frequency of about 1O-’ yielded carbenicillin-resistant transconju-
gants. It determined resistance to carbenicillin, gentamicin, streptomycin, sulfonamide, tobramycin, and mercuric chloride and was Tra-. The plasmid interfered with the propagation of phages B3, B39, D3, and GlOl (Table 5). As determined by cycling phase through pMG73+ and pMG73- hosts, a restriction-modification system was involved. Since for phages D3 and G 101 the efficiency of inhibition was reduced after growth on an R+ host, more than a single mechanism of phage inhibition may be involved, like the effect of FPl 10 on phage B39. Relationship between the Three RestrictionModification Systems Each of these plasmid-determined restriction-modification systems effected the prop agation of a different group of phages, but all inhibited the growth of phages B3, D3, and GlOl . B3 modified by growth on a PaeR7 host was still restricted by FPl 10 or pMG73. Similarly B3 modified by growth on FPl 10 was restricted by a PaeR7 plasmid or by pMG73 and B3 modified by growth on pMG73 was restricted by FPl 10 or by a PaeR7 plasmid. The three restriction modification systems are thus distinct. The designations PaeFPl 10 and PaeR73 are proposed for the systems produced by FPI 10 and pMG73, respectively. Compatibility crosses also indicated that the PaeFP110 and PaeR7 systemsare not the same. As shown in Table 6 the transfer of FPl 1O::Tnl to a pMG32+ recipient was re-
146
JACOBY AND SUTTON
duced 10e6-fold, but the rare (FPllO::Tnl) (pMG32) transconjugants could donate PPllO::TnZ to a pMG32+ or R- host with equal efficiency indicating that PPl 10, like the related plasmid RPl- 1 (Jacoby and Sutton, 1971), is restricted and modified by the PueR7 system. Since (FPl 1O::Tnl) (pMG32) derivatives from these crosseswere stable and FPllO::TnZ could be outcrossed independently, the plasmids belong to different incompatibility groups. In contrast, transfer of PP 1lO::Tn 7 into a recipient carrying pMG73 was not diminished. DISCUSSION
menus plasmids. In our plasmid collection PueR7 is more common and to date has been found on Tra- plasmids that are related by incompatibility. ACKNOWLEDGMENTS We thank D. Bradley, D. Goldmann, and G. Miller for supplying Pseudomonas strains from which plasmids were isolated, R. V. Miller and D. Comb for unpublished data, and G. Bahnam for performing phage titrations. This work was supported by Grant PCM-79 12052 from the National Science Foundation.
REFERENCES BARTH, P. T., DATTA, N., HEDGES,R. W., AND GRINTER,N. J. (1976). Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistance from R483 to other replicons. J. Bacterial 125, 800-810. CLEWELL,D. B., AND HELLINSKI, D. R. (1969). Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open circular form. Proc. Nat. Acad. Sci. USA 62, 11591166. CROSA,J. H., AND FALKOW, S. (1981). Plasmids. In “Manual of Methods for General Bacteriology” (P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips, eds.), pp. 266-282. American Society for Microbiology, Washington, D. C. DAVIS, B. D., AND M~NGIOLI,E. S. (1950). Mutants of Escherichia coli requiring methionine or vitamin Br2. J. Bacterial. 60, 17-28. FALKINER,F. R., KEANE, C. T., DALTON, M., CLANCY, M. T., AND JACOBY,G. A. (1977). Cross infection in a surgical ward caused by Pseudomonas aeruginosa with transferable resistance to gentamicin and tobramycin. J. Clin. Pathol. 30, 73 l-737. HAAS, D., AND HOLLOWAY,B. W. (1976). R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol. Gen. Genet. 144, 243-25 1. HINKLE, N. F., AND MILLER, R. V. (1979). pMG7-mediated restriction of Pseudomonas aeruginosa phage DNAs is determined by a class I1 restriction endonuclease. Plasmid 2, 387-393. INGRAM, L., SYKES,R. B., GRINSTED,J., SAUNDERS, J. R., AND RICHMOND,M. H. (1972). A transmissible resistance element from a strain of Pseudomonas aeruginosa containing no detectable extrachromosomal DNA. J. Gen. Microbial. 72, 269-279. IYOBE,S., HASUDA,K., FUSE,A., AND MITXJHASHI, S. (1974). Demonstration of R factors from Pseudo-
Hinkle and Miller ( 1979) have shown that PaeR7 is a class II restriction endonuclease that recognizes specific nucleotide sequences in the DNA of phage subject to restriction unless these sequences have been modified. The recognition sequence for this enzyme is related to the X/z01sequenceS..CTCGAG..3 (D. Comb, personal communication). Attempts to isolate an analogous endonuclease activity from a pMG70 or pMG73 host have so far been unsuccessful (Miller and Hinkle, Comb and Schildkraut, personal communications). Perhaps a new type of restrictionmodification systemis involved (Yuan, 1981). Plasmid I?110 has two effects on the propagation of phages such as B39. B39 is restricted and modified by PPllO but also its development is inhibited by an FPl lO::Tn7 derivative, such as pMG7 1, in which the Pa&P1 10 system has been inactivated by Tn7 integration. This second mechanism of phage interference, which is shared by other Pseudomonas plasmids (Jacoby, 1979), can be overcome by phage mutation. We have not succeeded in isolating Tnl or Tn7 derivatives of PPl 10 or Tnl derivatives of pMG7 1 devoid of B39 inhibition, although such derivatives might be more suitable for biochemical characterization of the presumed PueFP 110 endonuclease. monas aeruginosa. Antimicrob. Agents Chemother. 5, PueFP 110 and PueR73 are the second and 547-552. third plasmid-determined restriction-modiJACOBY,G. A. (1974). Properties of R plasmids determining gentamicin resistance by acetylation in Pseufication systems determined by Pseudo-
Pseudomonas RESTRICTION-MODIFICATION domonas aeruginosa. Antimicrob. Agents Chemother. 6,239-252.
JACOBY,G. A. (1979). Plasmids of Pseudomonas aeruginosa. In “Pseudomonas aeruginosa. Clinical Manifestations of Infection and Current Therapy” (R. G. Doggett, ed.), pp. 271-309. Academic Press, New York. JACOBY,G. A., AND SUTTON,L. (1977). Restriction and modification determined by a Pseudomonas R plasmid. Plasmid 1, 115-l 16. JACOBY,G. A., WEISS,R., KORFHAGEN,T. R., KRISHNAPILLAI, V., JACOB, A. E., AND HEDGES, R. W. (1976). An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression. J. Bacterial. 136, 1159-l 164. KRISHNAPILLAI, V. (1974). The use of bacteriophages for differentiating plasmids of Pseudomonas aeruginosa. Genet. Res. 23, 327-334. LEDERBERG,E. N., AND COHEN, S. N. (1974). Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bacterial. 119, 1072- 1074. MERCER,A. A., AND LOUTIT, J. S. (1979). Transformation and transfection of Pseudomonas aeruginosa: Effects of metal ions. J. Bacterial. 140, 37-42.
SYSTEMS
147
MEYERS,J. A., SANCHEZ,D., ELWELL,L. P., AND FALKOW, S. (1976). Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacterial. 127, 1529-1537. MICHEL-BRIAND, Y., STANISICH, V. A., AND JouVENOT, M. (1977). Pseudomonas aeruginosa strain isolated in France that carries a plasmid determining carbenicillin resistance. Antimicrob. Agents Chemother. 11, 589-593. ROYLE, P. L., AND HOLLOWAY, B. W. (1980). Relationship between R and FP plasmids in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 17, 293297.
ROYLE, P. L., MATSUMOTO, H., AND HOLLOWAY, B. W. (198 1). Genetic circularity of the Pseudomonas aeruginosa PA0 chromosome. J. Bacterial. 145, 145155.
YUAN, R. (1981). Structure and mechanism of multifunctional restriction endonucleases.Annu. Rev. Biothem. 50, 285-315. WEPPELMAN,R. M., AND BRINTON, C. G., JR. (1970). Infection of Pseudomonas aeruginosa spheroplasts by RNA from a pilus phage. Virology 41, 116-134.
8,
141-147 (1982)
Restriction-Modification Systems Determined by Pseudomonas Plasmids G. A. JACOBY AND L. SUTTON Massachusetts General Hospital, Boston, Massachusetts 021I4 Received March 10, 1982 Four additional Pseudomonas R plasmids determining the PaeR7 restriction-modification system have been detected. All are transfer deficient and appear to belong to the same incompatibility group. The Pseudomonas fertility plasmid FP 110 determines a different restrictionmodification system and also inhibits the propagation of phage B39 by a separate mechanism. Pseudomonas R plasmid pMG73 has a third distinct restriction-modification specificity. PaeFPl 10 and PaeR73 are proposed as designations for these new plasmid-determined systems for restriction and modification.
Although a variety of plasmids found in Pseudomonas aeruginosa interfere with the propagation of DNA bacteriophages (reviewed by Jacoby, 1979), to date only a single plasmid-determined restriction-modification system, PaeR7, has been described (Jacoby and Sutton, 1979). We have detected four additional R plasmids that determine PaeR7 and have characterized two more restriction-modification systems determined by Pseudomonas plasmids. One of these is determined by the FP1 10 fertility plasmid described by Royle and Holloway ( 1980). MATERIALS
AND METHODS
Bacterial strains, plasmids, and phages. The following R- Fp- P. aeruginosa strains were used: PA038 (leu-38) (Haas and Holloway, 1976); PA038 Rip, a spontaneous rifampin-resistant mutant; PA0303 (argBI8 chl-2) (Haas and Holloway, 1976); and PU2 1 (ilv ieu str rif) (Jacoby, 1974). Plasmid FPI 10 was obtained from D. Bradley in PA02 (ser-3) (Royle et al., 1981). RP4::Tn7 was transferred to PU21 by conjugation from E. coli W3 1lOT- (RP4::TnCl) (Barth et al., 1976). RPl-1 (Ingram et al., 1972) and pMG7 (Falkiner et al., 1977) have been described previously. Multiresistant 141
clinical P. aeruginosa isolates in which pMG32, pMG33, pMG5 1, and pMG67 were detected were supplied by G. Miller (Schering Corporation, Bloomfield, N. J.). D. Goldmann (Children’s Hospital Medical Center, Boston) provided the clinical isolate that yielded pMG73. The sources of phages B3, B39, C5, D3, E79, F116, GlOl, M6, PBl, and PR4 have been described (Jacoby, 1974). Media. Phage was propagated using ZC agar overlays on Z agar plates and harvested and diluted in Z broth (Weppelman and Brinton, 1970). Lysates were sterilized with chloroform except for chloroform-susceptible C5, F116, and PR4 which were passed through 0.45-pm pore size membrane filters (Millipore Corp.). Phage titrations were performed using the same media. For liquid cultures P. aeruginosa strains were grown in nutrient broth (Difco) containing 4 mg of potassium nitrate (NNB)/ml (Jacoby, 1974). Selection plates contained minimal medium A (Davis and Mingioli, 1950), supplemental growth factors as required, 2% agar (Difco), and 0.5% glucose. Supplements were added at the following concentrations (in rg/ml): L-arginine, 100; L-leucine, 80; L-isoleucine, 70; L-set-me,100, and L-valine, 117. Antibiotics were added at the following concentrations (in rg/ml): car0 147-619X/82/050 I4 l-07$02.00/0 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
142
JACOBY AND SUTTON TABLE 1 PROPERTIESOFFLASMIDSDETERMININGTHEPU~R~
SYSTEM
Plasmid
Origin
Properties”
Molecular weight (X106)
pMG7 pMG32 pMG33 pMG5 1 pMG67
Dublin, Ireland Chicago, Illinois Chicago, Illinois Rochester, New York Newark, New Jersey
Gm Km Su Tm Hg PueR7 TraGm Km Sm Su Tm PueR7 TmCb Gm Km Sm Su Tm PaeR7 TmHg PaeR7 TraGm Km Sm Su PaeR7 Tm-
28 23 26 64 28
’ Resistance abbreviations: carbenicillin (Cb), gentamicin (Gm), kanamycin (Km), streptomycin (Sm), sulfonamide (Su), tobramycin (Tm), and mercuric chloride (Hg). Tm- indicates transfer deficient.
benicillin, 500; gentamicin, 20; kanamycin, 500; rifampin, 100; streptomycin, 100; sulfadiazine, 5000; tobramycin, 10; and trimethoprim, 2000. For selecting or scoring Hg2+ resistance 0.1 mM HgC12was added to appropriately supplemented minimal plates. Mating. Matings were performed in NNB medium by mixing equal volumes of exponentially growing cultures of the donor at 10’ cells/ml and the recipient at 5 X lo8 cells/ml at 37°C usually for 2 h (Jacoby, 1974). The frequency of transfer was calculated with respect to the number of donor cells at the end of mating. Transformation. Plasmid DNA was isolated by cesium chloride-ethidium bromide gradient centrifugation (Clewell and Helinski, 1969; Jacoby et al., 1978). Transformation was performed as described by Lederberg and Cohen (1974) with MgCl, substituted for CaC12(Mercer and Loutit, 1979) in some experiments. Gel electrophoresis. Agarose gel electrophoresis was performed as described by Meyers et al. ( 1976) or Crosa and Falkow ( 1981). Plasmid molecular weight was estimated from the mobility in the same gel of standard plasmids of known size. Plasmid detection and characterization. Plasmids pMG32, pMG33, and pMG73 were detected in overnight crosses between multiresistant P. aeruginosa clinical isolates and PA038 Rif by plating on antibiotic-containing media with rifampin for counterselection. Plasmids pMG51 and pMG67 were
detected by transformation of PA038 with DNA purified from plasmid-containing multiresistant P. aeruginosa isolates selecting for resistance to Hg2+ or gentamicin. PA038 R+ derivatives were scored for unselected resistances by spotting on antibiotic or He contaming plates, tested for lysogeny by spotting a supematant of overnight growth on a lawn of PA038, crossedwith PA0303 to establish transfer proficiency, spotted with phage lysatesto screen for inhibition of phage prop agation, and their plasmid content determined by agarosegel electrophoresis. Construction of FPllO transposon derivatives. PA02 (FPllO) (RP4::Tn7) was constructed by mating PA02 (FP 110) with PU2 1 (RP4::Tn7). A phage-resistant derivative in which RP4::Tn7 became Tm- was selected with the donor-specific phage PR4 on Z plates containing 500 &ml carbenicillin to circumvent phage resistance from plasmid loss. This strain transferred resistance to carbenicillin or streptomycin and trimethoprim at a frequency of about lo-’ to PA038. Transconjugants resistant to carbenicillin were susceptible to streptomycin and trimethoprim and vice versa indicating that RP4::Tn7 had not been mobilized. RESULTS
Properties of PaeR7 Plasmids Four additional plasmids producing the PaeR7 restriction-modification system have been identified. Their properties are listed in
Pseudomonas RESTRICTION-MODIFICATION
Table 1 together with those of the original PaeR7 plasmid, pMG7, for comparison. Plasmids pMG32 and pMG33 came from strains isolated at the same hospital in Chicago and differed only in resistance to carbenicillin. The other PaeR7 plasmids had diverse geographical origins and less uniformity of resistance markers and molecular weights. All were transfer deficient (Tra-). Plasmids pMG5 1 and pMG67 were obtained by transformation of strain PA038 to mercuric chloride or gentamicin resistance, respectively, using DNA isolated from plasmid-containing P. aeruginosa clinical isolates. Plasmids pMG32 and pMG33 were detected in overnight crossesbetween clinical isolates and PA038 Rif’ that at a frequency of about 1O-’ yielded gentamicin- and rifampin-resistant transconjugants. Tra- plasmids are not uncommonly obtained in such crosses (Iyobe et al., 1974). The mechanism of plasmid transfer is not known, but the PA038 R+ derivatives were not lysogenized and TABLE 2 EFFICIENCYOF PLATINGON PueR7 STRAINS’ StEiiIl Phage B3.RB3.R+.RD3.RD3.R+.RFl16.RFll6.R+.RGlOl.RGlOl.R+.R-
PA038 (pMG32)
PA038 (PMG~ 1)
PA038 (pMG67)
2x 2x 2x 2x 1x 7x 3x 6X
5x 3x 4x 1x 7x 1x 1x 2x
3x 4x 5x 3x 7x 7x 3x 1x
lo-* 10-Z 1o-4 10-2 lo-’ 10-S lo-’ IO-*
1o-4 1o-3 10-5 10-j 10-6 10-S 10-5 lo-’
1o-2 lo-* 1o-4 10-2 10-s 10-7 Io-8 10-S
a The indicated phage was grown on PA038 (designated phage.R-) and titered on PA038 and PA038(R+) to determine the effect of plasmid-caniage on plating efficiency. Phage from a plaque on PA038(R+) was regrown on PA038(R+) and titered on PA038 and PA038(R+) where the efficiency of plating was identical (data not shown). Finally phage from a plaque on PA038 from this titration was regrown on PA038 (designated phage.R+.R-) and again titered on PA038 and PAO38(R+). Propagation of phages B39, C5, E79, M6, and PBl was not effected.
143
SYSTEMS TABLE 3
EFFKIENCYOF PLATINGON FP 110 DERIVATIVES’ Strain
Phage B3.FP B3.pMG70.FPB39.FP B39.pMG70.FP D3.FP D3.pMG70.FP GlOl.FIGlOl.pMG70.FP M6.FP PBlFI-
PA0303 (pMG70) 4x 3x 3x 8x 3x 4x 8x 7x <7 x 12 x
lo-’ 1o-6 lo-’ 1O-3 10-6 10-7 1o-5 1o-5 10-S 10-a
PA0303 (pMG7 1) 1.0 3 x lo+ 1.0 1.0 1.0 1.0
(1Plasmid pMG70 is an FPl lO::Tn7 derivative that retains the broad phage inhibiting properties of FPl 10. Plasmid pMG7 1is an FPI 10::Tn7derivative that effects only phage B39 propagation. The same convention for describing the host for phage growth shown in Table 2 has been followed. Propagation of phages C5, E79, or F116 was not effected.
demonstrated only a single plasmid band on agarosegel electrophoresis. When pMG7 was transformed into PA038(pMG32) or PA038(pMG67) by selecting for mercury resistance, streptomycin resistance determined by the resident plasmid was lost. When pMG7 was transformed ,into PA038(pMG51) by selecting for gentamicin resistance, the 64 X 1O6band of plasmid pMG51 was no longer present on agarose gel electrophoresis. These results suggest that in addition to determining the PaeR7 restriction-modification system, these plasmids also belong to the same incompatibility group.
The pattern of phage inhibition produced by plasmids pMG32, pMG5 1, and pMG67 is illustrated in Table 2. Like pMG7 (Jacoby and Sutton, 1979) these plasmids inhibited the propagation of phagesB3, D3, F116, and G 101. Phage from rare plaques on the R+ host could subsequently grow on an R+ or R- strain with equal efficiency, but once propagated on an R- strain the phage showed the samelow efficiency of propagation on the
144
JACOBY
AND SUTTON
tested) or FP 1lO::Tn7 (four of 40 tested) derivatives had this property, but the majority, EFFICIENCY OF B39 PLATING ON FP 110 DERIVATIVES’ like FP 110, effectedthe propagation of phages B3, D3, GlOl, M6, and PBl as well (Table Strain 3). For convenience an FPl lO::Tn7 derivaPA0303 PA0303 tive with broad phage inhibition properties Phage (pMG70) (PMG’J 1) will be termed pMG70 and a derivative effecting only B39 propagation will be desigB39.FP 3 x 1o-8 5 x 10-6 nated pMG7 1. For phages M6 and PB 1 inB39.pMG70 1.0 1.0 hibition was complete, but for the other B39.pMG7 1 8 x 1om3 1.0 B39.pMG70.FP 4 x 10-3 1.0 phages effected by pMG70 plaques were obB39.pMG7 l.FI5 x 10-3 1.0 served at low ( 1O-5 to lo-‘) frequency. Phagesfrom these plaques plated with equal a See footnote to Table 2 for the convention used to efficiency on pMG70 or FPl lo- hosts, and describe the hosts for phage growth. phages repropagated on an FPl lo- strain R+ host as the original stock thus indicating showed the same low efficiency of propagathat restriction and modification was re- tion as the original stock for phages B3, D3, sponsible for phage inhibition. Strain and G 101 indicating that a restriction-modPA038(pMG33) gave a similar pattern of ification system was involved. Phage B39, phage inhibition. In addition phage modified however, plated with a 104-fold greater effiby growth on PA038 containing each of the ciency after passage through pMG70 and five plasmids shown in Table 1 plated with FPl lo- hosts. Royle and Holloway (1980) showed that high efficiency on PA038 containing any of FP 110 as the resident plasmid is incompatthe other plasmids demonstrating that their ible with R plasmid RPl-1 (also known as restriction-modification systems were idenRI 8-l) or R56Be, an observation that we tical. have confirmed. These plasmids are known to inhibit the propagation of phage B39 Identification of a SecondPseudomonas (Krishnapillai, 1974; Michel-Briand et al., Restriction-ModiJication System 1977) by a process different from restriction Plasmid FP 110 is a fertility plasmid iso- since when rare B39 phage that have overlated by Royle and Holloway (1980) from a come plasmid interference are retested after clinical strain of P. aeruginosa.We observed TABLE 5 that PA02 (FPl 10) inhibited the propagation of phages B3, B39, D3, GlOl, M6, and EFFICIENCY OF PLATING ON PA038 (pMG73y PB 1. FP 110 carries no antibiotic resistances. To facilitate genetic manipulations Strain PA038 (pMG73) Phage FPI 1O::Tnl and F’PI lO::Tn7derivatives were constructed by outcrossing from a donor 1 x lo-’ B3.Rcontaining FPl 10 and a Tra- derivative of 2 x lo-’ B3.R+.RRP4 carrying Tnl and Tn7 selecting for car4 x lo-’ B39.Rbenicillin-resistant (Tnl) or streptomycin1 x lo-’ B39.Rf.Rand trimethoprim-resistant (Tn7) transcon9 x lo-6 D3.R4 x lo-3 D3.Rf.Rjugants. By agarose gel electrophoresis these 6 x lo-’ GlOl.Rderivatives were larger than FP 110 by the 1 x lo-2 GlOl.R+.Rexpected sizes of Tnl or Tn7. Royle and Holloway (1980) reported that a See footnote to Table 2 for the convention used to FP 110 inhibits the propagation of phage B39. describe the hosts for phage growth. Propagation of About 10% of the FPllO::Tnl (two of 19 phages C3, E79, Fl16, M6, and PBl was not effected. TABLE 4
Pseudomonas BESTRICTION-MODIFICATION
145
SYSTEMS
TABLE 6 EF’F’EC~ OFDMG~~ ON FPl lO::Tn7 TRANSFER
Crosses PU21 (FPl 10: :Tnl) X PU21 (FPI 1O::Tnl) X PA0303 (FPl 10: :Tnl) PA0303 (FPI 1O::Tnl)
PA0303 PA0303 (pMG32) (pMG32) X PA038 (pMG32) X PA038 (pMG32)
Frequency of carbenicillin-resistant transconjugants 1x 2x 1x 9x
10-l 10-7 1o-3 lo-4
Property of transconjugants”
0 Gm’/20 Cb’ 20 Gm’/20 Cb’
e The frequency of carbenicillin-resistant (Cb’) transconjugants also resistant to gentamicin (Grn? is indicated.
growth on an R- host, they plate with normal efficiency on a RP 1-1 host as though hostrange phage mutants had been selected. Phagesfrom plaques of B39 on a host carrying pMG7 1, the FP llO::Tn 7 derivative resistant only to this phage, had this same property. They plated with high efficiency on a pMG71 host whether grown on that strain or an FPl lo- host (Table 4). Such phage were, however, restricted 10e3-fold by a pMG70 strain. Phages from plaques of B39 that survived growth on a pMG70 host plated with high efficiency on a pMG7 I host whether or not they had been propagated on an FPl 10 derivative indicating that they also were host-range mutants but with a 1OV3-fold reduction in efficiency after growth on an FPl lo- strain indicating that their susceptibility to the FP 110 restriction system had not been altered. The 1O-’ to 1O-*-fold reduction in B39 plating seen with a pMG70 host is thus the combined effect of restriction and a requirement for phage host-range mutation. Furthermore, since B39 grown on pMG71 is still restricted by pMG70, modification has been lost as well as restriction in this FPl lO::Tn7 derivative. Characterization of a Third Pseudomonas Plasmid Restriction-Modification System Plasmid pMG73 was detected in an overnight cross between a multiresistant P. aeruginosa clinical isolate from Boston and PA038 Rif r that at a frequency of about 1O-’ yielded carbenicillin-resistant transconju-
gants. It determined resistance to carbenicillin, gentamicin, streptomycin, sulfonamide, tobramycin, and mercuric chloride and was Tra-. The plasmid interfered with the propagation of phages B3, B39, D3, and GlOl (Table 5). As determined by cycling phase through pMG73+ and pMG73- hosts, a restriction-modification system was involved. Since for phages D3 and G 101 the efficiency of inhibition was reduced after growth on an R+ host, more than a single mechanism of phage inhibition may be involved, like the effect of FPl 10 on phage B39. Relationship between the Three RestrictionModification Systems Each of these plasmid-determined restriction-modification systems effected the prop agation of a different group of phages, but all inhibited the growth of phages B3, D3, and GlOl . B3 modified by growth on a PaeR7 host was still restricted by FPl 10 or pMG73. Similarly B3 modified by growth on FPl 10 was restricted by a PaeR7 plasmid or by pMG73 and B3 modified by growth on pMG73 was restricted by FPl 10 or by a PaeR7 plasmid. The three restriction modification systems are thus distinct. The designations PaeFPl 10 and PaeR73 are proposed for the systems produced by FPI 10 and pMG73, respectively. Compatibility crosses also indicated that the PaeFP110 and PaeR7 systemsare not the same. As shown in Table 6 the transfer of FPl 1O::Tnl to a pMG32+ recipient was re-
146
JACOBY AND SUTTON
duced 10e6-fold, but the rare (FPllO::Tnl) (pMG32) transconjugants could donate PPllO::TnZ to a pMG32+ or R- host with equal efficiency indicating that PPl 10, like the related plasmid RPl- 1 (Jacoby and Sutton, 1971), is restricted and modified by the PueR7 system. Since (FPl 1O::Tnl) (pMG32) derivatives from these crosseswere stable and FPllO::TnZ could be outcrossed independently, the plasmids belong to different incompatibility groups. In contrast, transfer of PP 1lO::Tn 7 into a recipient carrying pMG73 was not diminished. DISCUSSION
menus plasmids. In our plasmid collection PueR7 is more common and to date has been found on Tra- plasmids that are related by incompatibility. ACKNOWLEDGMENTS We thank D. Bradley, D. Goldmann, and G. Miller for supplying Pseudomonas strains from which plasmids were isolated, R. V. Miller and D. Comb for unpublished data, and G. Bahnam for performing phage titrations. This work was supported by Grant PCM-79 12052 from the National Science Foundation.
REFERENCES BARTH, P. T., DATTA, N., HEDGES,R. W., AND GRINTER,N. J. (1976). Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistance from R483 to other replicons. J. Bacterial 125, 800-810. CLEWELL,D. B., AND HELLINSKI, D. R. (1969). Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open circular form. Proc. Nat. Acad. Sci. USA 62, 11591166. CROSA,J. H., AND FALKOW, S. (1981). Plasmids. In “Manual of Methods for General Bacteriology” (P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips, eds.), pp. 266-282. American Society for Microbiology, Washington, D. C. DAVIS, B. D., AND M~NGIOLI,E. S. (1950). Mutants of Escherichia coli requiring methionine or vitamin Br2. J. Bacterial. 60, 17-28. FALKINER,F. R., KEANE, C. T., DALTON, M., CLANCY, M. T., AND JACOBY,G. A. (1977). Cross infection in a surgical ward caused by Pseudomonas aeruginosa with transferable resistance to gentamicin and tobramycin. J. Clin. Pathol. 30, 73 l-737. HAAS, D., AND HOLLOWAY,B. W. (1976). R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol. Gen. Genet. 144, 243-25 1. HINKLE, N. F., AND MILLER, R. V. (1979). pMG7-mediated restriction of Pseudomonas aeruginosa phage DNAs is determined by a class I1 restriction endonuclease. Plasmid 2, 387-393. INGRAM, L., SYKES,R. B., GRINSTED,J., SAUNDERS, J. R., AND RICHMOND,M. H. (1972). A transmissible resistance element from a strain of Pseudomonas aeruginosa containing no detectable extrachromosomal DNA. J. Gen. Microbial. 72, 269-279. IYOBE,S., HASUDA,K., FUSE,A., AND MITXJHASHI, S. (1974). Demonstration of R factors from Pseudo-
Hinkle and Miller ( 1979) have shown that PaeR7 is a class II restriction endonuclease that recognizes specific nucleotide sequences in the DNA of phage subject to restriction unless these sequences have been modified. The recognition sequence for this enzyme is related to the X/z01sequenceS..CTCGAG..3 (D. Comb, personal communication). Attempts to isolate an analogous endonuclease activity from a pMG70 or pMG73 host have so far been unsuccessful (Miller and Hinkle, Comb and Schildkraut, personal communications). Perhaps a new type of restrictionmodification systemis involved (Yuan, 1981). Plasmid I?110 has two effects on the propagation of phages such as B39. B39 is restricted and modified by PPllO but also its development is inhibited by an FPl lO::Tn7 derivative, such as pMG7 1, in which the Pa&P1 10 system has been inactivated by Tn7 integration. This second mechanism of phage interference, which is shared by other Pseudomonas plasmids (Jacoby, 1979), can be overcome by phage mutation. We have not succeeded in isolating Tnl or Tn7 derivatives of PPl 10 or Tnl derivatives of pMG7 1 devoid of B39 inhibition, although such derivatives might be more suitable for biochemical characterization of the presumed PueFP 110 endonuclease. monas aeruginosa. Antimicrob. Agents Chemother. 5, PueFP 110 and PueR73 are the second and 547-552. third plasmid-determined restriction-modiJACOBY,G. A. (1974). Properties of R plasmids determining gentamicin resistance by acetylation in Pseufication systems determined by Pseudo-
Pseudomonas RESTRICTION-MODIFICATION domonas aeruginosa. Antimicrob. Agents Chemother. 6,239-252.
JACOBY,G. A. (1979). Plasmids of Pseudomonas aeruginosa. In “Pseudomonas aeruginosa. Clinical Manifestations of Infection and Current Therapy” (R. G. Doggett, ed.), pp. 271-309. Academic Press, New York. JACOBY,G. A., AND SUTTON,L. (1977). Restriction and modification determined by a Pseudomonas R plasmid. Plasmid 1, 115-l 16. JACOBY,G. A., WEISS,R., KORFHAGEN,T. R., KRISHNAPILLAI, V., JACOB, A. E., AND HEDGES, R. W. (1976). An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression. J. Bacterial. 136, 1159-l 164. KRISHNAPILLAI, V. (1974). The use of bacteriophages for differentiating plasmids of Pseudomonas aeruginosa. Genet. Res. 23, 327-334. LEDERBERG,E. N., AND COHEN, S. N. (1974). Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bacterial. 119, 1072- 1074. MERCER,A. A., AND LOUTIT, J. S. (1979). Transformation and transfection of Pseudomonas aeruginosa: Effects of metal ions. J. Bacterial. 140, 37-42.
SYSTEMS
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MEYERS,J. A., SANCHEZ,D., ELWELL,L. P., AND FALKOW, S. (1976). Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacterial. 127, 1529-1537. MICHEL-BRIAND, Y., STANISICH, V. A., AND JouVENOT, M. (1977). Pseudomonas aeruginosa strain isolated in France that carries a plasmid determining carbenicillin resistance. Antimicrob. Agents Chemother. 11, 589-593. ROYLE, P. L., AND HOLLOWAY, B. W. (1980). Relationship between R and FP plasmids in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 17, 293297.
ROYLE, P. L., MATSUMOTO, H., AND HOLLOWAY, B. W. (198 1). Genetic circularity of the Pseudomonas aeruginosa PA0 chromosome. J. Bacterial. 145, 145155.
YUAN, R. (1981). Structure and mechanism of multifunctional restriction endonucleases.Annu. Rev. Biothem. 50, 285-315. WEPPELMAN,R. M., AND BRINTON, C. G., JR. (1970). Infection of Pseudomonas aeruginosa spheroplasts by RNA from a pilus phage. Virology 41, 116-134.