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R1767, an example of the evolution of resistance plasmids
PLASMID
13, 163-172 (1985)
R1767, An Example of the Evolution of Resistance BERTHOLDA.NIES,JOACHIM
F. MEYER,JOHANNES JQATZ,AND
Plasmids
BERND~IEDEMANN
Institut ftir Medizinische Mikrobiologie und Immunologic, der Universitiit Bonn, 5300 Bonn, West Germany Received September 24, 1984; revised January 30, 1985 The Salmonella R-factor system R1767 undergoes frequent rearrangement of its plasmid components. The flux of genetic material within this plasmid system depends on a combination of illegitimate and homologous recombination. The presence of several copies of IS160 and two multiresistance transposons,Tn2410 and Tn2411, are substantial reasonsfor the observed variations. 0 1985 Academic FTes, Inc.
Plasmid-encoded antibiotic resistance among bacterial pathogens causing infectious diseasesis a major problem worldwide. However by studying the emergence and distribution of resistance genes in bacteria several mechanisms, such as transposition, inversion and deletion, mediated by illegitimate and generalized recombination have been found to participate in the macroevolution of bacterial plasmids (Cohen et al., 1978; Kopecko, 1980). Despite this, only rarely has the concerted action of several different mechanisms been examined in one and the same system. In 1974 we demonstrated the reassortment of the R-factor system RI767 and speculated on the possible mechanisms involved (Richmond and Wiedemann, 1974). Since then the system has been analyzed in more detail and it is now possible to propose credible explanation for this reassortment. This paper describes how a combination of several molecular processes provides a basis for the generation of new genetic structures. Accordingly, we believe that this Rfactor system may serve as an excellent example of the evolution of antibiotic resistance plasmids. MATERIAL
AND
METHODS
All methods, including DNA preparations, cloning experiments, transformation, conjugation, phage Pl transduction, and electron microscope analysis have been described bc-
fore (Meyer et al., 1983; Kratz et al., 1983a). Plasmids and bacterial strains are listed in Table 1. RESULTS
Phenotypic Derivatives of RI 767 and Its Plasmid Components
The conjugative R-factor system R1767 was derived from a clinical isolate of Salmonella typhimurium (Richmond and Wiedemann, 1974). This plasmid systemmediates resistance toward ampicillin by the production of an OXA- &lactamase, to streptomycin by the production of an APH-(3”) enzyme and an AAD-(3”) enzyme (the latter enzyme also confers resistance to spectinomycin), and to tetracycline, chloramphenicol, sulfonamides, mercuric chloride, arsenate, and carries genesfor the production of colicin I. R1767 is intrinsically unstable even in recA background (Wiedemann, 198I). Different bacterial hosts which harbored R1767 usually showed four plasmid bands. Two smaller replicons encoded Tc’ resistance (12 kb, designated pBP 15) and APH-( 3”)-mediated streptomycin resistance (8 kb, desig’ Abbreviations used: Ap, ampicillin; Cm, chloramphenicol; Su, sulfonamides; Hg, mercuric chloride; Sm, streptomycin; Sp, $rectinomycin; Tc, tetracycline; As, arsenate; Pm, paromomycin; ColI, colicin I; Km, kanamycin; Tra, transfer factor: aadA, gene encoding the AAD(3”) enzyme; oxa, gene encoding the OXA- /3lactamase.
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0147-619X/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
164
NIES ET AL. TABLE 1 BACTERIALSTRAINSAND~LASMIDS
Bacterial strains E. coli JC2926 E. coli SK1 592 E. coli W3110 Proteus mirabilis Pm 13 Plasmids pUB307 pSCIO5 pHS1 pACYC184::Tn21 R1767
Sm’, recA, thi, thr, arg, his, leu, lac, ma1 Tl r, gal, thi, sbcBl5, hsr4, hsm+ Nx’, lacf
Km’, Tc’, Tm+ Km’, Tc’ Tc’, replication defective at 42°C Cm’, Hg’, Sp’/Sm’ Su’ Ap’, Su’, Sm’, Sp’/Sm’, Hg’, Cm’, Tc’, As’, Colt, Tra+
nated pBP 14) respectively. Both resistance phenotypes were also encoded by tet or aph genes,respectively, integrated into larger replicons. The two other plasmid bands, comprising replicons of 90 and 120- 150 kb, could not be related to defined resistance patterns. Conjugation and transformation experiments with R1767 from ret+ or recA donor strains (the recipient strain always was Escherichia coli SK1592 in transformation and E. coli W3 110 in conjugation experiments) yielded a broad variety of phenotypes among the trascipients and these carried only some of the R1767 resistance markers, in different combinations. The Ret state of the donor did not influence the statistical distribution of the phenotypes significantly. The marker combinations found in these experiments are summarized in Table 2. In order to relate the resistance phenotype to plasmid content, different transformants were examined to determine what plasmids were carried. Ampicillin resistanceand sulfonamide resistance, sometimes linked to Sm’ [APH-(3”)] and/or Tc’ and/or Tra functions were encoded by plasmids varying in size from 11.5 to 60 kb. Some representative Ap’Su’ transformants were analyzed in detail and are described below (Fig. 1). Stability tests with two of these transformants-pBP 16 (Ap%TTc’Sm’As’Tra+, about 60 kb) and pBPl8 (Ap’Su’-
Bachmann ( 1972) Kushner (1978) Grinsted ( 1972) Coetzee ( 1963) Bennett et a/. (1975) Cohen et al. (1973) Hashimoto and Sekiguchi ( 1976) This paper Richmond and Wiedemann ( 1974)
Tc’AsTra+, also about 60 kb)-involving conjugation and mobilization experiments, revealed that plasmids indistinguishable from pBP 15 and pBP14 can dissociate, even in recA, at high frequency from these larger replicons. In contrast, a conjugative plasmid of about 90 kb, encoding Cm’Hg’Su’Sm’/Sp%olI and transfer functions, designated pBP 17, was comparatively stable (in ret+ and recA) when harbored in absence of other R1767 derivatives. At a frequency of about low4 it undergoes deletions of the Cm or the Sm/Sp resistance genes and thus provides the basis for deletion derivatives encoding either Cm’ or Co11production. Summarizing all the data concerning the genotypes of different derivatives and taking the results of the stability tests into account, it can be deduced that two main lines of plasmids have been derived from R1767. One line arises from pBP17 (Cm’Hg’Su’Sm’/ Sp%olI Tra+), whereas the second line comprises Ap’Su’ plasmids and is represented by pBP 16 (Ap’Su’Tc’Sm’As’Traf). Homology Studies of Ap’Su’ Plasmids In order to elucidate the molecular processesresponsible for the plasmid variability, e.g., see Table 2, we first analyzed pBP18, an Ap’Sul;c’As”Tra+ plasmid. This replicon
EVOLUTION
OF RESISTANCE PLASMIDS
TABLE 2
DIFFERENT PHENOTYPES~SOLATEDAFTER CONJUGATION ORTRANSFORMATION WITH THER-FACTOR SYSTEM R1167 AprTcrCmrSmrSurColI Tra+ CmrSmrSurColI Tra+ AP’ Ap’Tc%m’ Su’ColI Tra+ Ap’Tc’ Su%olI Tra+ Ap’ Cm’ Su%olI Tra+ Tc%m’ Su%olI Tra+ CmrSmrSurColI Tra+ Su%olI Tra+ Cm’ AprTcrCmrSmrSur Tra+ Ap’Tc’ Sm’Su’ Tra+/TraTra ‘/Tra AP' Cm ‘SmSu’ Sm ‘Su’ Tra+ AP’ Ap’Tc’ Su’ Tra+/TmAp’Tc%m’ Su’ Tra + Su’ Tra +/Tra AP’ TcrCmrSmrSur Tra+/TraTc%m’ Su’ Tra +/Tra CmSm’Su’ Tra+/TmCm’ Su’ Tra+/TraSm‘Su’ TmTc’ Su’ TraTc’ Sm’ Tra TraTc’ Sm’ TraPhenotypes of R I767 derivatives analyzed in detail Phenotype Plasmid CmrSmrSpecrSu’Hgr Co11Tra+ Sm’ Su’ As’ Tra+ Su’ As’ Tra+ Su’ As’ Tra+ Su' As' TraApr Su’ TmApr Tc’ TraSm' TraAp’Tc’ Ap’Tc’ AP’
pBP17 pBP16 pBP18 pBPl0 pBPl1 pBPl2
pBPI5 pBP14
No&. Sp, Hg, and As were not tested in these experiments.
was chosen, because it represents the most frequent derivative of R1767 arising from selection for ampicillin resistanceand because it evolves from pBP16 (Ap’SuTTc’Sm’As’ Tra+, the prototype of the second main line) at high frequencies by the loss of streptomycin resistance. Furthermore pBP 18 shows a close relationship to R46 (Brown and Willetts, 1981; Brown et al., 1984) in its restriction map. Restriction analysis of pBPl8 DNA
165
from different transformants revealed that there are at least two isomeric forms (pBPl81 and pBPl8-2; Fig. 1) that are equal in size but differ in their restriction pattern. This difference is due to an inversion of the region accommodating the Tc and As resistance genes mediated via 0.85-kb inverted repeat sequenceswhich flank these resistance genes. Transformation experiments with pBPl81 and pBPl8-2 DNA showed that from both forms of pBP18 the Tc resistance gene is deleted at a frequency of about 2 X lop3 in recA background. By restriction and heteroduplex analysis these Tc-deletion derivatives of pBPl8 could be divided into two groups. The first group contained derivatives all of which had lost the samediscrete region (about 4 kb), e.g., pBP107 (ApSu’AsTra+). Members of the second group also showed deletions; these always started at one sequence bordering the Tc’ region but ended at different distances from this point. Thus, this group contained plasmids encoding different phenotypes (Ap’Su’As’-e.g., pBP 106; Ap’Su’Tra+-e.g., pBPlO5; Ap%‘-e.g., pBP 12; Table 3). Heteroduplex analysis was used to determine the endpoints of the deletions more precisely. Figure 2 shows electron micrographs of self-annealed molecules of pBP181 and pBPl8-2. Both variants of pBP18 showed inverted repeats-IRI and IR2 in pBP18-1; IRla, IRlb, and IR2 in pBP18-2. In additional homoduplex structures the hybridization of IRla and -b sequencesrevealed that these are homologous (data not shown). Both pBPl8- 1 and pBPl8-2 carry four copies of the IRl sequence. The positions of these sequences,both in pBP18 and in its derivatives were mapped by length measurements, using different restriction sites and the inverted repeats themselves as reference points (Fig. 1). The analysis of the deletion derivatives of pBP18 indicated that in all those examined one of the four copies of IRl forms one endpoint of the deletion (Fig. 1). In pBP107 (ApSu’AsTTra+) the Tc’ region and one copy of IRl have been deleted. In the parental plasmid, pBP18, the Tc’ region is
IO
20
R.p, ----U”P -----
30
------
T.0 ------
--
40
-------_
PiI”‘ --_----
50
--
R*pZ ----I
60
-2
---.
-_-
--.
- -
- .
- -
.
_
FIG. 1. Schematic representation of the homology between different RI767 Ap’Su’ derivatives. The coordinates are given in kilobases. The positions of the resistance genes and transfer functions are indicated. Black boxes represent IS160, arrows indicate the relative orientation of IS160. The R46 map is derived from Brown and Willett.s, 1981.
0
I
Su Ap --_-___
105
pBP15
PBPll
pEaPI
pBPlOI
p(lPI0
pciP104
pBP18
pBPlR-I
n 46
pllP107
pBP
PDP12
EVOLUTION
OF RESISTANCE PLASMIDS
TABLE 3 R 1767 DERIVATIVES ENKIDING THE MARKER COMBINATION Ap’ Su’
Plasmids
Relevant markers
Size (kb)
Origin
pBP16 pBP18 pBP107 pBP104 pBPl0 pBPlO1 pBP106 pBPl1 pBPIO5 pBP12
Ap, Su, Tc, Sm, As, Tra Ap, Su, Tc, As, Tra Ap, Su, As, Tra Ap, Su, As, Tra Ap, Su, As, Tra Ap, Su, As Ap, Su, As Ap, Su, As Ap, Su, Tra AP, Su
60 60 56 50 45 32.0 17 26.3 41 Il.5
RI787 pBP16 pBP18 pBP18 R1767 pBP18 pBP18 RI767 pBP18 pBP18
167
DNA of these clones was subjected to heteroduplex and restriction analysis. The structure of pBP12 183 provides a representative example for several cointegrates that were examined. The cointegrates contained two IRl copies in direct orientation, one at each junction between pHS1 and pBP12 (Fig. 3), indicative of transposition. Duplication of the IRl sequence, as well as the location of the direct repeated elements at the junctions of the donor and the recipient molecules provide good evidence that this DNA sequence is an IS element. With the agreement of the plasmid reference center the IRl sequence has been designated IS160.
flanked by two direct copies of the IRl Isolation of Multiresistance Transposons sequence. In the second group deletions alfrom R1767 ways started at one of the four IRl sequences and ended at different distances from these Recent transposition experiments involving points, thus forming the heterogeneousgroup R1767 and using pUB307 (Km’Tc’) as the of plasmids (Fig. 1). transposon recipient, together with outcrossing to Proteus mirabilis, resulted in the isolation of two multiresistance transposons, Identification of the IRI Sequence designated Tn2410 and Tn2411 (R1767 is as an IS Element not transmissable to P. mirabilis; Kratz et From these observations we concluded that al., 1983a,b). Plasmid pUB307 belongs to the sequence designated IRl probably rep the incompatibility group IncPl and transfers resents an IS element. Its ability to transpose efficiently to P. mirabilis. Tn2411 (18.3 kb; was tested in a system described by Ohtsubo harbored by pBPl7) confers resistance to et al. ( 1980; Fig. 3). Plasmid pBPl2 (Fig. 1, mercuric chloride, sulfonamides, and strep Table 2) was chosen as donor plasmid, as it tomycin/spectinomycin [AAD-(3”)]. Tn2410 carries only one copy of IRl. The recipient is 18.5 kb and encodes resistance to mercuric plasmid was pHS1, a Tc’ plasmid that is chloride, sulfonamides, and ampicillin (OXAdeficient in replication at 42°C but which 2 P-lactamase;Fig. 4). These transposons are replicates normally at 30°C. homologous except for the regions encoding Both plasmids were transferred into E. coli the aad gene (0.9 kb) and the oxa gene (1.1 JC2926 by successive transformation. The kb), respectively. Furthermore the sequence resulting ApSuTTc strain was grown over- of Tn2412 is completely contained within night in Tc-free medium at 30°C and after- Tn2l (Kratz et al., 1983a); the latter harbors ward plated on Tc-agar plates and incubated an additional DNA sequence of 1.45 kb at 42°C. Only those cells which harbor coin- (Fig. 4). tegrates comprising pHS 1 and pBP12 were able to grow under these conditions. We Replacement of the aad Genefrom Tn21 detected Tc’ colonies at a frequency of about by the oxa Genefrom pBPl1 1 X 10e6.Plasmid DNA isolated from some The strategy leading to the experiments of these colonies was transferred by transformation to E. coli JC2926. Transformants described below was based on the following were selectedfor Ap’Tc’ at 42°C. The plasmid observations:
x174
FIG. 2. Electron micrographs and tracings of self-reannealed single-stranded DNA molecules of pBP18I (a) and pBPI8-2 (b) confirming the inverted orientation of IS160 (IRl) and IR2. 168
EVOLUTION
OF RESISTANCE PLASMIDS
Tc
IRl
COINTEGRATE
REC A-
FORMATION
TC - SELECTION
42O C
FIG. 3. Scheme of the cointegrate formation between pBPl2 and pHS1. The restriction sites for Sal1 and PstI are indicated by S and P.
169
(i) Both multiresistance transposons isolated from R1767 are very closely related despite the differences in the aadA and the oxu genes (Fig. 4). (ii) In derivatives of R1767, resistance to mercuric chloride was linked to Cm’ and Sp’ (pBPl7, carrying Tn2411) and not to ampicillin resistance (pBP 16). (iii) AprSu’ plasmids, like pBP 11 or pBP 12, share homology with Tn2410 in the region of the ApSu resistance genesover a length of about 4.5 kb (Kratz et al., 1983a). (iv) Cloning experiments revealed that in Tn2410 and Tn2411 the ml and aadA or oxu gene, respectively, form a transcription unit (data not shown). These facts led us to the conclusion that Tn2410 may have evolved from Tn2411 by homologous recombination with an Ap*Su’ plasmid. To test this conclusion, the promoter region of the ApSu transcription unit, located on the 0.3-kb BamHI fragment (Fig. 4) was deleted by cloning the adjoining 4. I-kb BarnHI fragment from pBPl1 into the single BamHI site of pSC105, which is located within the td gene. The resulting plasmid, pBP78, mediated only paromomycin resis-
FIG. 4. Schematic maps of different Tn24I1-like transposons. All elements are shown in relation to Tn2411. Black boxes represent the flanking inverted repeats of the structures. The insertion of DNA segmentsis indicated by triangles. Substitution of DNA segmentsare drawn with broken lines. Restriction sites are indicated by symbols. 9 = EcoRI, 4 = BumHI, T = SmaI, v = SalI, Q = BglII, lr = &I.
170
NIES ET AL.
tance, as encoded by the vector plasmid; the cloned ApSu genes, including the region homologous to Tn2410, were inactive. This plasmid was used in homologous recombination experiments with pACYC 184::Tn21. Substitution of the aadA gene from Tn21 by the oxa gene from pBPl1 would be expected to result in a “switching on” of ampicillin resistance. This “switching on” will depend on the recombination of the promoter-deficient oxa gene into the position of the aadAgene, i.e., placing the oxa gene under the control of the intact ml-aadA promoter present in Tn21. Ampicillin resistance arose with a frequency of lop3 ( 10e7in recA). All recombinants were Sp”. Restriction and heteroduplex analysis of the Tn21-derived Ap’ transposon, designated Tn2427, revealed the substitution of the aadA gene by the oxa gene (Fig. 4). DISCUSSION
From our experiments the observed reassortment of RI 767 can be reduced to the activity of two independent site-specific recombination systems: (i) rearrangements involving the class II transposons Tn2410 and Tn2411 and (ii) rearrangements involving the class I transposon IS160 (characterization according to Kleckner, 198l), together with the recA-dependent recombination system of the host. The involvement of these mechanisms leads to a steady “flux” of genetic material within and between the two main lines (pBP16 and pBP17) of R1767 and thereby results in the system’s variability. The identification of IS160 and its presence in pBP16 derived plasmids represents one substantial reason for the instability of R1767. From the homology between pBP18, a derivative of pBP16 that mediates no Sm resistance, and R46 (Brown and Willetts, 1981, Fig. 1) it can be concluded that IS160 is closely related, if not identical to the recently isolated element IS46 (Brown et al., 1984). In pBPl8 IS160 is present in four copies. Repetitions of an IS element on a DNA molecule can, by homologous or site-specific recombination, result in intra- or intermolec-
ular recombination (Ghosal et al., 1979; Saedler et al., 1981). These events comprise on the one-hand deletions of DNA sequences located between two direct repeated copies of IS160, like the deletion of the tet region to generate pBP107, or the formation of pBPl5 by the deletion of tet plus the adjacent region which apparently encodes replication functions. On the other hand, inverted repeated copies lead to inversions of the flanked sequence, as demonstrated in the cases of pBP18-1 and pBP18-2, as well as in the inversion of the As’ gene(s)(data not shown). Besides reassortment involving two copies of IS160, transformation experiments with pBPl8 and R1767 yielded a number of deletion derivatives (pBP 106, pBP 101, pBP 104, pBP105, and pBP12) in which IS160 forms one endpoint of the deletion. A reasonable explanation for these results is the intramolecular transposition of IS160, resulting in a deletion of the intervening sequence(between one end of IS160 and the target sequence), as was demonstrated for IS903 by Weinert et al. (1983; seealso Reif and Saedler, 1975). The data obtained for ISI60-mediated deletions seem to differ from the data obtained by Brown et al. (1984) for the resolution of IS46-mediated cointegrates and also from those obtained by Brau and Piepersberg (1983, and personal communication) for the resolution of cointegrates mediated by IS140, another IS element which apparently is closely related to IS46 and IS160. Since pBP18-2 can be considered as a cointegrate composed of two plasmids-pBP10 and pBPl5-the deletion of the tet region (or the tet region plus the replication region-pBP 15) from pBPl8-2 represents a cointegrate resolution event (Fig. 1). In contrast to the authors cited above, who only found cointegrate resolution in E. coli ret” strains, in our experiments the Tc resistance was deleted with a frequency of 2 X 10e3in E. coli JC2926 which is recA deficient. In more than half of all casesthe tet region was deleted between two direct repeated copies of IS160, forming, for example, either pBP107 from pBPl8- 1 or pBPl0 from pBPl8-2 (Fig. 1). In another context the behavior of IS160-
EVOLUTION
171
OF RESISTANCE PLASMIDS
mediated cointegrates concerning resolution was analyzed in more detail (Nies et al., in preparation). In these experiments different E. coli recA strains were used and in all cases cointegrates were resolved at frequencies ranging from 5 X 10m2to 3 X low3 depending on the composition of the cointegrates, but independent from the strain used. The resolution frequencies in E. coli ret+ strains were 1 to 2 orders of magnitude higher (ranging from 1 X 10-l to 7 X 10-l) than in recA background. As demonstrated during the transposition experiments involving IS160 (Fig. 3) and in agreement with the results of Brown et al. (1984) obtained for IS element IS46, IS160mediated cointegrate formation would seem to be a likely route for the formation of clones harboring resistance markers derived from the different main lines of R1767 combined on one replicon (Table 2). The 120to 150-kb plasmids that were observed in plasmid DNA preparations from some of the clones, showing the R1767 phenotype, might be considered as cointegrates joining the two main lines of R 1767. Besides IS160, the transposons Tn2410 and Tn2411 represent a second site-specific recombination system within R 1767 (Kratz et al., 1983a,b). Like other transposons Tn2410 and Tn2411 are able to promote different kinds of rearrangement, frequently reported for several transposable elements (for review, see Kleckner, 1981). A ret-dependent mechanism of joining or exchanging genes from the two main lines of R1767 is the replacement of the aadA gene from Tn2411 by the oxa gene of pBPl6 derivatives, e.g., pBPl1. Due to this mechanism R1767 harbors two transposable elements-Tn2410 and Tn2411. This replacement depicts the possibility for the evolution of new transposable resistance genesthat can contribute to the dissemination of Ap and Sp resistance genesbetween the R1767 replicons. All data concerning the variability of R1767 revealed that this R-factor system comprises a concerted action of illegitimate and homologous recombination between
plasmids or plasmid components and thus provides an excellent example for the evolution of resistance plasmids. ACKNOWLEDGMENTS This work was supported by a grant of the Deutsche Forschungsgemeinschaftto Bemd Wiedemann. REFERENCES ARTHUR, A., AND SHERRATT, D.(l979).
Dissection of the transposition process: A transposon-encoded sitespecific recombination system. Mol. Gen. Genet. 175, 267-274. BACHMANN, B. J. (1972). Pedigrees of some mutant strains of Escherichia coli K-12. Bacterial. Rev. 36, 525-527. BENNETT,P. M., GRINSTED,J., AND RICHMOND,M. H. (1975). Transposition of TnA does not generate deletions. Mol. Gen. Genet. 154, 205-212. BRKu, B., AND PIEPERSBERG, W. (1983). Cointegrational transduction and mobilization of gentamycin resistance plasmid pWPl4a is mediated by IS140. Mol. Gen. Genet. 189, 298-303. BROWN,A. M. C., COUPLAND,G. M., AND WILLETTS, N. S. (1984). Characterization of IS46, an insertion sequence found on two IncN plasmids. J. Bacterial. 159, 472-48 1. BROWN, A. M. C., AND WILLETTS, N. S. (1981). A physical and genetic map of the IncN plasmid R46. Plasmid 5, 188-20 1. COETZEE,J. N., (1963).Transduction of swarming Proteus mirabilis. J. Gen. Microbial.
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COHEN, S. N., BREVET, J., CABELLO, F., CHANG, A. C. Y., CHOW,J., KOPECKO,D. J., KRETSCHMER, P. J., NISEN, P., AND TIMMIS, K. (1978). Macro- and microevolution of bacterial plasmids. In “Microbiology-1978” (D. Schlessinger,ed.), pp. 217-220. Sot. for Microbial., Washington, D. C. COHEN, S. N., CHANG, A. C. Y., BOYER, H. W., AND HELLING, R. B. (1973). Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70, 3240-3244.
GHOSAL, D., SOMMER,H., AND SAEDLER,H. (1979). Nucleotide sequence of the transposable element 1%‘. Nucleic Acids Res. 6, 111l-l 122. GRINSTEDT,J., SAUNDERS,J. R., INGRAM,L. C., SYKES, R. B., AND RICHMOND,M. H. (1972). Properties of a R factor which originated in Pseudomonas aeruginosa 1822. J. Bacterial. 110, 529-537. HASHIMOTO,T., AND SEKIGUCHI,M. (1976). Isolation of temperature-sensitive mutants of R-plasmids by in vitro mutagenesis with hydroxylamine. J. Bacterial. 127, 1561-1563. KLECKNER,N. (198 1). Transposable elements in procaryontes. Annu. Rev. Genet. 15, 341-404. KOPECKO,D. J. ( 1980).Specializedgenetic recombination systems in bacteria: their involvement in gene expres-
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KRATZ, J., SCHMIDT,F., AND WIEDEMANN,B. (1983a). Characterization of Tn2411 and Tn2410, two transposons derived from R plasmid RI767 and related to Tn2603 and Tn21. J. Bacterial. 155, 1333-I 342. KRATZ, J., SCHMIDT,F., AND WIEDEMANN,B. (1983b). Transposition of a gene encoding OXA- @lactamase. J. Gen. Microbial.
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KUSHNER,S. R. (1978). An improved method for transformation of E. coli with ColEl derived plasmids. In “International Symposium on Genetic Engineering” (H. W. Boyer and S. Nicosia, eds.),pp. 17-23. Elsevier/ North-Holland, Amsterdam. MEYER,J. F., NIES, B. A., AND WIEDEMANN,B. (1983). Amikacin resistancemediated by multiresistance transposon Tn2424. J. Bacterial. 155, 755-760. OHTSUBO,E., ZENILMAN,M., AND OHTSUBO,H. (1980). Plasmids containing insertion elements are potential transposons. Proc. Natl. Acad. Sci. USA 77, 750-754.
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RICHMOND,M. H., AND WIEDEMANN,B. (1974). Plasmids and bacterial evolution. In “Evolution in the Microbial World” (Sot. for Gen. Microbial.), pp. 5985. Cambridge Univ. Press,Cambridge. SAEDLER,H., CORNELIS,G., CULLUM,J., SCHUMACHER, B., AND !SOMMER,H. (1981). ISZ-mediated DNA rearrangements. Cold Spring Harbor Symp. Quant. Biol. 45, 93-98.
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13, 163-172 (1985)
R1767, An Example of the Evolution of Resistance BERTHOLDA.NIES,JOACHIM
F. MEYER,JOHANNES JQATZ,AND
Plasmids
BERND~IEDEMANN
Institut ftir Medizinische Mikrobiologie und Immunologic, der Universitiit Bonn, 5300 Bonn, West Germany Received September 24, 1984; revised January 30, 1985 The Salmonella R-factor system R1767 undergoes frequent rearrangement of its plasmid components. The flux of genetic material within this plasmid system depends on a combination of illegitimate and homologous recombination. The presence of several copies of IS160 and two multiresistance transposons,Tn2410 and Tn2411, are substantial reasonsfor the observed variations. 0 1985 Academic FTes, Inc.
Plasmid-encoded antibiotic resistance among bacterial pathogens causing infectious diseasesis a major problem worldwide. However by studying the emergence and distribution of resistance genes in bacteria several mechanisms, such as transposition, inversion and deletion, mediated by illegitimate and generalized recombination have been found to participate in the macroevolution of bacterial plasmids (Cohen et al., 1978; Kopecko, 1980). Despite this, only rarely has the concerted action of several different mechanisms been examined in one and the same system. In 1974 we demonstrated the reassortment of the R-factor system RI767 and speculated on the possible mechanisms involved (Richmond and Wiedemann, 1974). Since then the system has been analyzed in more detail and it is now possible to propose credible explanation for this reassortment. This paper describes how a combination of several molecular processes provides a basis for the generation of new genetic structures. Accordingly, we believe that this Rfactor system may serve as an excellent example of the evolution of antibiotic resistance plasmids. MATERIAL
AND
METHODS
All methods, including DNA preparations, cloning experiments, transformation, conjugation, phage Pl transduction, and electron microscope analysis have been described bc-
fore (Meyer et al., 1983; Kratz et al., 1983a). Plasmids and bacterial strains are listed in Table 1. RESULTS
Phenotypic Derivatives of RI 767 and Its Plasmid Components
The conjugative R-factor system R1767 was derived from a clinical isolate of Salmonella typhimurium (Richmond and Wiedemann, 1974). This plasmid systemmediates resistance toward ampicillin by the production of an OXA- &lactamase, to streptomycin by the production of an APH-(3”) enzyme and an AAD-(3”) enzyme (the latter enzyme also confers resistance to spectinomycin), and to tetracycline, chloramphenicol, sulfonamides, mercuric chloride, arsenate, and carries genesfor the production of colicin I. R1767 is intrinsically unstable even in recA background (Wiedemann, 198I). Different bacterial hosts which harbored R1767 usually showed four plasmid bands. Two smaller replicons encoded Tc’ resistance (12 kb, designated pBP 15) and APH-( 3”)-mediated streptomycin resistance (8 kb, desig’ Abbreviations used: Ap, ampicillin; Cm, chloramphenicol; Su, sulfonamides; Hg, mercuric chloride; Sm, streptomycin; Sp, $rectinomycin; Tc, tetracycline; As, arsenate; Pm, paromomycin; ColI, colicin I; Km, kanamycin; Tra, transfer factor: aadA, gene encoding the AAD(3”) enzyme; oxa, gene encoding the OXA- /3lactamase.
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0147-619X/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
164
NIES ET AL. TABLE 1 BACTERIALSTRAINSAND~LASMIDS
Bacterial strains E. coli JC2926 E. coli SK1 592 E. coli W3110 Proteus mirabilis Pm 13 Plasmids pUB307 pSCIO5 pHS1 pACYC184::Tn21 R1767
Sm’, recA, thi, thr, arg, his, leu, lac, ma1 Tl r, gal, thi, sbcBl5, hsr4, hsm+ Nx’, lacf
Km’, Tc’, Tm+ Km’, Tc’ Tc’, replication defective at 42°C Cm’, Hg’, Sp’/Sm’ Su’ Ap’, Su’, Sm’, Sp’/Sm’, Hg’, Cm’, Tc’, As’, Colt, Tra+
nated pBP 14) respectively. Both resistance phenotypes were also encoded by tet or aph genes,respectively, integrated into larger replicons. The two other plasmid bands, comprising replicons of 90 and 120- 150 kb, could not be related to defined resistance patterns. Conjugation and transformation experiments with R1767 from ret+ or recA donor strains (the recipient strain always was Escherichia coli SK1592 in transformation and E. coli W3 110 in conjugation experiments) yielded a broad variety of phenotypes among the trascipients and these carried only some of the R1767 resistance markers, in different combinations. The Ret state of the donor did not influence the statistical distribution of the phenotypes significantly. The marker combinations found in these experiments are summarized in Table 2. In order to relate the resistance phenotype to plasmid content, different transformants were examined to determine what plasmids were carried. Ampicillin resistanceand sulfonamide resistance, sometimes linked to Sm’ [APH-(3”)] and/or Tc’ and/or Tra functions were encoded by plasmids varying in size from 11.5 to 60 kb. Some representative Ap’Su’ transformants were analyzed in detail and are described below (Fig. 1). Stability tests with two of these transformants-pBP 16 (Ap%TTc’Sm’As’Tra+, about 60 kb) and pBPl8 (Ap’Su’-
Bachmann ( 1972) Kushner (1978) Grinsted ( 1972) Coetzee ( 1963) Bennett et a/. (1975) Cohen et al. (1973) Hashimoto and Sekiguchi ( 1976) This paper Richmond and Wiedemann ( 1974)
Tc’AsTra+, also about 60 kb)-involving conjugation and mobilization experiments, revealed that plasmids indistinguishable from pBP 15 and pBP14 can dissociate, even in recA, at high frequency from these larger replicons. In contrast, a conjugative plasmid of about 90 kb, encoding Cm’Hg’Su’Sm’/Sp%olI and transfer functions, designated pBP 17, was comparatively stable (in ret+ and recA) when harbored in absence of other R1767 derivatives. At a frequency of about low4 it undergoes deletions of the Cm or the Sm/Sp resistance genes and thus provides the basis for deletion derivatives encoding either Cm’ or Co11production. Summarizing all the data concerning the genotypes of different derivatives and taking the results of the stability tests into account, it can be deduced that two main lines of plasmids have been derived from R1767. One line arises from pBP17 (Cm’Hg’Su’Sm’/ Sp%olI Tra+), whereas the second line comprises Ap’Su’ plasmids and is represented by pBP 16 (Ap’Su’Tc’Sm’As’Traf). Homology Studies of Ap’Su’ Plasmids In order to elucidate the molecular processesresponsible for the plasmid variability, e.g., see Table 2, we first analyzed pBP18, an Ap’Sul;c’As”Tra+ plasmid. This replicon
EVOLUTION
OF RESISTANCE PLASMIDS
TABLE 2
DIFFERENT PHENOTYPES~SOLATEDAFTER CONJUGATION ORTRANSFORMATION WITH THER-FACTOR SYSTEM R1167 AprTcrCmrSmrSurColI Tra+ CmrSmrSurColI Tra+ AP’ Ap’Tc%m’ Su’ColI Tra+ Ap’Tc’ Su%olI Tra+ Ap’ Cm’ Su%olI Tra+ Tc%m’ Su%olI Tra+ CmrSmrSurColI Tra+ Su%olI Tra+ Cm’ AprTcrCmrSmrSur Tra+ Ap’Tc’ Sm’Su’ Tra+/TraTra ‘/Tra AP' Cm ‘SmSu’ Sm ‘Su’ Tra+ AP’ Ap’Tc’ Su’ Tra+/TmAp’Tc%m’ Su’ Tra + Su’ Tra +/Tra AP’ TcrCmrSmrSur Tra+/TraTc%m’ Su’ Tra +/Tra CmSm’Su’ Tra+/TmCm’ Su’ Tra+/TraSm‘Su’ TmTc’ Su’ TraTc’ Sm’ Tra TraTc’ Sm’ TraPhenotypes of R I767 derivatives analyzed in detail Phenotype Plasmid CmrSmrSpecrSu’Hgr Co11Tra+ Sm’ Su’ As’ Tra+ Su’ As’ Tra+ Su’ As’ Tra+ Su' As' TraApr Su’ TmApr Tc’ TraSm' TraAp’Tc’ Ap’Tc’ AP’
pBP17 pBP16 pBP18 pBPl0 pBPl1 pBPl2
pBPI5 pBP14
No&. Sp, Hg, and As were not tested in these experiments.
was chosen, because it represents the most frequent derivative of R1767 arising from selection for ampicillin resistanceand because it evolves from pBP16 (Ap’SuTTc’Sm’As’ Tra+, the prototype of the second main line) at high frequencies by the loss of streptomycin resistance. Furthermore pBP 18 shows a close relationship to R46 (Brown and Willetts, 1981; Brown et al., 1984) in its restriction map. Restriction analysis of pBPl8 DNA
165
from different transformants revealed that there are at least two isomeric forms (pBPl81 and pBPl8-2; Fig. 1) that are equal in size but differ in their restriction pattern. This difference is due to an inversion of the region accommodating the Tc and As resistance genes mediated via 0.85-kb inverted repeat sequenceswhich flank these resistance genes. Transformation experiments with pBPl81 and pBPl8-2 DNA showed that from both forms of pBP18 the Tc resistance gene is deleted at a frequency of about 2 X lop3 in recA background. By restriction and heteroduplex analysis these Tc-deletion derivatives of pBPl8 could be divided into two groups. The first group contained derivatives all of which had lost the samediscrete region (about 4 kb), e.g., pBP107 (ApSu’AsTra+). Members of the second group also showed deletions; these always started at one sequence bordering the Tc’ region but ended at different distances from this point. Thus, this group contained plasmids encoding different phenotypes (Ap’Su’As’-e.g., pBP 106; Ap’Su’Tra+-e.g., pBPlO5; Ap%‘-e.g., pBP 12; Table 3). Heteroduplex analysis was used to determine the endpoints of the deletions more precisely. Figure 2 shows electron micrographs of self-annealed molecules of pBP181 and pBPl8-2. Both variants of pBP18 showed inverted repeats-IRI and IR2 in pBP18-1; IRla, IRlb, and IR2 in pBP18-2. In additional homoduplex structures the hybridization of IRla and -b sequencesrevealed that these are homologous (data not shown). Both pBPl8- 1 and pBPl8-2 carry four copies of the IRl sequence. The positions of these sequences,both in pBP18 and in its derivatives were mapped by length measurements, using different restriction sites and the inverted repeats themselves as reference points (Fig. 1). The analysis of the deletion derivatives of pBP18 indicated that in all those examined one of the four copies of IRl forms one endpoint of the deletion (Fig. 1). In pBP107 (ApSu’AsTTra+) the Tc’ region and one copy of IRl have been deleted. In the parental plasmid, pBP18, the Tc’ region is
IO
20
R.p, ----U”P -----
30
------
T.0 ------
--
40
-------_
PiI”‘ --_----
50
--
R*pZ ----I
60
-2
---.
-_-
--.
- -
- .
- -
.
_
FIG. 1. Schematic representation of the homology between different RI767 Ap’Su’ derivatives. The coordinates are given in kilobases. The positions of the resistance genes and transfer functions are indicated. Black boxes represent IS160, arrows indicate the relative orientation of IS160. The R46 map is derived from Brown and Willett.s, 1981.
0
I
Su Ap --_-___
105
pBP15
PBPll
pEaPI
pBPlOI
p(lPI0
pciP104
pBP18
pBPlR-I
n 46
pllP107
pBP
PDP12
EVOLUTION
OF RESISTANCE PLASMIDS
TABLE 3 R 1767 DERIVATIVES ENKIDING THE MARKER COMBINATION Ap’ Su’
Plasmids
Relevant markers
Size (kb)
Origin
pBP16 pBP18 pBP107 pBP104 pBPl0 pBPlO1 pBP106 pBPl1 pBPIO5 pBP12
Ap, Su, Tc, Sm, As, Tra Ap, Su, Tc, As, Tra Ap, Su, As, Tra Ap, Su, As, Tra Ap, Su, As, Tra Ap, Su, As Ap, Su, As Ap, Su, As Ap, Su, Tra AP, Su
60 60 56 50 45 32.0 17 26.3 41 Il.5
RI787 pBP16 pBP18 pBP18 R1767 pBP18 pBP18 RI767 pBP18 pBP18
167
DNA of these clones was subjected to heteroduplex and restriction analysis. The structure of pBP12 183 provides a representative example for several cointegrates that were examined. The cointegrates contained two IRl copies in direct orientation, one at each junction between pHS1 and pBP12 (Fig. 3), indicative of transposition. Duplication of the IRl sequence, as well as the location of the direct repeated elements at the junctions of the donor and the recipient molecules provide good evidence that this DNA sequence is an IS element. With the agreement of the plasmid reference center the IRl sequence has been designated IS160.
flanked by two direct copies of the IRl Isolation of Multiresistance Transposons sequence. In the second group deletions alfrom R1767 ways started at one of the four IRl sequences and ended at different distances from these Recent transposition experiments involving points, thus forming the heterogeneousgroup R1767 and using pUB307 (Km’Tc’) as the of plasmids (Fig. 1). transposon recipient, together with outcrossing to Proteus mirabilis, resulted in the isolation of two multiresistance transposons, Identification of the IRI Sequence designated Tn2410 and Tn2411 (R1767 is as an IS Element not transmissable to P. mirabilis; Kratz et From these observations we concluded that al., 1983a,b). Plasmid pUB307 belongs to the sequence designated IRl probably rep the incompatibility group IncPl and transfers resents an IS element. Its ability to transpose efficiently to P. mirabilis. Tn2411 (18.3 kb; was tested in a system described by Ohtsubo harbored by pBPl7) confers resistance to et al. ( 1980; Fig. 3). Plasmid pBPl2 (Fig. 1, mercuric chloride, sulfonamides, and strep Table 2) was chosen as donor plasmid, as it tomycin/spectinomycin [AAD-(3”)]. Tn2410 carries only one copy of IRl. The recipient is 18.5 kb and encodes resistance to mercuric plasmid was pHS1, a Tc’ plasmid that is chloride, sulfonamides, and ampicillin (OXAdeficient in replication at 42°C but which 2 P-lactamase;Fig. 4). These transposons are replicates normally at 30°C. homologous except for the regions encoding Both plasmids were transferred into E. coli the aad gene (0.9 kb) and the oxa gene (1.1 JC2926 by successive transformation. The kb), respectively. Furthermore the sequence resulting ApSuTTc strain was grown over- of Tn2412 is completely contained within night in Tc-free medium at 30°C and after- Tn2l (Kratz et al., 1983a); the latter harbors ward plated on Tc-agar plates and incubated an additional DNA sequence of 1.45 kb at 42°C. Only those cells which harbor coin- (Fig. 4). tegrates comprising pHS 1 and pBP12 were able to grow under these conditions. We Replacement of the aad Genefrom Tn21 detected Tc’ colonies at a frequency of about by the oxa Genefrom pBPl1 1 X 10e6.Plasmid DNA isolated from some The strategy leading to the experiments of these colonies was transferred by transformation to E. coli JC2926. Transformants described below was based on the following were selectedfor Ap’Tc’ at 42°C. The plasmid observations:
x174
FIG. 2. Electron micrographs and tracings of self-reannealed single-stranded DNA molecules of pBP18I (a) and pBPI8-2 (b) confirming the inverted orientation of IS160 (IRl) and IR2. 168
EVOLUTION
OF RESISTANCE PLASMIDS
Tc
IRl
COINTEGRATE
REC A-
FORMATION
TC - SELECTION
42O C
FIG. 3. Scheme of the cointegrate formation between pBPl2 and pHS1. The restriction sites for Sal1 and PstI are indicated by S and P.
169
(i) Both multiresistance transposons isolated from R1767 are very closely related despite the differences in the aadA and the oxu genes (Fig. 4). (ii) In derivatives of R1767, resistance to mercuric chloride was linked to Cm’ and Sp’ (pBPl7, carrying Tn2411) and not to ampicillin resistance (pBP 16). (iii) AprSu’ plasmids, like pBP 11 or pBP 12, share homology with Tn2410 in the region of the ApSu resistance genesover a length of about 4.5 kb (Kratz et al., 1983a). (iv) Cloning experiments revealed that in Tn2410 and Tn2411 the ml and aadA or oxu gene, respectively, form a transcription unit (data not shown). These facts led us to the conclusion that Tn2410 may have evolved from Tn2411 by homologous recombination with an Ap*Su’ plasmid. To test this conclusion, the promoter region of the ApSu transcription unit, located on the 0.3-kb BamHI fragment (Fig. 4) was deleted by cloning the adjoining 4. I-kb BarnHI fragment from pBPl1 into the single BamHI site of pSC105, which is located within the td gene. The resulting plasmid, pBP78, mediated only paromomycin resis-
FIG. 4. Schematic maps of different Tn24I1-like transposons. All elements are shown in relation to Tn2411. Black boxes represent the flanking inverted repeats of the structures. The insertion of DNA segmentsis indicated by triangles. Substitution of DNA segmentsare drawn with broken lines. Restriction sites are indicated by symbols. 9 = EcoRI, 4 = BumHI, T = SmaI, v = SalI, Q = BglII, lr = &I.
170
NIES ET AL.
tance, as encoded by the vector plasmid; the cloned ApSu genes, including the region homologous to Tn2410, were inactive. This plasmid was used in homologous recombination experiments with pACYC 184::Tn21. Substitution of the aadA gene from Tn21 by the oxa gene from pBPl1 would be expected to result in a “switching on” of ampicillin resistance. This “switching on” will depend on the recombination of the promoter-deficient oxa gene into the position of the aadAgene, i.e., placing the oxa gene under the control of the intact ml-aadA promoter present in Tn21. Ampicillin resistance arose with a frequency of lop3 ( 10e7in recA). All recombinants were Sp”. Restriction and heteroduplex analysis of the Tn21-derived Ap’ transposon, designated Tn2427, revealed the substitution of the aadA gene by the oxa gene (Fig. 4). DISCUSSION
From our experiments the observed reassortment of RI 767 can be reduced to the activity of two independent site-specific recombination systems: (i) rearrangements involving the class II transposons Tn2410 and Tn2411 and (ii) rearrangements involving the class I transposon IS160 (characterization according to Kleckner, 198l), together with the recA-dependent recombination system of the host. The involvement of these mechanisms leads to a steady “flux” of genetic material within and between the two main lines (pBP16 and pBP17) of R1767 and thereby results in the system’s variability. The identification of IS160 and its presence in pBP16 derived plasmids represents one substantial reason for the instability of R1767. From the homology between pBP18, a derivative of pBP16 that mediates no Sm resistance, and R46 (Brown and Willetts, 1981, Fig. 1) it can be concluded that IS160 is closely related, if not identical to the recently isolated element IS46 (Brown et al., 1984). In pBPl8 IS160 is present in four copies. Repetitions of an IS element on a DNA molecule can, by homologous or site-specific recombination, result in intra- or intermolec-
ular recombination (Ghosal et al., 1979; Saedler et al., 1981). These events comprise on the one-hand deletions of DNA sequences located between two direct repeated copies of IS160, like the deletion of the tet region to generate pBP107, or the formation of pBPl5 by the deletion of tet plus the adjacent region which apparently encodes replication functions. On the other hand, inverted repeated copies lead to inversions of the flanked sequence, as demonstrated in the cases of pBP18-1 and pBP18-2, as well as in the inversion of the As’ gene(s)(data not shown). Besides reassortment involving two copies of IS160, transformation experiments with pBPl8 and R1767 yielded a number of deletion derivatives (pBP 106, pBP 101, pBP 104, pBP105, and pBP12) in which IS160 forms one endpoint of the deletion. A reasonable explanation for these results is the intramolecular transposition of IS160, resulting in a deletion of the intervening sequence(between one end of IS160 and the target sequence), as was demonstrated for IS903 by Weinert et al. (1983; seealso Reif and Saedler, 1975). The data obtained for ISI60-mediated deletions seem to differ from the data obtained by Brown et al. (1984) for the resolution of IS46-mediated cointegrates and also from those obtained by Brau and Piepersberg (1983, and personal communication) for the resolution of cointegrates mediated by IS140, another IS element which apparently is closely related to IS46 and IS160. Since pBP18-2 can be considered as a cointegrate composed of two plasmids-pBP10 and pBPl5-the deletion of the tet region (or the tet region plus the replication region-pBP 15) from pBPl8-2 represents a cointegrate resolution event (Fig. 1). In contrast to the authors cited above, who only found cointegrate resolution in E. coli ret” strains, in our experiments the Tc resistance was deleted with a frequency of 2 X 10e3in E. coli JC2926 which is recA deficient. In more than half of all casesthe tet region was deleted between two direct repeated copies of IS160, forming, for example, either pBP107 from pBPl8- 1 or pBPl0 from pBPl8-2 (Fig. 1). In another context the behavior of IS160-
EVOLUTION
171
OF RESISTANCE PLASMIDS
mediated cointegrates concerning resolution was analyzed in more detail (Nies et al., in preparation). In these experiments different E. coli recA strains were used and in all cases cointegrates were resolved at frequencies ranging from 5 X 10m2to 3 X low3 depending on the composition of the cointegrates, but independent from the strain used. The resolution frequencies in E. coli ret+ strains were 1 to 2 orders of magnitude higher (ranging from 1 X 10-l to 7 X 10-l) than in recA background. As demonstrated during the transposition experiments involving IS160 (Fig. 3) and in agreement with the results of Brown et al. (1984) obtained for IS element IS46, IS160mediated cointegrate formation would seem to be a likely route for the formation of clones harboring resistance markers derived from the different main lines of R1767 combined on one replicon (Table 2). The 120to 150-kb plasmids that were observed in plasmid DNA preparations from some of the clones, showing the R1767 phenotype, might be considered as cointegrates joining the two main lines of R 1767. Besides IS160, the transposons Tn2410 and Tn2411 represent a second site-specific recombination system within R 1767 (Kratz et al., 1983a,b). Like other transposons Tn2410 and Tn2411 are able to promote different kinds of rearrangement, frequently reported for several transposable elements (for review, see Kleckner, 1981). A ret-dependent mechanism of joining or exchanging genes from the two main lines of R1767 is the replacement of the aadA gene from Tn2411 by the oxa gene of pBPl6 derivatives, e.g., pBPl1. Due to this mechanism R1767 harbors two transposable elements-Tn2410 and Tn2411. This replacement depicts the possibility for the evolution of new transposable resistance genesthat can contribute to the dissemination of Ap and Sp resistance genesbetween the R1767 replicons. All data concerning the variability of R1767 revealed that this R-factor system comprises a concerted action of illegitimate and homologous recombination between
plasmids or plasmid components and thus provides an excellent example for the evolution of resistance plasmids. ACKNOWLEDGMENTS This work was supported by a grant of the Deutsche Forschungsgemeinschaftto Bemd Wiedemann. REFERENCES ARTHUR, A., AND SHERRATT, D.(l979).
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