Gene, 3 (1978) 87--95
87
© Elsevier/North-Holland Biomedical Press Amsterdam -- Printed in The Netherlands
MAPPING OF FUNCTIONS IN THE R-PLASMID R388 BY EXAMINATION OF DELETION MUTANTS GENERATED IN VITRO
(E. Coli; TnA (Tn801) insertions; BamHI and BglII; localization of genes)
JOHN M. WARD and JOHN GRINSTED
Department of Bacteriology, Medical School, University of Bristol, University Walk, BS8 1TD, Bristol (Great Britain) (Received September 12th, 1977) (Accepted December 20th, 1977)
SUMMARY
Mutant plasmids in which large segments of R388 DNA are deleted were constructed in vitro from two R388: :TnA (Tn801) plasmids, using the BamHI site of TnA and the BamHI and BglII sites of R388. These deletion mutants permitted mapping of genetic functions into the restriction map of R388. INTRODUCTION
Mapping of function in the R-plasmid R388 by examination of deletion mutants generated in vitro R388 is a plasmid of the W incompatibility group which codes for resistance to trimethoprim and mlphonamides (Datta and Hedges, 1972) and has a molecular weight of approximately 22 million daltons (Bennett and Richmond, 1976). The plasmid has been used as a recipient for transposon TnA (Bennett and Richmond, 1976) and for a study of certain aspects of the mechanism of transposition (Robinson et al., 1977). This paper describes the generation of deletions in two R388: :TnA derivatives using the endonucleases BamHI (Roberts et ai., 1977, Wilson and Young, 1975)and BglII (Pirotta, 1976). The insertion of TnA generates an extra BamHI site (Rubens et al., 1976) which can be used, along with the three BamHI sites and two BglII sites in R388 (this paper), for construction, in vitro, of reduced derivatives of R388. This method is analogous to that used by Figurski et al. (1976), who used the HindIII sites of phage Mu to generate deletions in several Mu insertion-derivatives of the broad host range plasmid RK2. MATERIALS AND METHODS
(a) Bacterial strains and plasmids. Table I shows the strains and the plasmids u s e J
83
(b) Plasmid DNA preparation. Plasmid DNA was prepared as previously described (Cornelis et al., 1976), and stored in 10 mM Tris -- HCI (pH 7.5) at 4°C. (c) Enzymes. HindHI :~nd T4 ~ a s e were p u r c h ~ from Miles Biochemicals. All other restriction endonucleases were prepared from the appropriate strains by standard procedures. (d) Generation o f deletions. Plasmid DNA (approximately 5/Jg) was digested with a mixture of BamHI and BglII in 50 mM Tris -- HCI (pH 7.5), 50 mM NaCI, 5 mM MgCI2 (total volume 300/~1). Th~ mixture was then heated for 5 rain at 65°C and dialysed against 100 mM NaCI, 100 mM T r i s HCI (pH 7). One tenth volume of 0.66 M T r i s - HCI (pH 7.5), 0.01 M EDTA, 0.1 M MgCI2, I mg/ml B.S.A., 0.1 M dithiothreitol, 0.001 M ATP was then added along with 1/zl of T4 ligase and the mixture incubated at 4 ° C for 24 h, followed by 0° C for 4 days. The reaction was terminated by heating at 65°C for 5 min and the mixture then dialysed overnight against 20 mM T r i s - HCI (pH 8), 1ram EDTA, 20 mM NaCI. (e) Transformation and selection. Transformation of E. coU C600 (see Table I) was essentially as described by Cohen et al. (1972). Transformants were selected on minimal agar containing the appropriate supplements with either trimethoprim (50/~g/ml), carbenicillin (400 #g/ml) or both antibiotics. TABLE I BACTERIAL STRAINS AND PLASMIDS USED Bacteria and Plasmids
Phenotypic markers
Ref.
Bacteria E. coil C600 E. coli UB1832
Thr- Leu- ThiHis- Lys- Tryp- Strep r rifr
Achtman et ai. (1971) Rifampiein-resistant derivative of JC3272 Achtman et al. (1971)
Plasmids R388 360/1(e) 351/3 (e)
Tpr a Su r b Tra÷ d Tpr Sur Apr e Tra' Tp r Sur Ap r Tra÷
Datta and Hedges (1972) This paper e This paper
Tp r, trimethoprim-resistant. u r, suiphonamide-resistant. Ap r, ampicillin/carbenieillin'resistant" Tra+, ability to mediate self transfer. eThe plasmid~ 360/1 and 351/3 were constructed by Dr. P.M. Bennett using the TnA from RPI. This TnA unit has now been designated Tn801.
(f) Mating. Equal vviu~, es of exponentially growing donor and recipient cells were mixed and incubated statically at 37 ° C for 30 min. E. coli UB3272 (see Table I) was used as recipient and exconjugants were selected on minimal agar plates with appropriate supplements plus streptomycin sulphate
89
(20 #g/ml) and trimethoprim (30 #g/ml) or carbenicfllin (500 #g/ml). The number of donor cells added was calculated by plating dilutions of the donor culture on nutrient agar plates containing carbenicfllin (500 #g/ml) or trimethoprim (30 #g/ml). (g) Sulphonamide resistance. Mueller-Hinton agar (Difco Manual of Dehydrated Culture Media and Reagents, 9th ed.) supplemented with lysed horseblood (4%) and sulphadimidine (500/~g/ml) was used and resistance tested by streaking for single colonies. 9 (h) DNA digestion for restriction enzyme mapping. Digestions.were carfled out in 100 mM T r i s - HCI pH 7.5 (at 37 ° C) 10 mM MgCl2 for I to 16 h. Usually, 40 #l of DNA solution was used. Reactions were terminated by the addition of 10 #l of a solution of sucrose (40%), bromophenol blue (0.15 rag/ ml) in 0.1 M (Nab EDTA (pH 8). Agarose slab gels with a Tris --borate buffer system containing 0.5 #g/ml of ethidium bromide have been described (Petrocheflou et al., 1976). Agarose concentrations of 0.7% to 2% were used. Lambda C I 857 int-2 DNA digested with HindIII (Murray and Murray, 1975) EcoRI (Thomas and Davis, 1975) or HindIII plus EcoRI (Murray and Murray, 1975; J. Grinsted, unpublished) were used as molecular weight markers. (i) Recovery o f fragments from agarose gels. Bands were cut from gels with a scalpel and subjected to two cycles of freezing (at --18 c C overnight) and thawing. The gel slice was then placed on a prewet 0.45 #m filter (Oxoid) in the bottom of a 2 ml syringe barrel. By placing this inside a test tube and spinning at top speed on a bench centrifuge for 5 min, the agatose was spread thinly over the surface of the filter while the DNA band was collected in the test tube. The enzyme BglI (Pirotta, 1976) is active in the eluate from a T r i s - borate ethidium bromide buffer provided 100 mM Tris -- HCI (pH 7.5), 10 mM MgCI2 is added (EeoRI will not work in this combined buffer). RESULTS AND DISCUSSION
The plasmids 351/3 and 360/1 have a molecular weight of 25 million daltons (P.M. Bennett, personal communication) and were constructed by transposition of TnA from RPI to R388 (Bennett and Richmond, 1976). A restriction enzyme map of R388 and the position and orientation of TnA in the two derivatives, are shown in Fig. 1. Introduction of TnA results in the acquisition of another BamHI site (Rubens et al., 1976) and three new PstI sites (J. Grinsted and Chung Lit Choi, unpublished) as shown in Fig. lb. The plasmids 360/1 and 351/3 were digested with a mixture of BamHI and BglII. The digests were incubated with T4 ligase and used to transform E. coil C600. Transformants fell into 4 phenotypic classes; an example of 3 of the classes was chosen for further study (see Table II). The 4th class, with the phenoWpe tra÷ Apr Tp r Su s, was the only representative of this class out of all the clones isolated. Whey studied it was clear that this was not a simple addition to or substraction from R360/1, but a more complex rearrangement of sites which did not permit definitive restriction analysis.
90
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E 0~21.5 0121.5 Fig. 1. Restriction endonuclease map of R388, 36011 and 351/3. The maps are from unpublished data of J. Grinsted, J. Ward and Chung Lit Choi. The letters A, B, C, D etc., refer to fragments produced in a double digest using BamHI plus BglH and are lettered from the largest fragment (A) to the smallest (F). The enzymes Sinai and E¢oRH have no effect on R388. In Fig. la all sizes are in Megadaltons. The Single EcoRI site is taken as the zero point. In Fig. lb and lc the BamHI and BglII sites indicated are given values corresponding to distance from the single E¢oRI site as in Fig. la.
The enzymes BamHI and BglII p r o d u c e identical "sticky ends", so fragments produced b y either e n z y m e can be ligated together. A hybrid BamHI/ BglII site is resistant to the action o f either e n z y m e (Roberts, 1976). Plasmid p U B 5 5 0 2 (see Table II) confers resistance to t r i m e t h o p r i m only, it has a molecular weight of 4.7 million daltons as determined by electron microscopy and agarose gel electrophoresis. This size suggests t h a t it is comprised of fragments C and D ,of 351/3 (see Fig. l c ) , F r a g m e n t C is excised by BamHI at one end and BglH at t h e o t h e r end; fragment D is excised b y BamHI at each end. So, joining and circularization of C and D will produce a molecule with one BamHI site a n d no BglII site. The fragm ent patterns obtained with pUB5502 are shown i n Fig. 2, There is indeed a single BamHI site and
91
T A B L E II P R O P E R T I E S O F PLASMIDS F O U N D A F T E R T R A N S F O R M A T I O N Plasmid
Ap r
Tp r
Su r
Transfer ability a
Mol. wt. • 10 TM b
pUB5501 pUB5502 pUB5520
+ 0 +
0 + 0
0 0 0
÷ 0 +
14.9 4.7 22.1
Abbreviations as in legend t o Table I. aTransfer ability was carried o u t as in M A T E R I A L S AND METHODS ~:f). bSize m e a s u r e m e n t s were d e t e r m i n e d by electron microscope analysis and are a personal c o m m u n i c a t i o n from Dr. P.M. Bennett. pUB5501 and pUB5502 are derived from 3 5 1 / 3 ; p U B 5 5 2 0 is derived from 360/1.
no BglII site. There is only a single EcoRI site which is indicative of fragment D of 351/3 (or fragment C of R388 see Figs. l a and lc), and the distance between the BamHI site and the EeoRI site (1.1 million) shows that fragments C and D of 351/3 have not been inverted. Confirmation that this plasmid is comprised of fragments C and D is provided with the BglI patterns obtained from pUB5502 and isolated C and D fragments of 360/1. Fragment C, of 360/1, contains 3 BglI sites, fragment D, of 360/1, contains one (see Fig. 2). Two of the BglI fragments from within fragment C (0.78 and 0.42 million) are contained within pUB5502; furthermore, a double digest of pUB5502 with BglI and BamHI yields another of the fragments seen in the BglI digest of isolated C fragment from 360/1. The remaining BglI fragments and the effect of EeoRI on one of these fragments are all consistent with pUB5502 simply being that part of R388 covered by fragments B and C in Fig. la (or fragments C and D of Figs. l b and lc). Several plasmids identical to pUB5502 were obtained from 360/1 in these experiments. Clearly, the DNA coding for trimethoprim resistance must be in this region of R388; regions of DNA required for plasmid maintenance must also be here. Plasmid pUB5520 (see Table II) confers resistance to carbenicillin, is selftransmissible and has a molecular weight of 22.1 million daltons from electron microscopy. Clearly, the capacity to code for carbenicillin resistance indicates that at least part of TnA is still present, pUB5520 does not contain an EcoRI site or a HindIII site (data not shown). This indicates that fragments D and E of Fig. l b are not present. Because of the small size of fragment F in 360/1 we cannot rule out the possibility that pUB5520 contains this small fragment; but the size, as determined from electron microscopy, would tend to rule this out. The PstI fragments (Fig. 2) are those expected to arise from within TnA (J. Grinsted and Chung Lit Choi, unpublished). Clearly pUB5520 contains fragments A,B and C of 360/1 (Fig. lb) and has lost fragments D,E and probably F. Since pUB5520 is sulphonamide-sensitive and trimethoprimsensitive the regions coding for these resistances must be contained within
92
pUB5502
c f~
d~
"1 ( p ss2o
Js, so,
5-0
-]
.~. 102
. . B
BBX P
_,
E BE B[ BE; E l l BI
/ R1r 8
BX ~BZ P BP B
BI[ EB][ P
BP
Enzymes used (consuit legend}
Fig. 2. SummmT of fragmentation patterns obtained by treatment of reduced plasmids with restriction enzymes. Plasmid DNA was digested with enzymes as described in MATERIALS AND METHODS (h). Abbreviations: B, BamHI, BI, BglI, BH, BSIH, E, EcoRI, P, PstI. 360/1 C and D fragments are lettered as in Fig. l b and were excised from a gel according to MATERL~LS AND METHODS (i).
fragments D,E and F or span the BamHI sites at each end of these fragments. Plasmid pUB5501 confers resistance to cazbenicfllin, is self transmissible and has a molecular weight of 14.9 million (see Table II). Some or all of TnA must be present to confer carbenicillin-resistance. There is no EcoRI site or HindIII site, indicating that fragments D and E of Fig. lc are not present. The size of pUB5501, together with the fragment patterns in Fig. 2 indicate that fragments A and C of 351/3 are present. The BamHI to PstI distance is 0.59 million, exactly that contained in TnA (J. Grinsted and Chung Lit Choi, unpublished). No other PstI sites are present indicating that only the short ann of TnA, created by the single off-centre BamHI cut, is present. This region is known to contain the ~-lactamase gene (Heffzon et al., 1977). pUB5501 contains fragments A and C of 351/3 and is trander-positive. The plasmid transfers at approximately the same fzequency as 351/3 (data not shown). This means that all of the necessary transfer genes, as well as the genes necessary for plasmid maintenance, are contained within this 14.9 million dalton plasmid.
The three reduced derivatives of R388 described in this paper permit localization of function onto the restriction map of R388. The regions contained within the reduced derivatives are shown in Fig. 3. pUB5502 is the only reduced plasmid to confer trimethopnm resistance on its host and is the only reduced plasmid to contain the R388 C fragment (Fig. la). This places the tfimethopfim resistance gene(s) within this fragment or spanning the BamHI s~te in pUB5502. Only 351/3, 360/1 and R388, Le, t h e complete parental molecules confer
93
°-
\
/
lnA
pUB55 2
puBss20
Fig. 3. Position of reduced plasmids on the map of R388 and localization of function. The map of R388 is oriented as in Fig. la, b, c. In all three reduced derivatives the ends indicated by bars in the figure are joined to each other to form CCC DNA in vivo. Abbreviations as in legend to Table I. rep, region(s) involved in plasmid maintenance and segregation. pUB5501 contains 0.9 million daltons of T n A DNA, this is indicated by the raised end of pUB5501.
sulphonamide resistance to their host. Since fragments D and E of R388 (E and F of 360/1 and 351/3 Figs. la,b and c) are the only fragments not represented in any of the reduced plasmids and none of the reduced plasmids carry sulphonamide resistance, the sulphonamide resistance gene(s) must be within fragments D and E of R388 (Fig. la) or span the ends of these fragments. When R388 is cut with BglII alone, and the digest transformed into C600 (manuscript in preparation), plasmids coding for Tp r and Su r are obtained. This confirms the above data on localization of the Su r determinant. All of the deleted plasmids contain the R388 B fragment (Fig. la); this 3.4 million BamHI to BglII fragment must therefore contain the genes necessaw for plasmid replication, maintenance and segregation as well as an origin of replication. Our data cannot rule out the possibility of R388 containing more than one origin of replication. The transfer region of R388 is contained within the plasmid pUB5501 which transfers at approximately the same frequency as its parent 351/3. R388 transfers between E. coU K12 strains at approximately 10- 2 per donor (data not shown) whereas 351/3, 360/1, pUB5501 and pUB5520 all transfer at approximately 10- 4 per donor cell, so the TnA is exerting an effect on transfer frequency in both R388: :TnA derivatives. Since fragment B of R388 contains the plasmid maintenance region, the A fragment of 351/3 (Fig, lc) must contain the transfer region of R388. The transfer genes could extend into the B fragment of R388 (Fig. la) such that integrity of the BglII
94
site is necessary for function. This transfer region is much smaller than the transfer region of F and F-like plasmids. (Helmuth and Achtman, 1975). Fig. 3 summarizes the above findings. There is a region at the left side of R388 (orientation as in Fig. la) of roughly 5 million daltons with no known function. The two r e s i s t a n c e - d e t e r m ~ regions are a d j a c ~ t to one another and contain all but one of the restriction enzyme sites for those enzymes used here. This clustering of sii:es associated with resistance determinants is also seen in RPI (Figurski et al., 1976; Barth and Grinter, 1977; Grinsted et al., 1977). The plasmid maintenance genes of R388 are adjacent to the two resistance determinants. ACKNOWLEDGEMENTS
This work was supported by a programme grant from the Medical Research Council to Professor Mark Richmond and by a Science Research Council Grant to John Ward for training in the methods of research. REFERENCES
Achtman, M., Willetts, N. and Clark, A.J., Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherich~ coli by isolation and characterization of transfer defective mutants J. Bacteriol., 106 (1971) 50-9--538. Barth, P.T. and Grinter, N.J., Map of plasmid RP4 derived by insertionof transposon C, J. MoI. Biol., 113 (1977) 445--474. Bennett, P.M. and Richmond, M.H., Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicon in Eschevfch~ coJi, J. Bacteriol., 126 (1976) 1--6. Cohen, S.N., Chang, A.C.Y. and Hsu, L., Non~hromosomal antibiotic resistance in bacteria, VII. Genetic transformation of EscherJch~z coU by R-factor DNA, Proc. Natl. Acad. Sei. USA, 69 (1972) 2110--2114. Cornelis, G., Bennett, P.M. and Grinsted, J., Properties of pGCI a/~c plasmid originating in Yersinia enterocolitica 842, J. Bacteriol., 127 (1976) 1058--1062. Datta, N. and Hedges, R.W., Trimethoprim resistance conferred by W plasmids in Enterobacteriucea, J. Gen. Microbiol., 72 (1972) 349--355. Figurski, D., Meyer, R., Miller, D.S,, and Helinski, D.R., Generation, in vitro, of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease, Gene, 1 (1976) 107--:[19. Grinsted, J., Bennett, P.M. and Richmond, M.H., A restriction enzyme map of the R-faetor RPI, Plasmid, (1977)in (press) Heffron, F., Bedinger, P,, Champoux, J.J. and Falkow, S,, Deletions affection the transposition of an antibiotic resistance gene, Proc. Natl, Acad. Sei, USA, 74 (1977) 702--706.
Helmuth, R, and Achtman, M., Operon structure of DNA transfer cistrons on the F sex factor, Nature, 257 (1975) 652--656, . . . . . Murray, K. and Murray, N.E., Phage lambda receptor chromosomes for DNAfragments made with restriction endonucle~ IIIofHaemophilus influenzae, J, MoLBiol., 98 (1975)551--564;i ~ • Petrocheilou, V.. G~nsted, J,: and Richmond,M.H., R - p l a s m i d t r ~ e r in vivo inthe absence of antibiotic selection p r e ~ r ~ , Antimicrob. Agents Chemother., 10 (19.76) 753--761.
95 Pirrotta, V., Two restriction endonucleases from Bacillus globiggi, Nucl. Acid. Rest, 3 (1976) 1747--1760. Roberts, R.J., Restriction endonucleases, Crit. Rev. Biochem., 4 (1976) 123--164. Roberts, R.J., Wilson, G. and Young, F., Recognition sequence of specific endonuclease BamHI from Bacillus amyloliquefaciens H, Nature, 265 (1977) 82--83. Robinson, M., Bennett, P.M. and Richmond, M.H., Inhibition of TnA translocation by TnA, J. Bacteriol., 129 (1977) 407--417. Rubens, C., Heffron, F. and Faikow, S., Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion, J. Bacteriol., 128 (1976) 425--434. Thonias, M. and Davies, R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91 (1975) 315--328. Wilson, G. and Young, F., Isolation of a sequence specific endonuclease from Bacillus amyloliquefaciens H, J. Mol. Biol., 97 (1975) 123. Communicated by D.R. Helinski.
87
© Elsevier/North-Holland Biomedical Press Amsterdam -- Printed in The Netherlands
MAPPING OF FUNCTIONS IN THE R-PLASMID R388 BY EXAMINATION OF DELETION MUTANTS GENERATED IN VITRO
(E. Coli; TnA (Tn801) insertions; BamHI and BglII; localization of genes)
JOHN M. WARD and JOHN GRINSTED
Department of Bacteriology, Medical School, University of Bristol, University Walk, BS8 1TD, Bristol (Great Britain) (Received September 12th, 1977) (Accepted December 20th, 1977)
SUMMARY
Mutant plasmids in which large segments of R388 DNA are deleted were constructed in vitro from two R388: :TnA (Tn801) plasmids, using the BamHI site of TnA and the BamHI and BglII sites of R388. These deletion mutants permitted mapping of genetic functions into the restriction map of R388. INTRODUCTION
Mapping of function in the R-plasmid R388 by examination of deletion mutants generated in vitro R388 is a plasmid of the W incompatibility group which codes for resistance to trimethoprim and mlphonamides (Datta and Hedges, 1972) and has a molecular weight of approximately 22 million daltons (Bennett and Richmond, 1976). The plasmid has been used as a recipient for transposon TnA (Bennett and Richmond, 1976) and for a study of certain aspects of the mechanism of transposition (Robinson et al., 1977). This paper describes the generation of deletions in two R388: :TnA derivatives using the endonucleases BamHI (Roberts et ai., 1977, Wilson and Young, 1975)and BglII (Pirotta, 1976). The insertion of TnA generates an extra BamHI site (Rubens et al., 1976) which can be used, along with the three BamHI sites and two BglII sites in R388 (this paper), for construction, in vitro, of reduced derivatives of R388. This method is analogous to that used by Figurski et al. (1976), who used the HindIII sites of phage Mu to generate deletions in several Mu insertion-derivatives of the broad host range plasmid RK2. MATERIALS AND METHODS
(a) Bacterial strains and plasmids. Table I shows the strains and the plasmids u s e J
83
(b) Plasmid DNA preparation. Plasmid DNA was prepared as previously described (Cornelis et al., 1976), and stored in 10 mM Tris -- HCI (pH 7.5) at 4°C. (c) Enzymes. HindHI :~nd T4 ~ a s e were p u r c h ~ from Miles Biochemicals. All other restriction endonucleases were prepared from the appropriate strains by standard procedures. (d) Generation o f deletions. Plasmid DNA (approximately 5/Jg) was digested with a mixture of BamHI and BglII in 50 mM Tris -- HCI (pH 7.5), 50 mM NaCI, 5 mM MgCI2 (total volume 300/~1). Th~ mixture was then heated for 5 rain at 65°C and dialysed against 100 mM NaCI, 100 mM T r i s HCI (pH 7). One tenth volume of 0.66 M T r i s - HCI (pH 7.5), 0.01 M EDTA, 0.1 M MgCI2, I mg/ml B.S.A., 0.1 M dithiothreitol, 0.001 M ATP was then added along with 1/zl of T4 ligase and the mixture incubated at 4 ° C for 24 h, followed by 0° C for 4 days. The reaction was terminated by heating at 65°C for 5 min and the mixture then dialysed overnight against 20 mM T r i s - HCI (pH 8), 1ram EDTA, 20 mM NaCI. (e) Transformation and selection. Transformation of E. coU C600 (see Table I) was essentially as described by Cohen et al. (1972). Transformants were selected on minimal agar containing the appropriate supplements with either trimethoprim (50/~g/ml), carbenicillin (400 #g/ml) or both antibiotics. TABLE I BACTERIAL STRAINS AND PLASMIDS USED Bacteria and Plasmids
Phenotypic markers
Ref.
Bacteria E. coil C600 E. coli UB1832
Thr- Leu- ThiHis- Lys- Tryp- Strep r rifr
Achtman et ai. (1971) Rifampiein-resistant derivative of JC3272 Achtman et al. (1971)
Plasmids R388 360/1(e) 351/3 (e)
Tpr a Su r b Tra÷ d Tpr Sur Apr e Tra' Tp r Sur Ap r Tra÷
Datta and Hedges (1972) This paper e This paper
Tp r, trimethoprim-resistant. u r, suiphonamide-resistant. Ap r, ampicillin/carbenieillin'resistant" Tra+, ability to mediate self transfer. eThe plasmid~ 360/1 and 351/3 were constructed by Dr. P.M. Bennett using the TnA from RPI. This TnA unit has now been designated Tn801.
(f) Mating. Equal vviu~, es of exponentially growing donor and recipient cells were mixed and incubated statically at 37 ° C for 30 min. E. coli UB3272 (see Table I) was used as recipient and exconjugants were selected on minimal agar plates with appropriate supplements plus streptomycin sulphate
89
(20 #g/ml) and trimethoprim (30 #g/ml) or carbenicfllin (500 #g/ml). The number of donor cells added was calculated by plating dilutions of the donor culture on nutrient agar plates containing carbenicfllin (500 #g/ml) or trimethoprim (30 #g/ml). (g) Sulphonamide resistance. Mueller-Hinton agar (Difco Manual of Dehydrated Culture Media and Reagents, 9th ed.) supplemented with lysed horseblood (4%) and sulphadimidine (500/~g/ml) was used and resistance tested by streaking for single colonies. 9 (h) DNA digestion for restriction enzyme mapping. Digestions.were carfled out in 100 mM T r i s - HCI pH 7.5 (at 37 ° C) 10 mM MgCl2 for I to 16 h. Usually, 40 #l of DNA solution was used. Reactions were terminated by the addition of 10 #l of a solution of sucrose (40%), bromophenol blue (0.15 rag/ ml) in 0.1 M (Nab EDTA (pH 8). Agarose slab gels with a Tris --borate buffer system containing 0.5 #g/ml of ethidium bromide have been described (Petrocheflou et al., 1976). Agarose concentrations of 0.7% to 2% were used. Lambda C I 857 int-2 DNA digested with HindIII (Murray and Murray, 1975) EcoRI (Thomas and Davis, 1975) or HindIII plus EcoRI (Murray and Murray, 1975; J. Grinsted, unpublished) were used as molecular weight markers. (i) Recovery o f fragments from agarose gels. Bands were cut from gels with a scalpel and subjected to two cycles of freezing (at --18 c C overnight) and thawing. The gel slice was then placed on a prewet 0.45 #m filter (Oxoid) in the bottom of a 2 ml syringe barrel. By placing this inside a test tube and spinning at top speed on a bench centrifuge for 5 min, the agatose was spread thinly over the surface of the filter while the DNA band was collected in the test tube. The enzyme BglI (Pirotta, 1976) is active in the eluate from a T r i s - borate ethidium bromide buffer provided 100 mM Tris -- HCI (pH 7.5), 10 mM MgCI2 is added (EeoRI will not work in this combined buffer). RESULTS AND DISCUSSION
The plasmids 351/3 and 360/1 have a molecular weight of 25 million daltons (P.M. Bennett, personal communication) and were constructed by transposition of TnA from RPI to R388 (Bennett and Richmond, 1976). A restriction enzyme map of R388 and the position and orientation of TnA in the two derivatives, are shown in Fig. 1. Introduction of TnA results in the acquisition of another BamHI site (Rubens et al., 1976) and three new PstI sites (J. Grinsted and Chung Lit Choi, unpublished) as shown in Fig. lb. The plasmids 360/1 and 351/3 were digested with a mixture of BamHI and BglII. The digests were incubated with T4 ligase and used to transform E. coil C600. Transformants fell into 4 phenotypic classes; an example of 3 of the classes was chosen for further study (see Table II). The 4th class, with the phenoWpe tra÷ Apr Tp r Su s, was the only representative of this class out of all the clones isolated. Whey studied it was clear that this was not a simple addition to or substraction from R360/1, but a more complex rearrangement of sites which did not permit definitive restriction analysis.
90
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B]I18.g5
1.0 02 ";
E 0~21.5 0121.5 Fig. 1. Restriction endonuclease map of R388, 36011 and 351/3. The maps are from unpublished data of J. Grinsted, J. Ward and Chung Lit Choi. The letters A, B, C, D etc., refer to fragments produced in a double digest using BamHI plus BglH and are lettered from the largest fragment (A) to the smallest (F). The enzymes Sinai and E¢oRH have no effect on R388. In Fig. la all sizes are in Megadaltons. The Single EcoRI site is taken as the zero point. In Fig. lb and lc the BamHI and BglII sites indicated are given values corresponding to distance from the single E¢oRI site as in Fig. la.
The enzymes BamHI and BglII p r o d u c e identical "sticky ends", so fragments produced b y either e n z y m e can be ligated together. A hybrid BamHI/ BglII site is resistant to the action o f either e n z y m e (Roberts, 1976). Plasmid p U B 5 5 0 2 (see Table II) confers resistance to t r i m e t h o p r i m only, it has a molecular weight of 4.7 million daltons as determined by electron microscopy and agarose gel electrophoresis. This size suggests t h a t it is comprised of fragments C and D ,of 351/3 (see Fig. l c ) , F r a g m e n t C is excised by BamHI at one end and BglH at t h e o t h e r end; fragment D is excised b y BamHI at each end. So, joining and circularization of C and D will produce a molecule with one BamHI site a n d no BglII site. The fragm ent patterns obtained with pUB5502 are shown i n Fig. 2, There is indeed a single BamHI site and
91
T A B L E II P R O P E R T I E S O F PLASMIDS F O U N D A F T E R T R A N S F O R M A T I O N Plasmid
Ap r
Tp r
Su r
Transfer ability a
Mol. wt. • 10 TM b
pUB5501 pUB5502 pUB5520
+ 0 +
0 + 0
0 0 0
÷ 0 +
14.9 4.7 22.1
Abbreviations as in legend t o Table I. aTransfer ability was carried o u t as in M A T E R I A L S AND METHODS ~:f). bSize m e a s u r e m e n t s were d e t e r m i n e d by electron microscope analysis and are a personal c o m m u n i c a t i o n from Dr. P.M. Bennett. pUB5501 and pUB5502 are derived from 3 5 1 / 3 ; p U B 5 5 2 0 is derived from 360/1.
no BglII site. There is only a single EcoRI site which is indicative of fragment D of 351/3 (or fragment C of R388 see Figs. l a and lc), and the distance between the BamHI site and the EeoRI site (1.1 million) shows that fragments C and D of 351/3 have not been inverted. Confirmation that this plasmid is comprised of fragments C and D is provided with the BglI patterns obtained from pUB5502 and isolated C and D fragments of 360/1. Fragment C, of 360/1, contains 3 BglI sites, fragment D, of 360/1, contains one (see Fig. 2). Two of the BglI fragments from within fragment C (0.78 and 0.42 million) are contained within pUB5502; furthermore, a double digest of pUB5502 with BglI and BamHI yields another of the fragments seen in the BglI digest of isolated C fragment from 360/1. The remaining BglI fragments and the effect of EeoRI on one of these fragments are all consistent with pUB5502 simply being that part of R388 covered by fragments B and C in Fig. la (or fragments C and D of Figs. l b and lc). Several plasmids identical to pUB5502 were obtained from 360/1 in these experiments. Clearly, the DNA coding for trimethoprim resistance must be in this region of R388; regions of DNA required for plasmid maintenance must also be here. Plasmid pUB5520 (see Table II) confers resistance to carbenicillin, is selftransmissible and has a molecular weight of 22.1 million daltons from electron microscopy. Clearly, the capacity to code for carbenicillin resistance indicates that at least part of TnA is still present, pUB5520 does not contain an EcoRI site or a HindIII site (data not shown). This indicates that fragments D and E of Fig. l b are not present. Because of the small size of fragment F in 360/1 we cannot rule out the possibility that pUB5520 contains this small fragment; but the size, as determined from electron microscopy, would tend to rule this out. The PstI fragments (Fig. 2) are those expected to arise from within TnA (J. Grinsted and Chung Lit Choi, unpublished). Clearly pUB5520 contains fragments A,B and C of 360/1 (Fig. lb) and has lost fragments D,E and probably F. Since pUB5520 is sulphonamide-sensitive and trimethoprimsensitive the regions coding for these resistances must be contained within
92
pUB5502
c f~
d~
"1 ( p ss2o
Js, so,
5-0
-]
.~. 102
. . B
BBX P
_,
E BE B[ BE; E l l BI
/ R1r 8
BX ~BZ P BP B
BI[ EB][ P
BP
Enzymes used (consuit legend}
Fig. 2. SummmT of fragmentation patterns obtained by treatment of reduced plasmids with restriction enzymes. Plasmid DNA was digested with enzymes as described in MATERIALS AND METHODS (h). Abbreviations: B, BamHI, BI, BglI, BH, BSIH, E, EcoRI, P, PstI. 360/1 C and D fragments are lettered as in Fig. l b and were excised from a gel according to MATERL~LS AND METHODS (i).
fragments D,E and F or span the BamHI sites at each end of these fragments. Plasmid pUB5501 confers resistance to cazbenicfllin, is self transmissible and has a molecular weight of 14.9 million (see Table II). Some or all of TnA must be present to confer carbenicillin-resistance. There is no EcoRI site or HindIII site, indicating that fragments D and E of Fig. lc are not present. The size of pUB5501, together with the fragment patterns in Fig. 2 indicate that fragments A and C of 351/3 are present. The BamHI to PstI distance is 0.59 million, exactly that contained in TnA (J. Grinsted and Chung Lit Choi, unpublished). No other PstI sites are present indicating that only the short ann of TnA, created by the single off-centre BamHI cut, is present. This region is known to contain the ~-lactamase gene (Heffzon et al., 1977). pUB5501 contains fragments A and C of 351/3 and is trander-positive. The plasmid transfers at approximately the same fzequency as 351/3 (data not shown). This means that all of the necessary transfer genes, as well as the genes necessary for plasmid maintenance, are contained within this 14.9 million dalton plasmid.
The three reduced derivatives of R388 described in this paper permit localization of function onto the restriction map of R388. The regions contained within the reduced derivatives are shown in Fig. 3. pUB5502 is the only reduced plasmid to confer trimethopnm resistance on its host and is the only reduced plasmid to contain the R388 C fragment (Fig. la). This places the tfimethopfim resistance gene(s) within this fragment or spanning the BamHI s~te in pUB5502. Only 351/3, 360/1 and R388, Le, t h e complete parental molecules confer
93
°-
\
/
lnA
pUB55 2
puBss20
Fig. 3. Position of reduced plasmids on the map of R388 and localization of function. The map of R388 is oriented as in Fig. la, b, c. In all three reduced derivatives the ends indicated by bars in the figure are joined to each other to form CCC DNA in vivo. Abbreviations as in legend to Table I. rep, region(s) involved in plasmid maintenance and segregation. pUB5501 contains 0.9 million daltons of T n A DNA, this is indicated by the raised end of pUB5501.
sulphonamide resistance to their host. Since fragments D and E of R388 (E and F of 360/1 and 351/3 Figs. la,b and c) are the only fragments not represented in any of the reduced plasmids and none of the reduced plasmids carry sulphonamide resistance, the sulphonamide resistance gene(s) must be within fragments D and E of R388 (Fig. la) or span the ends of these fragments. When R388 is cut with BglII alone, and the digest transformed into C600 (manuscript in preparation), plasmids coding for Tp r and Su r are obtained. This confirms the above data on localization of the Su r determinant. All of the deleted plasmids contain the R388 B fragment (Fig. la); this 3.4 million BamHI to BglII fragment must therefore contain the genes necessaw for plasmid replication, maintenance and segregation as well as an origin of replication. Our data cannot rule out the possibility of R388 containing more than one origin of replication. The transfer region of R388 is contained within the plasmid pUB5501 which transfers at approximately the same frequency as its parent 351/3. R388 transfers between E. coU K12 strains at approximately 10- 2 per donor (data not shown) whereas 351/3, 360/1, pUB5501 and pUB5520 all transfer at approximately 10- 4 per donor cell, so the TnA is exerting an effect on transfer frequency in both R388: :TnA derivatives. Since fragment B of R388 contains the plasmid maintenance region, the A fragment of 351/3 (Fig, lc) must contain the transfer region of R388. The transfer genes could extend into the B fragment of R388 (Fig. la) such that integrity of the BglII
94
site is necessary for function. This transfer region is much smaller than the transfer region of F and F-like plasmids. (Helmuth and Achtman, 1975). Fig. 3 summarizes the above findings. There is a region at the left side of R388 (orientation as in Fig. la) of roughly 5 million daltons with no known function. The two r e s i s t a n c e - d e t e r m ~ regions are a d j a c ~ t to one another and contain all but one of the restriction enzyme sites for those enzymes used here. This clustering of sii:es associated with resistance determinants is also seen in RPI (Figurski et al., 1976; Barth and Grinter, 1977; Grinsted et al., 1977). The plasmid maintenance genes of R388 are adjacent to the two resistance determinants. ACKNOWLEDGEMENTS
This work was supported by a programme grant from the Medical Research Council to Professor Mark Richmond and by a Science Research Council Grant to John Ward for training in the methods of research. REFERENCES
Achtman, M., Willetts, N. and Clark, A.J., Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherich~ coli by isolation and characterization of transfer defective mutants J. Bacteriol., 106 (1971) 50-9--538. Barth, P.T. and Grinter, N.J., Map of plasmid RP4 derived by insertionof transposon C, J. MoI. Biol., 113 (1977) 445--474. Bennett, P.M. and Richmond, M.H., Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicon in Eschevfch~ coJi, J. Bacteriol., 126 (1976) 1--6. Cohen, S.N., Chang, A.C.Y. and Hsu, L., Non~hromosomal antibiotic resistance in bacteria, VII. Genetic transformation of EscherJch~z coU by R-factor DNA, Proc. Natl. Acad. Sei. USA, 69 (1972) 2110--2114. Cornelis, G., Bennett, P.M. and Grinsted, J., Properties of pGCI a/~c plasmid originating in Yersinia enterocolitica 842, J. Bacteriol., 127 (1976) 1058--1062. Datta, N. and Hedges, R.W., Trimethoprim resistance conferred by W plasmids in Enterobacteriucea, J. Gen. Microbiol., 72 (1972) 349--355. Figurski, D., Meyer, R., Miller, D.S,, and Helinski, D.R., Generation, in vitro, of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease, Gene, 1 (1976) 107--:[19. Grinsted, J., Bennett, P.M. and Richmond, M.H., A restriction enzyme map of the R-faetor RPI, Plasmid, (1977)in (press) Heffron, F., Bedinger, P,, Champoux, J.J. and Falkow, S,, Deletions affection the transposition of an antibiotic resistance gene, Proc. Natl, Acad. Sei, USA, 74 (1977) 702--706.
Helmuth, R, and Achtman, M., Operon structure of DNA transfer cistrons on the F sex factor, Nature, 257 (1975) 652--656, . . . . . Murray, K. and Murray, N.E., Phage lambda receptor chromosomes for DNAfragments made with restriction endonucle~ IIIofHaemophilus influenzae, J, MoLBiol., 98 (1975)551--564;i ~ • Petrocheilou, V.. G~nsted, J,: and Richmond,M.H., R - p l a s m i d t r ~ e r in vivo inthe absence of antibiotic selection p r e ~ r ~ , Antimicrob. Agents Chemother., 10 (19.76) 753--761.
95 Pirrotta, V., Two restriction endonucleases from Bacillus globiggi, Nucl. Acid. Rest, 3 (1976) 1747--1760. Roberts, R.J., Restriction endonucleases, Crit. Rev. Biochem., 4 (1976) 123--164. Roberts, R.J., Wilson, G. and Young, F., Recognition sequence of specific endonuclease BamHI from Bacillus amyloliquefaciens H, Nature, 265 (1977) 82--83. Robinson, M., Bennett, P.M. and Richmond, M.H., Inhibition of TnA translocation by TnA, J. Bacteriol., 129 (1977) 407--417. Rubens, C., Heffron, F. and Faikow, S., Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion, J. Bacteriol., 128 (1976) 425--434. Thonias, M. and Davies, R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91 (1975) 315--328. Wilson, G. and Young, F., Isolation of a sequence specific endonuclease from Bacillus amyloliquefaciens H, J. Mol. Biol., 97 (1975) 123. Communicated by D.R. Helinski.
