Molec. gen. Genet. 140, 7--14 (1975) © by Springer-Verlag 1975

Susceptibility of a Hybrid Plasmid to Excision of Genetic Material J. N. Coetzee Department of Microbiology, Institute of Pathology, University of Pretoria, Republic of South Africa A. E. Jacob and R. W. Hedges Department of Bacteriology, Royal Postgraduate Medical School, London Received May 12, 1975 Sum~ary. A 5 Megadalton segment of DNA carrying a gene for kanamycin resistance from R447 b (a plasmid of group N of molecular weight 33 Megadaltons) has been inserted into Plac (a plasmid of the A-C complex of molecular weight 101 Megadaltons) to produce the recombinant plasmid Plac-R447b (Coetzee, 1974). The recombinant plasmid is a typical member of the A-C complex except that entry of an N group plasmid into a Plac-R447b + recipient frequently leads to the loss of 5 Megadaltons of DNA (including the kanamycin resistance determinant) from the resident plasmid. In those transcipients from which kanamycin resistance is not eliminated, both plasmids are stably inherited.

Introduction

When a plasmid is transferred b y conjugation into a recipient strain carrying a compatible plasmid, the latter is usually unaffected. However, certain pairs of plasmids show interactions which have been termed dislodgement (Coetzee, D a t t a and Hedges, 1972). These interactions m a y lead to the elimination of the resident plasmid, loss of some but not all of the phenotypie characteristics determined b y that plasmid or recombination between the two plasmids. The first reported ease concerned I~394, a plasmid of group T (Coetzee et al., 1972) which showed peculiar interactions with plasmids of group N. For example, transfer of R394 into an t~390 + recipient produced some transeipients carrying I~394 only (elimination of R390), some transeipients carrying I~394 plus an I~390 which had lost the ability to confer tetracycline resistance (excision of genetic material from R390) and some transcipients carrying apparently normM R394 and R390. I n the latter two classes of transcipient, both plasmids were stably inherited and separately traltsmissible b y conjugation with 1~- recipients. These observations lead Coetzee et al. (1972) to conclude t h a t during (or immediately after) conjugal transfer, a gene of t~394 (repressed during vegetative growth) was derepressed and produced an enzyme (perhaps a nuclease involved in recircularization of the linear transferring R394 DNA molecule) which could attack N plasmids, leading to loss of all or part of any such structures. Since R394 was able to dislodge only plasmids of group N (and all three N group plasmids tested) the agent responsible for dislodgement (whether a nuclease or not) has sufficient specificity to be able to 'reeognise' N plasmids and act only upon these.

8

J.N. Coetzee et al.

A second example, of dislodgement was provided by R62 (Guerry, Falkow and Datta, 1974). This plasmid, a member of compatibility group Ice ,was shown to contain a substantial amount of DNA capable of hybridizing with plasmids of group N and it determines a number of genetic characters typical of plasmids of this group but not so far found on other plasmids of group Is. I~62 was, thus, the product of recombination between an I s plasmid and an I~ factor of group N and the genes uncharacteristic of an I s plasmid were interpreted as markers of the N group derived DNA. R62 was susceptible to dislodgement by N plasmids. A particularly striking observation was t h a t entry of the N plasmid frequently lead to the elimination of the marker genes for the N-derived DNA segment. Coetzee, Datta, Hedges and Appelbaum (1973) and Coetzee (1974) have described a transductional technique whereby recombinant plasmids carrying genetic material from two unrelated I~ factors can be constructed and in this paper we describe experiments to define the susceptibility to dislodgement of a plasmid of the A-C compatibility complex (Hedges, 1974; Hedges, 1975; N. D a t t a personal communication) carrying a segment of DNA from an R factor of group N. Materials and Methods Bacteria. Proteus mirabilis Pro13 (Coetzee and Sacks, 1960) Providencia sp P29 (~ NCTC9295) (Coetzee, Smit and Prozesky, 1966) Escherichia coli K12 J53 F-, pro, met (Bachmann, 1972) J53-2 F , pro, met, ri] n mutant of J53 J62 F-, pro, his, trp, lac (Bachmann, 1972) J62-2 F-, pro, his, trp, lac, ri/n mutant of J62 W677 F-, thr, leu, thi, lac (Baehmann, 1972) W677-1 F-, thr, leu, thi, lac, hal R mutant of W677 W3110Thy F-, thy (Baehmann, 1972) Plasmids. see Table 1 Techniques. Conjugal transfer and compatibility properties o/ plasmids were studied by the techniques of Datta, Hedges, Shaw, Sykes, and Richmond (1971), Coetzee, Datta and Hedges (1972) and Dcnnison (1972). Radiolabelling o/ R + strains and isolation o/ plasmid DNA by caesium chloride-ethidinm bromide density gradient centri]ugation o/cleared lysates were by the technique of Clewell and Helinski (1969). Neutral sucrose gradient analysis and calculation o] plasmid molecular weight were by the method of Barth and Grinter (1974). Results 1. Genetic Properties o / P l a s m i d Plac-R447b Plac-I:C447b is a plasmid derived, b y Coetzce (1974), from recombination between Plac, a plasmid of the A-C compatibility complex (Hedges, 1975) and the N plasmid R447b (Hedges, Datta, Coetzee and Dennison, 1973). I t s properties in Proteus mirabilis and Providencia have been described (Coetzee, 1974). When transferred to Escherichia coli K12 it behaved as a stable self-transmissible plasmid with the typical properties of a member of the A-C complex (Datta and Hedges, 1972; Hedges, 1974; Hedges, 1975) in both exclusion and compatibility properties in crosses in which it was the transferred plasmid (Table 2

Susceptibility of ~ Hybrid Piasmid to Excision of Genetic Material Table 1. Plasmids used in this work Designation

Resistance and other genetic determinants

Cornpatibility group

Reference

Plac

R447 b

Su, lac A, S, C, K, Su A, S, C, K, Su A, K

A-C FII FII N

N3 N3T R136

S, T, Su T T

~ N FII

Plac-tCldrd19 Plac-Rldrdl9-2

A, S, C, K, Su, lac A, S, C, Su K, Su, lac

A-C A-C A-C

Hedges (1975) Hedges and Datta (1972) Meynell and Datta (1967) Hedges, Datta, Coetzee and Dennison (1973) Datta and Hedges (1971) Hedges (1972) Lawn, Meynell, Meynell and Datta (1967) Coetzee (1974) and this paper This paper Coetzec (1974) and this paper

R1 l~ldrd19

P/ac-R447b

A ~ ampicillin; S ~ streptomycin; T ~ tetracycline; C ~ chloramphenicol; K = kanamycin; Sn = sulphonamides; lac ~ lactose fermentation. See Datta (1975) for the classification into compatibility groups.

lines 5-8). W h e n Plac-R447b was transferred into J62-2(N3T) no exclusion and no elimination of tetracycline resistance was observed (Table 2, line 8). W h e n Plac-Rldrdl9-2 (a plasmid of the A - C complex [Table 2, lines 1 and 2]) was transferred to a Plac-l~447b + strain the resident plasmid was. eliminated (Table 2, line 3). This confirms the conclusion t h a t Plac-R447b is a m e m b e r of the A - C complex. W h e n the N plasmid N3T was transferred into a Plac-R447 b+ strain two types of transcipient were observed (Table 3). I n one (type A), resistance to k a n a m y c i n has been lost; however, both other observable phenotypic properties of PlacR 4 4 7 b (and of the parental Plac plasmid) were retained. I n the other transcipients (type B) all the phenotypic properties of Plac-Rd47 b were retained. B o t h types of transcipient were shown to carry two plasmids, which were stably inherited and separately transmissible (Table 3). F r o m one transcipient on t y p e A, a plasmid conferring lactose fermentation ability and sulphonamide resistance (Plac-R447b-K s) was transferred to strain J53. A clone lacking N3T was isolated and used as P l a c - R 4 4 7 b - K s donor to strain W3110Thy. Similarly, Plac-R447b was transferred from a transcipient of t y p e B into J53 and thence into W3110Thy. 2. Molecular Properties o/ Plac, Plac-R447b and P l a c - R 4 4 7 b - K s

The molecular weight of Plac DNA, radiolabelled with laC, was determined b y comparing its sedimentation rate t h r o u g h a neutral sucrose gradient with 3H-labelled plasmid R1 D N A as reference (Fig. 1). Sedimentation was from right to left. Plac was a single plasmid species; the covalently closed circular and open circular D N A tertiary forms gave peaks at fractions 13 and 27 respectively.

10

J. N. Coetzee et al. Table 2. Transfer and compatibility properties of the recombinant plasmids

Donor

1 2 3 4 5 6 7 8

J53-2 J53-2 J53-2 J53-2 J53-2 J53-2 J53-2 J53-2

iPlac-Rldrdl9-2) Plac-Rldrdl9-2) Plac-Rldrdl9.2) Plac-Rldrdl9-2) Plac-R447 b) Plac-R447 b) P/ac-R447b) Plac-R447b)

Recipient

Selective medium a

Efficiency Properties of of transfer transcipients

J62-2 J62-2 (Plac) J62-2 (Plac.I~447b) J62-2 (1~136) J62-2 J62-2 (Plac) J62-2 (Plac-Rldrdl9-2) J62-2 (N3T)

pro, his, trp + C 3 × 10-4 pro, his, trp ~-C ~10 -c pro, his, trp -~ C ~ 10-6 pro, his, trp ~-C 2 × 10-4 pro, met + K 2 × 10-4 pro, met ~- K ~ 10-6 pro, m e t ~ - K ~ 1 0 -~ pro, met ~-K 2 × 10-4

10 of i0 lac-, C1~ 10 of 10 lac-, KSC1~ 8 TRcI~b : 2 TSc 1~

10 of 10 CSKRlac+ 20 of 20 TI~KRb

a Abbreviations as for Table 1 and pro, proline; met, methionine; his, histidine; trp, trypto-

phan. b From these strains, two R factors were separately transmissible.

Table 3. Transcipicnts produced by transferring N3T into a strain carrying Plac-R447b Recipient

Donor

Selective medium •

Transcipient Number Plasmid-borne Designamarkers a tion TlClac+Sul~KS Type A TI~lac+SuRKI¢ Type B

1 J53 (N3T)

J62-2 (Plac-R447b)

pro, his, trp ~-T

18 2

2 Type A transeipient of Mating 1

J53

pro, met ~- T

15 T]CSus 5 TRSu~ 20 of 20 TSSu1~

pro, met-~ Su 3 Type B transcipient of Mating 1

J53

pro, met ~- T pro, met ~- Su pro, met ~- K

17 3 19 1 17 3

TgKSSu s T~K~Su~ TSK~tSug TRKlCSu~ TSKt~Su1~ T~K~Su~

Abbreviations as in Tables 1 and 2.

R1 covalently closed circular a n d open circular D N A gave peaks at fractions 19 a n d 31. The open circular forms of b o t h plasmids were formed b y SpOntaneous b r e a k d o w n of covalently closed circles. Using the value of 60 Megadaltons (Mdal) for the molecular weight of plasmid 1~1 (Clowes, 1972 a n d P. T. Barth, u n p u b lished), Plac molecular weight was calculated to be 100.8 Mdal. Plasmids Plac-R447b a n d P l a c - R 4 4 7 b - K s were also shown to be single plasmid species. W i t h Plac D N A as a s e d i m e n t a t i o n reference, their molecular weights were estimated as 105.5 Mdal a n d 100.8 Mdal respectively. A direct comparison of the s e d i m e n t a t i o n rate of t h e two plasmids is shown i n Fig. 2. Covalently closed circular, ~I-I-labelled, Plac-g447 b D N A sedimented 1 fraction further t h a n 14C-labelled, covalently closed circular P l a c - R 4 4 7 b - K s D N A (at fraction 13).

Susceptibility of a Hybrid Plasmid to Excision of Genetic Material

11

5

i 32

3

1 1

10

i0

io

Fractionnumber

4o

Fig. 1. Neutral sucrose gradient analysis of plasmid Plac DNA. 14C-labelled Plac DNA was mixed with all-labelled R1 DNA and sedimented through a 5 to 20% sucrose gradient at 100000 × g and 20° C, for 75 min. Fractions (0.1 ml) were collected directly onto glass-fibre discs and, after drying, washing and re-drying, were assayed for radioactivity. ,, all; ©, 1~C

7 51

~

6

3

~ ,4, 3

2

lj

2 1 lo

3o

Fractionnumber

io

Fig. 2. Comparison of the sedimentation of Plac-R447b DNA and Plac-R447b-K s DNA through a neutral sucrose gradient, atI-labelled Plac-R447b DNA was mixed with 14C-labelled Plac-R447b-K s DNA and analysed as described in the legend to Fig. 1 . . , ~H; ©, 1~C

Assuming the molecular weight of 100.8 Mdal for P l a c - R 4 4 7 b - K s, P l a c - R 4 4 7 b was calculated as 105.6 Mdal. W e have also d e t e r m i n e d t h e molecular weight of plasmid R 4 4 7 b to be 32.9 Mdal. Thus, the simplest i n t e r p r e t a t i o n of the d a t a is t h a t P l a c - R 4 4 7 b has been formed b y r e c o m b i n a t i o n between Plac a n d a D N A sequence of molecular weight 4.8 Mdal from plasmid R447 b. W e would like to suggest t h a t this sequence is dislodged b y plasmid N 3 T to form plasmid P l a c - R 4 4 7 b - K s, which is indistinguishable from Plac.

12

J. N: Coetzee et al.

3. Genetic Properties o/the Kanamycin Resistance Region o/Plac-R447b Our interpretation, of the above data suggests that when N3T enters a strain carrying Plac-R447b the kanamycin resistance region is usually excised; occasionally the resident plasmid is apparently unaffected. This could be a random event : if the incoming N3T produces only a small number of molecules of the dislodgement effector, there must be some probability that none of these will reach the resident plasmid. Alternatively, the undamaged Plac-R447b might be abnormally insusceptible to the dislodgement effector. This could be the result of spontaneous mutation of the plasmid whose effect is made manifest by the failure of N3T to excise the kanamycin region, or an alteration brought about by N3T, for example, specific excision of the target(s) for the dislodgement effector. We have distinguished between these possibilities, since a Plac.R447 b plusmid from a type B transcipient proved to be fully as susceptible to loss of kanamycin resistance as was the original Plac-R447b. The survival of intact hybrid plasmid therefore seems to be a matter of chance and not to reflect any permanent alteration. The structural characteristics of the kanamycin resistance region of PlacR447b (a piece of 'foreign' DNA carrying a resistance determinant inserted into the continuity of a plasmid) resemble those of an inserted transposon (Hedges and Jacob, 1974). We therefore constructed J62-2 derivatives carrying either R64drdll or R138drd6 and Plac-R447b, and incubated these with W677-1. The mixed cultures were plated on MacConkey agar plates containing kanamycin and nalidixic acid. All transcipients, more than 10~ from each mating, were lactose fermenting. We had, therefore, failed to transpose the kanamycin resistance region from Plac-R447 b onto either plasmid tested. A number of other experiments (not described here) also failed to show any evidence for transposibility. Discussion

The loss of the kanamycin resistance determinant of Plac-R447b under the influence of an incoming plasmid of group N has been shown to be associated with excision of a piece of DNA, equal (to the limits of accuracy of our techniques) to the size of the piece of DNA inserted during the construction of the hybrid plasmid. Since this loss is accompanied by the elimination of the only recognisable N-derived marker and the Plac-R447 b-K s plasmid is indistinguishable from the parental Plac, we believe that the DNA excised is primarily (and perhaps exclusively) the N-derived material. We therefore conclude that upon entering a newly infected cell a plasmid of group N produces an enzyme which directly or indirectly acts as a deoxyribonuclease recognising N plasmid DNA exclusively. Possibly this enzyme acts only upon circular DNA; thus the incoming linear plasmid would not be attacked by its own enzyme. Once the incoming plasmid has established itself, production of the nuclease would be repressed. Production of such a nuclease could have selective advantage for the producing plasmid in several ways.

Susceptibility of a Hybrid Plasmid to Excision of Genetic Material

13

i. I t could be a component of the DNA metabolising processes required for conjugal transfer. ii. If an N plasmid is transferred into a host already carrying an incompatible plasmid, there will be competition between these for ability to reproduce in step with the host cell. I t will be advantagous if the incoming plasmid can physically inactivate the resident. iii. Since two incompatible plasmids cannot coexist stably, there is little opportunity for genetic exchange. The only chance for recombination will be in unstable doubles produced when a plasmid is transferred into a host carrying a related resident plasmid. I t is plausible t h a t highly efficient recombination mechanisms should evolve to exploit these transitory opportunities and the N-specific nuclease m a y be part of such a system. We, thus, propose that dislodgement phenomena are side effects of a system whose evolutionary significance concerns interactions between N plasmids which in our experiments would be almost indistinguishable from incompatibility interactions. Where however, a plasmid of some other compatibility group has a DNA region derived from a N plasmid, the hybrid plasmid is susceptible to the N-specific nuclease. A hybrid plasmid, attacked by this nuclease, m~y suffer loss of the N DNA, m a y be irreversibly inactivated (perhaps, especially likely if the N DNA is near to an essential gene), m a y undergo recombination with the incoming plasmid, or m a y undergo repair. The identity of the nuclease responsible is unknown. Many N plasmids determine a characteristic restriction-modification specificity: hsplI (Hedges, 1972) but the restriction nuclease is not a plausible candidate since at least one hspII(non-restricting) N plasmid, I~447b, is effective in dislodgement (Guerry et al., 1974). J . T . Smith and colleagues have investigated the survival of the N group plasmid R1818 ( z R46) in thy- hosts. They found efficient curing of the plasmid b y thymine starvation and concluded that R46 determines a nuclease inducible by thymine starvation which preferentially degrades R46 DNA and is responsible for elimination of the plasmid under these conditions (Pinney and Smith, 1972; Tweats, Pinney and Smith, 1974 : Pinney, Bremer and Smith, 1974). The fact that this enzyme is not produced during normal growth makes this a possible dislodgemerit effector.

Acknowledgements. We thank Mrs. Janet Cresswell for help with the plasmid DNA isolation. A. E. Jacob was supported by a grant from the Medical Research Council of the United Kingdom. J. N. Coetzee is in receipt of grants from the South African Medical Research Council. References Bachmann, B. J. : Pedigrees of some mutant strains of Escherichia cell 1(12. Bact. Rev. 36, 525-557 (1972) Barth, P.T., Grinter, N. J. : Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulphonamides. J. Bact. 120, 618-630 (1974) Clewell, D. B., Helinski, D. R. : Supercoiled circular DNA-protein complex in Escherichia cell: purification and induced conversion to an open circular DNA form. Prec. nat. Acad. Sci. (Wash.) 62, 1159-1166 (1969)

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J . N . Coetzee et al.

Clowes, R. C.: Molecular structure of bacterial plasmids. Bact. Rev. 36, 361405 (1972) Coetzee, J. N. : Properties of Proteus and Providence strains harbouring recombinant plasmids between Plac, Rldrdl9 or 1%447b. J. gen. Microbiol. 80, 119-130 (1974) Coetzee, J. N., Datta, N., Hedges, R. W. : R factors from Proteus rettgeri. J. gen. Microbiol. 72, 543-552 (1972) Coetzee, J . N . , Datta, N., Hedges, R. W., Appelbaum, P. C. : Transduction of R factors in Proteus ~airabilis and P. rettgeri. J. gen. Microbiol. 76, 355 368 (1973) Coctzee, J . N . , Sacks, T. G. : Transduction of streptomycin resistance in Proteus mirabilis. J. gen. Microbiol. 23, 445-455 (1960) Coetzee, J. N., Smit, J. A., Prozesky, 0. W. : Properties of Providence and Proteus morganii transducing phages. J. gen. Microbiol. 44, 167-176 (1966) Datta, N.: Epidemiology and classification of plasmids. In: Microbiology--1974 (American Society for Microbiology), ed. D. Schlessinger, p. 8-17 (1975) Datta, N., Hedges, R. W. : Compatibility groups among /i- R factors. Nature (Lond.) 234, 222-223 (1971) Datta, N., Hedges, R. W. : R factors identified in Paris, some conferring gentamicin resistance, constitute a new compatibility group. Ann. Inst. Pasteur. 123, 879-883 (1972) Datta, N., Hedges, R. W., Shaw, E. J., Sykes, 1%. P., Richmond, M. H. : Properties of an 1% factor from Pseudomonas aeruginosa. J. Bact. 108, 1244-1249 (1971) Dennison, S. : Naturally occurring R factor, dcrepressed for Pilus Synthesis, belonging to the same compatibility group as the sex factor F of Escheriehia coli K12. J. Bact. 109, 416422 (1972) Guerry, P., Falkow, S., Datta, N. : R62, a naturally-occurring hybrid plasmid. J. Bact. 119, 144-151 (1974) Hedges , 1%. W. : Phenotypic characterization of ]i- 1%factors determining the restriction and modification hsplI specificity. Molec. gem Genet. llS, 225-233 (1972) Hedges, R . W . : R factors from Providence. J. gen. Microbiol. 81, 171-181 (1974) Hedges, R. W. : R factors from swarming Proteus strains. J. gen. Microbiol. (in press) 1975 Hedges, R. W., Datta, N. : R124, an fi + R factor of a new compatibility class. J. gen. Microbiol. 71, 403~/:05 (1972) Hedges, R. W., Jacob, A. E. : Transposition of ampicillin resistance from RP4 to other replieons. Molec. gen. Genet. 132, 31-40 (1974) Lawn, A. M., Meynell, E., Meynell, G, G, Datta, N. : Sex pili and the classification of sex factors in the Enterobacteriaceae. Nature (Lond.) 216, 343-346 (1967) Meynell, E., Datta, N. : Mutant drug resistant factors of high transmissibility. Nature (Lond.) 214, 885-887 (1967) Pinney, 1%. J., Bremer, K., Smith, J. T. : R factor elimination by inhibitors of thymidylate synthetase (fluorodeoxyuridine and showdomycin) and the occurrence of single strand breaks in plasmid DNA. Molec. gen. Genet. 133, 163-174 (1974) Pinney, 1%. J., Smith, J. T. : R factor elimination during thymine starvation: effects of inhibition of protein synthesis and readdition of thymine. J. Bact. l l l , 361-367 (1972) Tweats, D. J., Pinney, R. J., Smith, J. T. : R factor-mediated nuclease activity involved in thymineless elimination. J. Baet. 118, 790-795 (1974) Communicated by W. Arber Dr. J. N. Coetzee Department of Microbiology Institute of Pathology University of Prctoria Pretoria Republic of South Africa

Dr. A. E. Jacob Dr. R. W. Hedges Department of Bacteriology Royal Postgraduate Medical School Du Cane Road London W12 OHS England