Molec. gen. Genet. 157, 17-23 (1973) © by Springer-Verlag 1977
Mapping of the Drug Resistance Genes Carried by the r-Determinant of the R100.1 Plasmid D. L a n e * and M. Chandler D6partement de Biologie Mol6culaire, 30, quai Ernest-Ansermet, CH-1211 Gen6ve 4, Switzerland
Summary. We have cloned the E c o R I fragments of pLC1, a circular D N A element f o u n d in an E s c h e r i chia coli dnaAts strain integratively suppressed by R100.1 (Chandler etal., 1977a), using the plasmid vector pCR1. All the resistance genes k n o w n to be present on the r-determinant o f R100.1 were f o u n d to be present on pLC1. The isolation o f pCR1 derivatives carrying various E c o R I fragments o f either pLC1 or RI00.1 has allowed a m o r e precise m a p p i n g o f the position o f the resistance genes on the R100.1 molecule.
Introduction The antibiotic resistance plasmid R100.1 is a selftransmissible e x t r a c h r o m o s o m a l element which specifies resistance to tetracycline (Tc), chloramphenicol (Cm), sulphonamides (Su), streptemycin (Sm), mercury (Hg) and fusidic acid (Fu) 1. In c o m m o n with several R plasmids, R100.1 comprises two functionally distinct parts: the resistance transfer factor ( R T F ) which carries genetic information for replication and conjugal transfer o f the plasmid and also for resistance to tetracycline, and the r-determinant (r-det) which carries genes specifying resistance to the other drugs. In some situations these two parts dissociate f r o m each other ( R o w n d & Mickel, 1971 ; C h a n d l e r et al., 1977a). We have observed one example o f this dissociation in a dnaAts m u t a n t o f E s c h e r i c h i a coli in which the temperature-sensitive initiation defect in c h r o m o s o m e replication is suppressed by integration o f R100.1 (Bird et al., 1976). In m a n y such strains, integration is associated with the f o r m a t i o n o f closed c i r c u l a r In this report, Cm, Su, Sm, Hg and Fu are used to designate resistance phenotypes. The corresponding genes are designated cam, sul, str, met and fus * Present address: Department of Cell Biology, University of Auck-
land, Private Bag, Auckland, New Zealand For offprints contact." M. Chandler
D N A elements. We have investigated the structure of one o f these elements, pLC1 and have shown that it has physical properties identical to those o f the r-det (Chandler et al., 1977, a, b). Since we were unable to establish pLC1 in strains other than the parental strain, LC2633, we could not demonstrate that pLC1 carries all the resistance genes associated with the r-det region o f R100.1 In this report we show that, by cloning restriction enzyme fragments o f pLC1 o n t o a self-replicating plasmid vector, we can obtain plasmids containing all the k n o w n r-det resistance genes. This genetic evidence further demonstrates that pLC1 and t h e r-det o f R100.1 are identical. F r o m the k n o w n relative positions o f these fragments on both R100.1 and the pLC1 molecule (Tanaka et al., 1976; Chandler et al., 1977a) we can determine the order o f the resistance genes. The results agree with those o f D e m p s e y & Willets (1976) who established this order using deletion mutants of 2-R100 co-integrates. O u r assignment o f the genes to the various E c o R I fragments extends the results o f T a n a k a et al. (1976) to include m e r and fus.
Materials and Methods Bacterial Strains
Strains LC468 and LC2633 (Table 1), both derivatives of Escherichia coli K12 W1485 (Chandler et al., 1977a), served as sources of R100.1 and pLC1 DNA's respectively. Strain LC555, a derivative of E. coli C600 which carries the colEl-kan plasmid pCR1 (Covey et al., 1976), was used to prepare pCRI DNA. E. coli C600 was used as a recipient in all transformations except where fusidic acid was to be tested. For these experiments a fusidic acid-sensitive strain, LC582, was isolated, as follows. C600 was grown in L-broth to midlog phase and nitrosoguanidine was added to 0.1 mg/ml. After 5 min the cells were washed, resuspended in L-broth and grown for 2 h. Fusidic acid (a generous gift of Leo Pharmaceuticals) was added to 0.1 mg/mi, followed 10 min later by penicillin G to 5000 units/ml. After Iysis samples were plated on L-agar and incubated at 37° C. The colonies were replica-plated onto L-agar with and without fusidic acid (0.1 mg/ml). One fusidic acid sensitive clone was purified for further use. This strain was sensitive to concentrations of fusidic acid lower than 0.1 mg/ml and a concentration of 20 gg/ml was used in all further experiments.
18
D. Lane and M. Chandler: Resistance Genes of R100.1
Table 1. Strains of Escherichia coli
Results
Strain
Sex
Description
Construction o f Plasmids f r o m p L C l
LC468
R+
thi leu thy/RlO0.1 : a derivative of W1485
LC555
F
C600/pCR1
LC582
F-
a fusidate sensitive derivative of C600
LC2633
R. Hfr
thi leu thy (proB lac)del dnaAt~46 (ilv: :Mu-1), (2ind-) Transfers 0 proA proB ... araR (Bird et al., 1976; Chandler et al. 1977a)
Media L-broth, M9 glucose casamino-acids, and the corresponding solid media have been described (Bird et al., 1976). Resistance to kanamycin, streptomycin, chloramphenicol, HgCL2 and fusidic acid was tested on L-agar plates with 20 ~tg/mlof the appropriate drug; resistance to sulphonamide was tested on M9-glucose-casamino acids plates containing 100 gg/ml sulphamerazin.
Isolation of Plasmid DNA R100.1 and pLC1 closed circular DNAs were isolated from strains LC524 and LC2633 by centrifugation of cleared lysates in CsC1EtBr gradients (Clewell and Helinski, 1969) as described previously (Chandler et al., 1977a). pCR1 DNA was prepared by the same method from overnight cultures of LC555 which had been treated with 150 gg/ml chloramphenicol at a cell concentration of 5 x 108/ml for 16 h. The addition of chloramphenicol results in an amplification of colEl-derived plasmids (Clewell, 1972). Plasmid DNA from purified transformants (see below) was routinely prepared from cleared lysates (Clewelland Helinski, 1969) of 25 ml cultures grown in M9 glucose casamino acids medium. The cleared lysates were extracted twice with an equal volume of phenol (Merck analytical grade, saturated with 10 mM Tris, 1 mM EDTA pH 7.6) and the DNA precipitated with ethanol. The precipitates were redissolvedin 0.3~0.5 ml of H20 and dialyzed against several changes of 10 mM Tris, 1 mM EDTA pH 7.6. When necessary RNA was removed from the preparations by treatment with RNAase A (10 lag/ml) for 30 min at 37° C.
Enzyme Reactions Digestion of DNA by the restriction enzyme EcoRi (the generous gift of J. Hedgpeth and M. Ballivet) and electrophoresis of the products on 1% agarose slab gels have been described (Chandler et al., 1977a). Digestion with the enzyme HindII (Bethesda Research Laboratories) was carried out in a similar way in 60 mM NaCI, 6.6 mM MgC12, 6 mM 2-mercaptoethanoi and 10 mM Tris pH 7.9. Prior to ligation, the EcoRI digested preparations were incubated at 65°C for 10 rain to inactivate the endonuclease, then chilled on ice. Ligation was carried out in 10 mM dithiothreitol, 10 mM MgC12, 0.055 mM ATP and 10 mM Tris pH 7.6. T4 DNA ligase (Miles) was added to 0.1-1 units/ml and the mixture (50-100 gl) incubated at 11 °C for 3-6 days. The ligated DNA was added to 1 ml of Escherichia coli tRNA (50 gg/ml) and ethanol precipitated. The dried precipitate was dissolved in 0.1 ml of 20 mM Tris pH 8.0, 20 mM NaCI, and 1 mM EDTA. It was used directly for transformation according to the procedure of Cohen et al. (1972). NIH Pl level containment procedures were followed throughout these experiments.
It has previously been d e t e r m i n e d that pLC1 is f o r m e d f r o m R100.1 by reciprocal r e c o m b i n a t i o n between the two IS1 elements present o n the E c o R I fragments A a n d H, as illustrated in Figure 1 ( C h a n d l e r et al., 1977b). This results in fusion of part of the A a n d H fragments to yield a new fragment, AH, in pLC1. The pLC1 molecule also carries the six R100.1 fragm e n t s G, I, J, K, L a n d M. I n s e r t i o n of a pLC1 f r a g m e n t into the u n i q u e E c o R I site o n the pCR1 molecule (Covey et al., 1976) via the E c o R I generated " s t i c k y e n d s " should yield a n a u t o n o m o u s l y replicating p l a s m i d that expresses the pCR1 specified k a n a m y c i n resistance a n d a n y a n t i b i o t i c resistance carried by the pLC1 fragment. To make such plasmids we ligated a mixture of a complete E c o R I digest of pCR1 D N A (1 gg) a n d a n extensive b u t incomplete digest of pLC1 D N A (1 gg). The ligated mixture was used to t r a n s f o r m E. coli C600. Selection was m a d e for k a n a m y c i n resistance alone a n d for k a n a m y c i n resistance together with mercury, streptomycin, or chlora m p h e n i c o l resistance. In T a b l e 2 we show the relative frequencies of each class of t r a n s f o r m a n t s obtained. N o t e that the frequency of C m r K m r transform a n t s is a p p r o x i m a t e l y half, a n d that of H g r K m r t r a n s f o r m a n t s a p p r o x i m a t e l y one twentieth, of the frequency of S m r K m r t r a n s f o r m a n t s . The possible reason for these differences will be discussed later.
Analysis o f the Plasmids Several t r a n s f o r m a n t s of each class were purified by streaking o n L-agar s u p p l e m e n t e d with the a p p r o p r i ate d r u g , then tested directly for their resistance to mercury, c h l o r a m p h e n i c o l , s t r e p t o m y c i n a n d sulp h o n a m i d e . In order to determine whether a n y of the plasmids also carried the R100.1 fusidate resistance character, cleared lysates of cultures of selected clones were p r e p a r e d a n d used to t r a n s f o r m LC582, a fusidate sensitive derivative of C600. Selection in each case was m a d e for k a n a m y c i n resistance a n d the resulting t r a n s f o r m a n t s were screened for their resistance to fusidic acid. The results of this screening together with the p a t t e r n s of resistance exhibited by 12 of the original clones are shown in Table 3. We find that E c o R I fragments of pLC1 give rise to hybrid plasmids which carry all the resistance genes k n o w n to reside o n the r-det of R100.1. I n order to determine which E c o R I fragments carry the various resistance genes, cleared lysates of 20 of the original clones were digested with E c o R I
D. Lane and M. Chandler: Resistance Genes of R100.1
19 G
R I00.1
pLC I
J J M
ISI
RTF
ia C~ _ _ _ ~ E
~
a D
F
E
Fig. 1. Map of Rl00.1, after Tanaka et al. (1976), and its components, showing the sites of action of the restriction endonuclease EcoRI. Reciprocal recombination between the two ISI elements generates the RTF and pLC1 (the r-det). Exchange of D N A segments between fragments A and H of R100.1 results in the formation of fragments A' of RTF and AH of pLC1
Table 2. Transformation of C600 with ligated pCR1 and pLC1 DNAs
a
b
c
d
e
f
g
h
Selected resistance to:
Relative frequency of transformation
Km
K m + Hg
Km + Sm
K m + Cm
100
0.11
2.33
1.50
Table 3. Properties of the plasmids Plasmid
pLC3] pLC32 pLC34 pLC33 pLC36 pLC37 pLC38 pLC40 pLC41 pLC42 pLC43 pLC44
Confers resistance to :
Contains pLC1 EcoRI fragments :
Hg Sm Su C m F u
G
I
AH J
+ + + + --
+ + + + + + +
-+ + +
+ + + +
+
+ + + + + +
+ + + + + +
+
+
+
+
+
+
+ + + +
+ + + +
-
K
L
M
+ + + +
+ + . . . . + + + + + +
+ +
and the products separated by agarose gel electrophoresis. The fragment patterns of some pertinent plasmids are shown in Figure 2. The pattern of fragments was then correlated with the resistance pattern of each plasmid species. A summary of these results is presented in Table 3. Streptomycin and sulphonamide resistance always occur together. The genes specifying resistance to these
Pig. 2. Agarose gel electrophoresis of EcoRI digests of the plasmids made from pCR1 and pLC1. a) pLC43, b) pLC36, c) pLC33, d) pLC1, e) pCR1, f) pLC3t, g) pLC44, h) pLC1
20
antibiotics must both be located on fragment G since plasmids carrying only fragment G in addition to the pCR1 moiety (for example pLC36: Table 3 ; Fig. 2, channel b) confer resistance to both. All those plasmids conferring resistance to mercury (e.g. pLC31: Fig. 2, channel f), contain both fragments I and AH; we found no plasmids carrying I without AH. Plasmids which carry AH alone (e.g. pLC43: Fig. 2, channel a), do not confer mercury resistance. It is likely therefore that both I and AH are necessary for expression of mercury resistance. Each of the plasmids which confer resistance to chloramphenicol also confer resistance to fusidic acid. This phenotype is correlated with the presence of fragment AH since pLC43 (CmrFu r) carries only AH in addition to the pCR1 moiety. As discussed later, however, this result does not necessarily show that AH carries the entire cam and f u s genes. Of 11 transformants that had been selected for HgrKm r, and thus contained plasmids carrying the AH fragment, only 4 expressed resistance to chloramphenicol. When three of the HgrCmSKm r plasmids were transferred to LC582 they were unable to confer resistance to fusidate, whereas both the HgrCmrKm r plasmids tested in this way conferred resistance to the antibiotic. Hence the presence of AH within the plasmid is not in itself sufficient for the expression of cam and fus. We examine this phenomenon in more detail later.
Construction and Analysis o f Plasmids f r o m RIO0.1
The data presented above, together with the known location of the E c o R I fragments of pLC1 (Fig. 1), have allowed us to order the resistance genes on the pLC1 molecule. This order agrees with that determined by Dempsey and Willets (1976), although these authors concluded that the cam and mer genes are at opposite ends of the r-det in R100. In pLC1 they are both present, at least partially, on one fragment (AH). AH arisesby fusion of the r-det regions of R100.1 fragments A and H following a reciprocal recombination between the flanking IS1 elements (Fig. 1). Therefore it is likely that cam resides at least partially on fragment A and mer partially on fragment H of R100.1. To determine whether this is so, we constructed plasmids in the same way as for pLC1 but used a mixture of complete digests of pCR1 and R100.1 DNAs. The ligated DNA was used to transform C600; selection for HgrKm r yielded two transformants. Both plasmids proved to contain fragments H and I. One, pLC52 (Fig. 3, channel b), also contained fragment G and conferred resistance to strepto-
D. Lane and M. Chandler: Resistance Genes of R100.1
a
b
c
d
Fig. 3. Agarose gel electrophoresis of Ec oRI digests of plasmids made from pCR1 and R100.1 a) R100.1, b) pLC52, c) pLC54, d) pCR1. In R100.1 the H fragment, rather than the smaller AH fragment as in pLC1, adjoins the I fragment (see Fig. 1)
mycin and sulphonamide. Neither plasmid conferred resistance to chloramphenicol or fusidate. Since these plasmids were constructed from completely digested DNA, rather than a partial digest as was the case for the pLCl-derived plasmids, there can be little doubt that the mer gene spans the H-I junction. Selection for CmrKm r yielded 38 transformants. The plasmids from 13 of these were analyzed further; all contained the A fragment in addition to pCR1 (e.g. pLC54, Fig. 3, channel c), and only one, pLC55, carried an additional fragment (fragment K). Of the thirteen plasmids twelve conferred resistance to fusidic acid, none to mercury, streptomycin or sulphonamide.
Expression o f cam and f u s
As noted above, some of the pLCl-derived plasmids contained fragment AH but failed to express cam and fus. One explanation for this behaviour is that, of the two orientations in which AH can be inserted into pCR1, only one permits the expression of cam
D. Lane and M. Chandler: Resistance Genes of R100.1
A a
b
21
B c
d
a
b
c
d
Fig. 4. A Agarose gel electrophoresis of HindII-digested plasmids. (a) pCR1, (b) pLC43, (c) pLC31, (d) pLC44. B Agarose gel electrophoresis of restriction enzyme digests of plasmids expressing chloramphenicol and fusidic acid resistance. (a) pLC81 +HindII, (b) pLC43 + HindlI, (c) pLC81 + EcoRI, (d) pLC43 + EcoRI
and fus. If this notion is correct, digestion of each class of plasmid with a second restriction enzyme should produce dissimilar fragment patterns. In order to determine whether the orientation of AH in HgrCmSFu ~ plasmids is different from that in HgrCmrFu r plasmids, we compared the fragment patterns of a representative of each class following digestion with the HindII restriction enzyme. Digestion of pLC31 (HgrCmSFuS) and pLC44 (HgrCmrFur), plasmids which are composed of' pCR1 and pLC1 EcoRI fragments AH and I (Table 3), did indeed give rise to different fragment patterns. Comparison of these patterns with that obtained for pLC43 (CmrFur), which contains only the AH fragment in addition to pCR1 (Fig. 2, channel b), shows that both the CmrFu ~ plasmids, pLC43 and pLC44, have one fragment in common (Fig. 4A, channels b and d: arrow) which is not present in the CmSFu ~ plasmid, pLC31 (Fig. 4A, channel c), nor in pCR1 (Fig. 4A, channel a). This fragment must encompass one of the junctions between pCR1 and pLC1 D N A and
indicates a difference in orientation of the EcoRI fragments AH and I with respect to pCR1 in pLC31 and pLC44. If the expression of cam and fus is determined by the orientation of AH it should be possible to obtain CmrFu r derivatives of the CmSFu s plasmid, pLC31, by inverting the orientation of AH. Furthermore, such derivatives should exhibit the HindlI fragment common to the CmrFu r plasmids, pLC43 and pLC44, but not present in pLC31. A preparation of pLC31 was digested with EcoRI, ligated and used to transform C600. Several Cm r transformants were obtained. When the plasmids from three of these transformants were examined by digestion with EcoRI, all gave a gel pattern identical to that of pLC43 and carried only fragment AH in addition to the pCR1 moiety (Fig. 4B, channels c and d). One plasmid, pLC81, was examined in more detail. It was transferred to LC582 where it conferred resistance to fusidate as well as to chloramphenicol. The products of digestion with HindII were identical in molecular weight to those from pLC43 (Fig. 4B, channels and b). These results demonstrate that the orientation of the I-AH segment relative to pCR1 determines whether cam and fus are expressed. In all pLCl-derived plasmids that contained the AH fragment we found that cam and fus were either expressed together or both unexpressed. This suggests that the cam and fus genes are transcribed from the same promoter and is consistent with the conclusions of Miki et al. (1977) that cam and fus form part of a single operon. In the example given above we obtained simultaneous recovery of expression of cam andfus from a plasmid that previously expressed neither. However, as stated above, we obtained one R100.1-derived plasmid, pLC53, from a Cm r transformant that failed to exhibit fusidate resistance in LC582. We think it unlikely that this behaviour can be explained on the basis of the orientation of the A fragment in p C R I ; more probably some other, undetected genetic alteration in the plasmid is responsible. We are currently unable to provide an explanation for the behaviour of this plasmid.
Discussion
Cloning of EcoRI restriction enzyme fragments from pLC1 onto pCR1, together with a knowledge of the relative positions of the fragments (Tanaka et al., 1976), has allowed us to map the known antibiotic resistance genes of the R100.1 r-determinant with some precision. Our conclusions are summarized in Figure 5. The order of the genes agrees with that previously published by Tanaka et al. (1976) and by
22
D. Lane and M. Chandler: Resistance Genes of R100.1
cam , f u s
gig. g. Positions of the resistance genes in R100.1 and pLC1 relative to the EcoRI restriction fragment m a p
Dempsey and Willets (1976). We have not determined the relative positions of str and sul, and the order of these genes shown in Figure 5 is that reported by these two groups. The frequencies with which transformants selected for different drug resistances arose (Table 2) probably reflects the relation of the resistance genes to the E c o R I sites. SmrKm r transformants were the most frequent class obtained; str is entirely contained, presumably with its promoter, in fragment G. The lower frequency of CmrKm r transformants reflects the dependence of cam expression on the orientation of AH in the pCR1 molecule and results from the frequent insertion of AH into pCR1 in the orientation that does not permit expression of cam. Although we have obtained no plasmid which carries I but not A H, and have therefore been unable to rule out the possibility that mer is carried by fragment I, the finding that HgrKm r transformants occur at a much lower frequency than either SmrKm r or CmrKm r transformants presumably stems from the necessity of joining two pLC1 fragments in order to restore a structural gene or to connect such a gene with its promoter. That one of these fragments carries a promoter is suggested by the observation that the expression of rner is not dependent on the orientation of AH with respect to pCR1 (see below). We take the dependence of both cam and f u s expression on the orientation of the AH fragment within pCR1 to mean that part of the system for their expression lies outside AH. This part could be the promoter which in R100.1 might reside in the J fragment. A further reason for believing that at least part of the f u s and cam genes lies outside the AH fragment is
the observation that the region which is common to both AH and A, and is thus responsible for expression of cam a n d f u s in the pCR1 derived hybrid plasmids, is only 0.37 kilobases in length (Chandler etal., 1977b). Since the chloramphenicol transacetylase polypeptide is known to have a molecular weight of 20,000 daltons (Foster and Howe, 1973) and would alone require transcription of about 0.5 kilobases of DNA, it is difficult to imagine that both cam and f u s are located entirely on this small region of DNA. One possibility is that a portion of either the cam or the f u s gene product is coded by the AH-proximal portion of the J fragment and is replaceable by other non-related amino acid sequences coded for by the pCR1 molecule. Another possibility is suggested by the geneology of the vector plasmid pCR1. This plasmid, derived from colE1, carries a gene for resistance to kanamycin (kan). It was constructed by insertion of an E c o R I fragment of pSC105, itself a derivative of the plasmid R6.5 (Cohen et al., 1973), at the unique E c o R I site in colE1 (Hershfield et al., 1974); one of the two resulting E c o R I sites was subsequently removed (Covey et al., 1976). R6.5 is closely related to RI00.1 (Sharp etal., 1973). It carries all the resistance genes of R100.1 in addition to the kan gene. The R6.5 fragment carrying kan has been shown to map at the same position on the R6.5 molecule as does fragment J on R100.1 (Cabello etal., 1977; Tanaka etal., 1976). It has been demonstrated by Cabello et al. (1977) that the R6.5 fragment carrying kan also carries part of the cam gene. It is likely therefore that, in the hybrid plasmids described here, part of the cam and f u s genes of R6.5 are carried by the vector plasmid pCR1 and can substitute for those sequences present in the J fragment of R100.1. (We are grateful to R. Rownd for bringing this aspect of the construction of pCR1 to our attention.) This is in agreement with the observations of Miki et al. (1977) which demonstrate that when the colEl-amp derivative RSF 2124 (So et al., 1975) is used to clone various E c o R I fragments of NR1 (identical to R100.1) both fragments A and J are required for expression of cam and fus. In summary, our results demonstrate that the pLC1 closed circular molecule found in strain LC2633 contains all the information known to be encoded by the r-determinant of R100.1, and hence that pLC1 is, as far as can be determined, genetically identical to the r-determinant. As anticipated from the way in which pLC1 is generated by reciprocal recombination between IS1 elements (Chandler etal., 1977b) we find that the rner and cam genes, which in R100.1 are at opposite ends of the r-determinant, are adjacent in pLC1. The mer gene and its promoter span the
D. Lane and M. Chandler: Resistance Genes of R100.1
EcoRI site between the I and AH fragments. The str and sul genes are both contained in the G frag-
ment presumably with their promoters. The observation that cam and fus expression occur together or not at all suggests that these genes are part of an operon. This operon probably spans the EcoRI site between A and J on R100.1 and AH and J in pLC1. It should be noted that, on the basis of the size of the EcoRI fragments of R100.1 and pLC1 which carry known resistance genes, we have accounted for a maximum of only one quarter of the coding capacity of the r-determinant. The results presented here agree with those of Miki et al. (1977). They are also consistent with the results of Cabello et al. (1977) who have mapped the resistance genes of the closely related R plasmid R6.5. Acknowledgements. We would like to thask L. Caro, Lynn Silver
and R. Rownd for helpful discussions. We would also like to thank R. Rownd, F. Cabello and K. Timmis for communicating their results to us prior to publication. This work was supported by grant nr. 3.679.75 to L. Caro from the Swiss National Science Foundation.
References Bird, R.E., Chandler, M., Caro, L. : Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1 : change of chromosome origin in synchronized cultures. J. Bact. 126, 1215-1223 (I976) Cabello, F., Timmis, K., Cohen, S.N.: Cloning of Hind III and EcoRI fragments of the R6.5 plasmid by insertional inactivation. In: Microbiology-1977. Washington, D.C. : American Society for Microbiolo~ (in press) Chandler, M., Allet, B., Gallay, E., Boy de la Tour, E., Caro, L. : Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1. Molec. gen. Genet. 153, 289-295 (1977) Chandler, M., Silver, L., Frey, J., Caro, L.: Suppression of an E. coli dnaA mutation by the integrated R factor R100.1 : Generation of small plasmids after integration. J. Bact. 130, 303-311 (1977)
23 CleweI1, D.B.: Nature of colE1 plasmid replication in Escherichia coli in the presence of chlormaphenicol. J. Bact. 116, 667 676 (1972) Clewell, D.B., Helinski, D.R.: Supercoiled circular DNA-protein complex in E. coli: purification and induced conversion to an open circular form. Proc. nat. Acad. Sci. (Wash.) 62, 1159-1166 (1969) Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Helling, R.B.: Construction of biologically functional bacterial plasmids in v#vo. Proc. nat. Acad. Sci, (Wash.) 70, 3240 3244 (1973) Cohen, S.N., Chang, A.C.Y., Hsu, L.: Non-chromosomal antibiotic resistance in bacteria : genetic transformation of Escherichia coli by R-factor DNA. Proc. nat. Acad. Sci. (Wash.) 69, 2110 2114 (I972) Covey, C., Richardson, D., Carbon, J. : A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid. Molec. gen. Genet. 145, 155-158 (1976) Dempsey, W.B., Willets, N.S. : Plasmid co-integrates of prophage lambda and R factor R100. J. Bact. 126, 166-176 (1976) Foster, T.J., Howe, T.G.B. : Deletion map of the chloramphenicol resistance region of R1 and R100. I. J. Bact. 116, 1062-1063 (1973) Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A., Helinski, D.R.: Plasmid colE1 as a molecular vehicle for cloning and amplification of DNA. Proc. nat Acad. Sci. (Wash.) 71, 3455-3459 (1974) Miki, T., Easton, A.M., Rownd, R.H. : Mapping of the resistance genes of the R plasmid NR1. Submitted (1977) Rownd, R.H., Mickel, S.: Dissociation and reassociation of RTF and r-determinants of the R factor NR1 in Proteus mirabilis. Nature (Lond.) 234, 4 0 4 2 (1971) Sharp, P.A., Cohen, S.N., Davidson, N.: Electron microscopic heteroduplex studies of sequence relations among plasmids of E. coli. II Structure of drug resistance (R) factors and F factors. J. molec. Biol. 75, 235 255 (1973) So, M., Boyer, H.W., Betlach, M., Falkow, S.: Molecular cloning of an Escherichia coli plasmid determinant that encodes for the production of a heat stable enterotoxin. J. Bact. 128, 463-472 (1976) Tanaka, N., Cramer, J.H., Rownd, R.H.: EcoRI restriction endonuclease map of the composite R plasmid NR1. J. Bact. 127, 619-636 (1976)
Communicated by W. Arber Received June 14~August 10, 1977
Mapping of the Drug Resistance Genes Carried by the r-Determinant of the R100.1 Plasmid D. L a n e * and M. Chandler D6partement de Biologie Mol6culaire, 30, quai Ernest-Ansermet, CH-1211 Gen6ve 4, Switzerland
Summary. We have cloned the E c o R I fragments of pLC1, a circular D N A element f o u n d in an E s c h e r i chia coli dnaAts strain integratively suppressed by R100.1 (Chandler etal., 1977a), using the plasmid vector pCR1. All the resistance genes k n o w n to be present on the r-determinant o f R100.1 were f o u n d to be present on pLC1. The isolation o f pCR1 derivatives carrying various E c o R I fragments o f either pLC1 or RI00.1 has allowed a m o r e precise m a p p i n g o f the position o f the resistance genes on the R100.1 molecule.
Introduction The antibiotic resistance plasmid R100.1 is a selftransmissible e x t r a c h r o m o s o m a l element which specifies resistance to tetracycline (Tc), chloramphenicol (Cm), sulphonamides (Su), streptemycin (Sm), mercury (Hg) and fusidic acid (Fu) 1. In c o m m o n with several R plasmids, R100.1 comprises two functionally distinct parts: the resistance transfer factor ( R T F ) which carries genetic information for replication and conjugal transfer o f the plasmid and also for resistance to tetracycline, and the r-determinant (r-det) which carries genes specifying resistance to the other drugs. In some situations these two parts dissociate f r o m each other ( R o w n d & Mickel, 1971 ; C h a n d l e r et al., 1977a). We have observed one example o f this dissociation in a dnaAts m u t a n t o f E s c h e r i c h i a coli in which the temperature-sensitive initiation defect in c h r o m o s o m e replication is suppressed by integration o f R100.1 (Bird et al., 1976). In m a n y such strains, integration is associated with the f o r m a t i o n o f closed c i r c u l a r In this report, Cm, Su, Sm, Hg and Fu are used to designate resistance phenotypes. The corresponding genes are designated cam, sul, str, met and fus * Present address: Department of Cell Biology, University of Auck-
land, Private Bag, Auckland, New Zealand For offprints contact." M. Chandler
D N A elements. We have investigated the structure of one o f these elements, pLC1 and have shown that it has physical properties identical to those o f the r-det (Chandler et al., 1977, a, b). Since we were unable to establish pLC1 in strains other than the parental strain, LC2633, we could not demonstrate that pLC1 carries all the resistance genes associated with the r-det region o f R100.1 In this report we show that, by cloning restriction enzyme fragments o f pLC1 o n t o a self-replicating plasmid vector, we can obtain plasmids containing all the k n o w n r-det resistance genes. This genetic evidence further demonstrates that pLC1 and t h e r-det o f R100.1 are identical. F r o m the k n o w n relative positions o f these fragments on both R100.1 and the pLC1 molecule (Tanaka et al., 1976; Chandler et al., 1977a) we can determine the order o f the resistance genes. The results agree with those o f D e m p s e y & Willets (1976) who established this order using deletion mutants of 2-R100 co-integrates. O u r assignment o f the genes to the various E c o R I fragments extends the results o f T a n a k a et al. (1976) to include m e r and fus.
Materials and Methods Bacterial Strains
Strains LC468 and LC2633 (Table 1), both derivatives of Escherichia coli K12 W1485 (Chandler et al., 1977a), served as sources of R100.1 and pLC1 DNA's respectively. Strain LC555, a derivative of E. coli C600 which carries the colEl-kan plasmid pCR1 (Covey et al., 1976), was used to prepare pCRI DNA. E. coli C600 was used as a recipient in all transformations except where fusidic acid was to be tested. For these experiments a fusidic acid-sensitive strain, LC582, was isolated, as follows. C600 was grown in L-broth to midlog phase and nitrosoguanidine was added to 0.1 mg/ml. After 5 min the cells were washed, resuspended in L-broth and grown for 2 h. Fusidic acid (a generous gift of Leo Pharmaceuticals) was added to 0.1 mg/mi, followed 10 min later by penicillin G to 5000 units/ml. After Iysis samples were plated on L-agar and incubated at 37° C. The colonies were replica-plated onto L-agar with and without fusidic acid (0.1 mg/ml). One fusidic acid sensitive clone was purified for further use. This strain was sensitive to concentrations of fusidic acid lower than 0.1 mg/ml and a concentration of 20 gg/ml was used in all further experiments.
18
D. Lane and M. Chandler: Resistance Genes of R100.1
Table 1. Strains of Escherichia coli
Results
Strain
Sex
Description
Construction o f Plasmids f r o m p L C l
LC468
R+
thi leu thy/RlO0.1 : a derivative of W1485
LC555
F
C600/pCR1
LC582
F-
a fusidate sensitive derivative of C600
LC2633
R. Hfr
thi leu thy (proB lac)del dnaAt~46 (ilv: :Mu-1), (2ind-) Transfers 0 proA proB ... araR (Bird et al., 1976; Chandler et al. 1977a)
Media L-broth, M9 glucose casamino-acids, and the corresponding solid media have been described (Bird et al., 1976). Resistance to kanamycin, streptomycin, chloramphenicol, HgCL2 and fusidic acid was tested on L-agar plates with 20 ~tg/mlof the appropriate drug; resistance to sulphonamide was tested on M9-glucose-casamino acids plates containing 100 gg/ml sulphamerazin.
Isolation of Plasmid DNA R100.1 and pLC1 closed circular DNAs were isolated from strains LC524 and LC2633 by centrifugation of cleared lysates in CsC1EtBr gradients (Clewell and Helinski, 1969) as described previously (Chandler et al., 1977a). pCR1 DNA was prepared by the same method from overnight cultures of LC555 which had been treated with 150 gg/ml chloramphenicol at a cell concentration of 5 x 108/ml for 16 h. The addition of chloramphenicol results in an amplification of colEl-derived plasmids (Clewell, 1972). Plasmid DNA from purified transformants (see below) was routinely prepared from cleared lysates (Clewelland Helinski, 1969) of 25 ml cultures grown in M9 glucose casamino acids medium. The cleared lysates were extracted twice with an equal volume of phenol (Merck analytical grade, saturated with 10 mM Tris, 1 mM EDTA pH 7.6) and the DNA precipitated with ethanol. The precipitates were redissolvedin 0.3~0.5 ml of H20 and dialyzed against several changes of 10 mM Tris, 1 mM EDTA pH 7.6. When necessary RNA was removed from the preparations by treatment with RNAase A (10 lag/ml) for 30 min at 37° C.
Enzyme Reactions Digestion of DNA by the restriction enzyme EcoRi (the generous gift of J. Hedgpeth and M. Ballivet) and electrophoresis of the products on 1% agarose slab gels have been described (Chandler et al., 1977a). Digestion with the enzyme HindII (Bethesda Research Laboratories) was carried out in a similar way in 60 mM NaCI, 6.6 mM MgC12, 6 mM 2-mercaptoethanoi and 10 mM Tris pH 7.9. Prior to ligation, the EcoRI digested preparations were incubated at 65°C for 10 rain to inactivate the endonuclease, then chilled on ice. Ligation was carried out in 10 mM dithiothreitol, 10 mM MgC12, 0.055 mM ATP and 10 mM Tris pH 7.6. T4 DNA ligase (Miles) was added to 0.1-1 units/ml and the mixture (50-100 gl) incubated at 11 °C for 3-6 days. The ligated DNA was added to 1 ml of Escherichia coli tRNA (50 gg/ml) and ethanol precipitated. The dried precipitate was dissolved in 0.1 ml of 20 mM Tris pH 8.0, 20 mM NaCI, and 1 mM EDTA. It was used directly for transformation according to the procedure of Cohen et al. (1972). NIH Pl level containment procedures were followed throughout these experiments.
It has previously been d e t e r m i n e d that pLC1 is f o r m e d f r o m R100.1 by reciprocal r e c o m b i n a t i o n between the two IS1 elements present o n the E c o R I fragments A a n d H, as illustrated in Figure 1 ( C h a n d l e r et al., 1977b). This results in fusion of part of the A a n d H fragments to yield a new fragment, AH, in pLC1. The pLC1 molecule also carries the six R100.1 fragm e n t s G, I, J, K, L a n d M. I n s e r t i o n of a pLC1 f r a g m e n t into the u n i q u e E c o R I site o n the pCR1 molecule (Covey et al., 1976) via the E c o R I generated " s t i c k y e n d s " should yield a n a u t o n o m o u s l y replicating p l a s m i d that expresses the pCR1 specified k a n a m y c i n resistance a n d a n y a n t i b i o t i c resistance carried by the pLC1 fragment. To make such plasmids we ligated a mixture of a complete E c o R I digest of pCR1 D N A (1 gg) a n d a n extensive b u t incomplete digest of pLC1 D N A (1 gg). The ligated mixture was used to t r a n s f o r m E. coli C600. Selection was m a d e for k a n a m y c i n resistance alone a n d for k a n a m y c i n resistance together with mercury, streptomycin, or chlora m p h e n i c o l resistance. In T a b l e 2 we show the relative frequencies of each class of t r a n s f o r m a n t s obtained. N o t e that the frequency of C m r K m r transform a n t s is a p p r o x i m a t e l y half, a n d that of H g r K m r t r a n s f o r m a n t s a p p r o x i m a t e l y one twentieth, of the frequency of S m r K m r t r a n s f o r m a n t s . The possible reason for these differences will be discussed later.
Analysis o f the Plasmids Several t r a n s f o r m a n t s of each class were purified by streaking o n L-agar s u p p l e m e n t e d with the a p p r o p r i ate d r u g , then tested directly for their resistance to mercury, c h l o r a m p h e n i c o l , s t r e p t o m y c i n a n d sulp h o n a m i d e . In order to determine whether a n y of the plasmids also carried the R100.1 fusidate resistance character, cleared lysates of cultures of selected clones were p r e p a r e d a n d used to t r a n s f o r m LC582, a fusidate sensitive derivative of C600. Selection in each case was m a d e for k a n a m y c i n resistance a n d the resulting t r a n s f o r m a n t s were screened for their resistance to fusidic acid. The results of this screening together with the p a t t e r n s of resistance exhibited by 12 of the original clones are shown in Table 3. We find that E c o R I fragments of pLC1 give rise to hybrid plasmids which carry all the resistance genes k n o w n to reside o n the r-det of R100.1. I n order to determine which E c o R I fragments carry the various resistance genes, cleared lysates of 20 of the original clones were digested with E c o R I
D. Lane and M. Chandler: Resistance Genes of R100.1
19 G
R I00.1
pLC I
J J M
ISI
RTF
ia C~ _ _ _ ~ E
~
a D
F
E
Fig. 1. Map of Rl00.1, after Tanaka et al. (1976), and its components, showing the sites of action of the restriction endonuclease EcoRI. Reciprocal recombination between the two ISI elements generates the RTF and pLC1 (the r-det). Exchange of D N A segments between fragments A and H of R100.1 results in the formation of fragments A' of RTF and AH of pLC1
Table 2. Transformation of C600 with ligated pCR1 and pLC1 DNAs
a
b
c
d
e
f
g
h
Selected resistance to:
Relative frequency of transformation
Km
K m + Hg
Km + Sm
K m + Cm
100
0.11
2.33
1.50
Table 3. Properties of the plasmids Plasmid
pLC3] pLC32 pLC34 pLC33 pLC36 pLC37 pLC38 pLC40 pLC41 pLC42 pLC43 pLC44
Confers resistance to :
Contains pLC1 EcoRI fragments :
Hg Sm Su C m F u
G
I
AH J
+ + + + --
+ + + + + + +
-+ + +
+ + + +
+
+ + + + + +
+ + + + + +
+
+
+
+
+
+
+ + + +
+ + + +
-
K
L
M
+ + + +
+ + . . . . + + + + + +
+ +
and the products separated by agarose gel electrophoresis. The fragment patterns of some pertinent plasmids are shown in Figure 2. The pattern of fragments was then correlated with the resistance pattern of each plasmid species. A summary of these results is presented in Table 3. Streptomycin and sulphonamide resistance always occur together. The genes specifying resistance to these
Pig. 2. Agarose gel electrophoresis of EcoRI digests of the plasmids made from pCR1 and pLC1. a) pLC43, b) pLC36, c) pLC33, d) pLC1, e) pCR1, f) pLC3t, g) pLC44, h) pLC1
20
antibiotics must both be located on fragment G since plasmids carrying only fragment G in addition to the pCR1 moiety (for example pLC36: Table 3 ; Fig. 2, channel b) confer resistance to both. All those plasmids conferring resistance to mercury (e.g. pLC31: Fig. 2, channel f), contain both fragments I and AH; we found no plasmids carrying I without AH. Plasmids which carry AH alone (e.g. pLC43: Fig. 2, channel a), do not confer mercury resistance. It is likely therefore that both I and AH are necessary for expression of mercury resistance. Each of the plasmids which confer resistance to chloramphenicol also confer resistance to fusidic acid. This phenotype is correlated with the presence of fragment AH since pLC43 (CmrFu r) carries only AH in addition to the pCR1 moiety. As discussed later, however, this result does not necessarily show that AH carries the entire cam and f u s genes. Of 11 transformants that had been selected for HgrKm r, and thus contained plasmids carrying the AH fragment, only 4 expressed resistance to chloramphenicol. When three of the HgrCmSKm r plasmids were transferred to LC582 they were unable to confer resistance to fusidate, whereas both the HgrCmrKm r plasmids tested in this way conferred resistance to the antibiotic. Hence the presence of AH within the plasmid is not in itself sufficient for the expression of cam and fus. We examine this phenomenon in more detail later.
Construction and Analysis o f Plasmids f r o m RIO0.1
The data presented above, together with the known location of the E c o R I fragments of pLC1 (Fig. 1), have allowed us to order the resistance genes on the pLC1 molecule. This order agrees with that determined by Dempsey and Willets (1976), although these authors concluded that the cam and mer genes are at opposite ends of the r-det in R100. In pLC1 they are both present, at least partially, on one fragment (AH). AH arisesby fusion of the r-det regions of R100.1 fragments A and H following a reciprocal recombination between the flanking IS1 elements (Fig. 1). Therefore it is likely that cam resides at least partially on fragment A and mer partially on fragment H of R100.1. To determine whether this is so, we constructed plasmids in the same way as for pLC1 but used a mixture of complete digests of pCR1 and R100.1 DNAs. The ligated DNA was used to transform C600; selection for HgrKm r yielded two transformants. Both plasmids proved to contain fragments H and I. One, pLC52 (Fig. 3, channel b), also contained fragment G and conferred resistance to strepto-
D. Lane and M. Chandler: Resistance Genes of R100.1
a
b
c
d
Fig. 3. Agarose gel electrophoresis of Ec oRI digests of plasmids made from pCR1 and R100.1 a) R100.1, b) pLC52, c) pLC54, d) pCR1. In R100.1 the H fragment, rather than the smaller AH fragment as in pLC1, adjoins the I fragment (see Fig. 1)
mycin and sulphonamide. Neither plasmid conferred resistance to chloramphenicol or fusidate. Since these plasmids were constructed from completely digested DNA, rather than a partial digest as was the case for the pLCl-derived plasmids, there can be little doubt that the mer gene spans the H-I junction. Selection for CmrKm r yielded 38 transformants. The plasmids from 13 of these were analyzed further; all contained the A fragment in addition to pCR1 (e.g. pLC54, Fig. 3, channel c), and only one, pLC55, carried an additional fragment (fragment K). Of the thirteen plasmids twelve conferred resistance to fusidic acid, none to mercury, streptomycin or sulphonamide.
Expression o f cam and f u s
As noted above, some of the pLCl-derived plasmids contained fragment AH but failed to express cam and fus. One explanation for this behaviour is that, of the two orientations in which AH can be inserted into pCR1, only one permits the expression of cam
D. Lane and M. Chandler: Resistance Genes of R100.1
A a
b
21
B c
d
a
b
c
d
Fig. 4. A Agarose gel electrophoresis of HindII-digested plasmids. (a) pCR1, (b) pLC43, (c) pLC31, (d) pLC44. B Agarose gel electrophoresis of restriction enzyme digests of plasmids expressing chloramphenicol and fusidic acid resistance. (a) pLC81 +HindII, (b) pLC43 + HindlI, (c) pLC81 + EcoRI, (d) pLC43 + EcoRI
and fus. If this notion is correct, digestion of each class of plasmid with a second restriction enzyme should produce dissimilar fragment patterns. In order to determine whether the orientation of AH in HgrCmSFu ~ plasmids is different from that in HgrCmrFu r plasmids, we compared the fragment patterns of a representative of each class following digestion with the HindII restriction enzyme. Digestion of pLC31 (HgrCmSFuS) and pLC44 (HgrCmrFur), plasmids which are composed of' pCR1 and pLC1 EcoRI fragments AH and I (Table 3), did indeed give rise to different fragment patterns. Comparison of these patterns with that obtained for pLC43 (CmrFur), which contains only the AH fragment in addition to pCR1 (Fig. 2, channel b), shows that both the CmrFu ~ plasmids, pLC43 and pLC44, have one fragment in common (Fig. 4A, channels b and d: arrow) which is not present in the CmSFu ~ plasmid, pLC31 (Fig. 4A, channel c), nor in pCR1 (Fig. 4A, channel a). This fragment must encompass one of the junctions between pCR1 and pLC1 D N A and
indicates a difference in orientation of the EcoRI fragments AH and I with respect to pCR1 in pLC31 and pLC44. If the expression of cam and fus is determined by the orientation of AH it should be possible to obtain CmrFu r derivatives of the CmSFu s plasmid, pLC31, by inverting the orientation of AH. Furthermore, such derivatives should exhibit the HindlI fragment common to the CmrFu r plasmids, pLC43 and pLC44, but not present in pLC31. A preparation of pLC31 was digested with EcoRI, ligated and used to transform C600. Several Cm r transformants were obtained. When the plasmids from three of these transformants were examined by digestion with EcoRI, all gave a gel pattern identical to that of pLC43 and carried only fragment AH in addition to the pCR1 moiety (Fig. 4B, channels c and d). One plasmid, pLC81, was examined in more detail. It was transferred to LC582 where it conferred resistance to fusidate as well as to chloramphenicol. The products of digestion with HindII were identical in molecular weight to those from pLC43 (Fig. 4B, channels and b). These results demonstrate that the orientation of the I-AH segment relative to pCR1 determines whether cam and fus are expressed. In all pLCl-derived plasmids that contained the AH fragment we found that cam and fus were either expressed together or both unexpressed. This suggests that the cam and fus genes are transcribed from the same promoter and is consistent with the conclusions of Miki et al. (1977) that cam and fus form part of a single operon. In the example given above we obtained simultaneous recovery of expression of cam andfus from a plasmid that previously expressed neither. However, as stated above, we obtained one R100.1-derived plasmid, pLC53, from a Cm r transformant that failed to exhibit fusidate resistance in LC582. We think it unlikely that this behaviour can be explained on the basis of the orientation of the A fragment in p C R I ; more probably some other, undetected genetic alteration in the plasmid is responsible. We are currently unable to provide an explanation for the behaviour of this plasmid.
Discussion
Cloning of EcoRI restriction enzyme fragments from pLC1 onto pCR1, together with a knowledge of the relative positions of the fragments (Tanaka et al., 1976), has allowed us to map the known antibiotic resistance genes of the R100.1 r-determinant with some precision. Our conclusions are summarized in Figure 5. The order of the genes agrees with that previously published by Tanaka et al. (1976) and by
22
D. Lane and M. Chandler: Resistance Genes of R100.1
cam , f u s
gig. g. Positions of the resistance genes in R100.1 and pLC1 relative to the EcoRI restriction fragment m a p
Dempsey and Willets (1976). We have not determined the relative positions of str and sul, and the order of these genes shown in Figure 5 is that reported by these two groups. The frequencies with which transformants selected for different drug resistances arose (Table 2) probably reflects the relation of the resistance genes to the E c o R I sites. SmrKm r transformants were the most frequent class obtained; str is entirely contained, presumably with its promoter, in fragment G. The lower frequency of CmrKm r transformants reflects the dependence of cam expression on the orientation of AH in the pCR1 molecule and results from the frequent insertion of AH into pCR1 in the orientation that does not permit expression of cam. Although we have obtained no plasmid which carries I but not A H, and have therefore been unable to rule out the possibility that mer is carried by fragment I, the finding that HgrKm r transformants occur at a much lower frequency than either SmrKm r or CmrKm r transformants presumably stems from the necessity of joining two pLC1 fragments in order to restore a structural gene or to connect such a gene with its promoter. That one of these fragments carries a promoter is suggested by the observation that the expression of rner is not dependent on the orientation of AH with respect to pCR1 (see below). We take the dependence of both cam and f u s expression on the orientation of the AH fragment within pCR1 to mean that part of the system for their expression lies outside AH. This part could be the promoter which in R100.1 might reside in the J fragment. A further reason for believing that at least part of the f u s and cam genes lies outside the AH fragment is
the observation that the region which is common to both AH and A, and is thus responsible for expression of cam a n d f u s in the pCR1 derived hybrid plasmids, is only 0.37 kilobases in length (Chandler etal., 1977b). Since the chloramphenicol transacetylase polypeptide is known to have a molecular weight of 20,000 daltons (Foster and Howe, 1973) and would alone require transcription of about 0.5 kilobases of DNA, it is difficult to imagine that both cam and f u s are located entirely on this small region of DNA. One possibility is that a portion of either the cam or the f u s gene product is coded by the AH-proximal portion of the J fragment and is replaceable by other non-related amino acid sequences coded for by the pCR1 molecule. Another possibility is suggested by the geneology of the vector plasmid pCR1. This plasmid, derived from colE1, carries a gene for resistance to kanamycin (kan). It was constructed by insertion of an E c o R I fragment of pSC105, itself a derivative of the plasmid R6.5 (Cohen et al., 1973), at the unique E c o R I site in colE1 (Hershfield et al., 1974); one of the two resulting E c o R I sites was subsequently removed (Covey et al., 1976). R6.5 is closely related to RI00.1 (Sharp etal., 1973). It carries all the resistance genes of R100.1 in addition to the kan gene. The R6.5 fragment carrying kan has been shown to map at the same position on the R6.5 molecule as does fragment J on R100.1 (Cabello etal., 1977; Tanaka etal., 1976). It has been demonstrated by Cabello et al. (1977) that the R6.5 fragment carrying kan also carries part of the cam gene. It is likely therefore that, in the hybrid plasmids described here, part of the cam and f u s genes of R6.5 are carried by the vector plasmid pCR1 and can substitute for those sequences present in the J fragment of R100.1. (We are grateful to R. Rownd for bringing this aspect of the construction of pCR1 to our attention.) This is in agreement with the observations of Miki et al. (1977) which demonstrate that when the colEl-amp derivative RSF 2124 (So et al., 1975) is used to clone various E c o R I fragments of NR1 (identical to R100.1) both fragments A and J are required for expression of cam and fus. In summary, our results demonstrate that the pLC1 closed circular molecule found in strain LC2633 contains all the information known to be encoded by the r-determinant of R100.1, and hence that pLC1 is, as far as can be determined, genetically identical to the r-determinant. As anticipated from the way in which pLC1 is generated by reciprocal recombination between IS1 elements (Chandler etal., 1977b) we find that the rner and cam genes, which in R100.1 are at opposite ends of the r-determinant, are adjacent in pLC1. The mer gene and its promoter span the
D. Lane and M. Chandler: Resistance Genes of R100.1
EcoRI site between the I and AH fragments. The str and sul genes are both contained in the G frag-
ment presumably with their promoters. The observation that cam and fus expression occur together or not at all suggests that these genes are part of an operon. This operon probably spans the EcoRI site between A and J on R100.1 and AH and J in pLC1. It should be noted that, on the basis of the size of the EcoRI fragments of R100.1 and pLC1 which carry known resistance genes, we have accounted for a maximum of only one quarter of the coding capacity of the r-determinant. The results presented here agree with those of Miki et al. (1977). They are also consistent with the results of Cabello et al. (1977) who have mapped the resistance genes of the closely related R plasmid R6.5. Acknowledgements. We would like to thask L. Caro, Lynn Silver
and R. Rownd for helpful discussions. We would also like to thank R. Rownd, F. Cabello and K. Timmis for communicating their results to us prior to publication. This work was supported by grant nr. 3.679.75 to L. Caro from the Swiss National Science Foundation.
References Bird, R.E., Chandler, M., Caro, L. : Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1 : change of chromosome origin in synchronized cultures. J. Bact. 126, 1215-1223 (I976) Cabello, F., Timmis, K., Cohen, S.N.: Cloning of Hind III and EcoRI fragments of the R6.5 plasmid by insertional inactivation. In: Microbiology-1977. Washington, D.C. : American Society for Microbiolo~ (in press) Chandler, M., Allet, B., Gallay, E., Boy de la Tour, E., Caro, L. : Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1. Molec. gen. Genet. 153, 289-295 (1977) Chandler, M., Silver, L., Frey, J., Caro, L.: Suppression of an E. coli dnaA mutation by the integrated R factor R100.1 : Generation of small plasmids after integration. J. Bact. 130, 303-311 (1977)
23 CleweI1, D.B.: Nature of colE1 plasmid replication in Escherichia coli in the presence of chlormaphenicol. J. Bact. 116, 667 676 (1972) Clewell, D.B., Helinski, D.R.: Supercoiled circular DNA-protein complex in E. coli: purification and induced conversion to an open circular form. Proc. nat. Acad. Sci. (Wash.) 62, 1159-1166 (1969) Cohen, S.N., Chang, A.C.Y., Boyer, H.W., Helling, R.B.: Construction of biologically functional bacterial plasmids in v#vo. Proc. nat. Acad. Sci, (Wash.) 70, 3240 3244 (1973) Cohen, S.N., Chang, A.C.Y., Hsu, L.: Non-chromosomal antibiotic resistance in bacteria : genetic transformation of Escherichia coli by R-factor DNA. Proc. nat. Acad. Sci. (Wash.) 69, 2110 2114 (I972) Covey, C., Richardson, D., Carbon, J. : A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid. Molec. gen. Genet. 145, 155-158 (1976) Dempsey, W.B., Willets, N.S. : Plasmid co-integrates of prophage lambda and R factor R100. J. Bact. 126, 166-176 (1976) Foster, T.J., Howe, T.G.B. : Deletion map of the chloramphenicol resistance region of R1 and R100. I. J. Bact. 116, 1062-1063 (1973) Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A., Helinski, D.R.: Plasmid colE1 as a molecular vehicle for cloning and amplification of DNA. Proc. nat Acad. Sci. (Wash.) 71, 3455-3459 (1974) Miki, T., Easton, A.M., Rownd, R.H. : Mapping of the resistance genes of the R plasmid NR1. Submitted (1977) Rownd, R.H., Mickel, S.: Dissociation and reassociation of RTF and r-determinants of the R factor NR1 in Proteus mirabilis. Nature (Lond.) 234, 4 0 4 2 (1971) Sharp, P.A., Cohen, S.N., Davidson, N.: Electron microscopic heteroduplex studies of sequence relations among plasmids of E. coli. II Structure of drug resistance (R) factors and F factors. J. molec. Biol. 75, 235 255 (1973) So, M., Boyer, H.W., Betlach, M., Falkow, S.: Molecular cloning of an Escherichia coli plasmid determinant that encodes for the production of a heat stable enterotoxin. J. Bact. 128, 463-472 (1976) Tanaka, N., Cramer, J.H., Rownd, R.H.: EcoRI restriction endonuclease map of the composite R plasmid NR1. J. Bact. 127, 619-636 (1976)
Communicated by W. Arber Received June 14~August 10, 1977
