Integrative Compatibility: Stable Coexistence of Chromosomally Integrated and Autonomous Derivatives of Plasmid RP4

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1 JouRNAL OF BACTFRIOLOGY, May 1980, p /80/ /05$02.00/0 Vol. 142, No. 2 Integrative Compatibility: Stable Coexistence of Chromosomally Integrated and Autonomous Derivatives of Plasmid RP4 MARTIN D. WATSONt* AND J. G. SCAIFE Department ofmolecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland P group plasmid RP4Aatt has a novel feature. Its incompatibility function is phenotypically switched off when it integrates into the bacterial chromosome. Related plasmids are usually unable to coexist in the bacterial cell. This incompatibility is a basic criterion for relatedness between plasmids (2) and probably arises from the operation of the replication control mechanism. The plasmid F expresses incompatibility whether or not it is integrated in the chromosome (8). We show here that this is not always so. Plasmid RP4 is unable to mobilize the Escherichia coli chromosome, but this can be remedied by providing a means for efficient integration of the plasmid into the chromosome. For this purpose we used a derivative of RP4 containing the att region of phage A (sria2-3) inserted at the single EcoRI site of the vector (12; R. Pastrana, Ph.D. thesis, University of Edinburgh, Scotland, 1976). The derivative, RP4Xatt, forms Hfr's, which transfer the chromosome with a counterclockwise polarity from the A attachment site. An Hfr of this kind has been studied in detail (12). We show here that, unlike its autonomous progenitor, the integrated plasmid has become compatible with other P group plasmids; we call this new property integrative compatibility. We describe the recovery of the integrated plasmid by A-promoted site-specific recombination (4). The excised plasmid regains its incompatibility, confirming that integrative compatibility is a fundamental property of RP4. MATERILS AND METHODS Bacterial strains and plasmids. All bacterial Wtrains and plasmids are listed in Table 1. Phages. A phages used were A wild type and AcI857 xis-i, a kind gift of B. Fischer. Media. Media and growth conditions were as described previously (12). Final concentrations used were as follows: ampicillin, 50 ug/ml; kanamycin, 50 pg/ml; tetracycline, 10 Ag/ml; gentamicin, 25 gg/ml; streptomycin, 100 jag/ml; spectinomycin, 200 jig/ml; rifampin, 100 tsg/ml; and mercuric chloride, 10.5 yg/ml. Matings. Matings involving MW2006,, MW2021, and MW2023 were liquid matings performed t Present address: Department of Botany, University of Durham, Durham DH1 3LE, England. as described previously (12). Matings involving MW2050 and TC1-TC6 were performed by plate matings. These give better trnsfer of R26-8, which transfers poorly in liquid matings, and were performed as follows. Log phase L broth donor and recipient cultures (50 pl of each) were mixed and spotted onto a sterile membrane filter (Millipore Corp., Bedford, Mass.). The filter was then placed on the surface of a prewarmed L agar plate and incubated for 3 h at 370C. The bacteria were then washed off the filter by blending in a Vortex mixer in 1 ml of buffer. Suitable dilutions (0.1-ml samples) were then plated onto selective media and incubated at 37 C. Selection and scoring were made on suitably supplemented glucose minimal salts agar. Axis curing. MW2050 was mixed with serial dilutions of either wild-type A or Axis-1. The infected bacteria were plated onto L agar in a 2.5-ml 0.7% softagar overlay. The plates were then incubated overnight at either 370C for wild-type A or 30 C for AcI857 xis-i. Bacteria were then picked from the turbid centers of plaques and purified by two rounds of singlecolony isolation on L agar. Isolates were tested for drug resistance by streaking onto suitably supplemented L agar plates. Isolates that were sensitive to gentamicin and mercuric chloride but remained resistant to tetracycline were considered to have lost R26-8. Restriction analysis of plasmid DNA. The isolation of plasmid DNA and its restriction and subsequent analysis on 0.7% agarose gels have been described elsewhere (5, 6, 12). RESULTS AND DISCUSSION Transfer of IneP plasmids to Hfr- (RP4Aatt). Strain, the Hfr derivative of RP4Aatt (12), was shown to have integrated RP4Aatt at the bacterial A attachment site without concomitant loss of cytoplasmic plasmid. This situation is unlike that found for Hfr's of the sex factor F (8). The apparent lack of incompatibility shown between integrated and autonomous RP4Aatt extends to other IncP plasmids. The plasmid RP1-85 is closely related to RP4 (10). Unlike RP4, it carries the transposon Tn501, conferring mercury resistance, which is inserted into and inactivates the kan gene. RP1-85 was transferred to RP4Aatt strains, and the 462

2 VOL. 142, 1980 INTEGRATIVE COMPATIBILITY OF PLASMID RP4 463 fate of each plasmid was monitored by its characteristic resistance phenotype (kanamycin [Km] for RP4Aatt, mercuric chloride [Hg] for RP1-85). The strain MW2006 has an autonomous RP4Xatt (12). It yields Hgr colonies which on testing prove to have lost the resident plasmid. By contrast, the isogenic strain with an integrated RPXatt yields Hgr derivatives which retain the resistance phenotype of RP4Xatt (Kin TcO) as well as that of RP1-85 (Hgr). The same behavior was shown by another IncP plasmid, R26-8 (9), which carries gentamicin resistance (Gm'), as well as mercury resistance, but has an inactive tetracycline resistance gene (Tc8) (Table 2). These results suggest that RP1-85 and R26-8 coexist with RP4 Xatt in clones with the mixed resistance phenotype. Do such clones retain RP4Aatt in the autonomous or the integrated state? Our results show that they have lost the autonomous RP4Aatt, presumably by operation of the normal process of incompatibility between related plasmids. They retain the integrated RP4Xatt. MW2021, a derivative harboring RP1-85, has lost the ability to transfer kanamycin resistance (carried by RP4Xatt) at a high rate (Table 3) but transfers Pro' as an Hfr. Instead, it has acquired the ability to transfer mercury resistance characteristic of RP1-85. Most of the Pro' recombinants from the cross (70 out of 94 tested) were resistant to mercuric chloride, whereas all (94 out of 94) remained kanamycin sensitive. We conclude that KMr and Hg' are on separate plasmids. The derivative MW2021 carries Kmr on the integrated RP4Xatt and Hgr on an autonomous RP1-85, which has replaced the autonomous RP4Xatt TABLE 1. Bacterial strains and plasmids Strain/plasmid Genotype/description Source or reference Strain MW2006 rpsl supe (RP4Xatt) 12 Hfr(RP4Xatt) rpsl supe (RP4Xatt) 12 MW2021 Hfr(RP4Xatt) rpsl supe (RP1-85) Hg' Kmr Str' transconjugant JC6650 x MW2023 Hfr(RP4Aatt) rpsl supe (R26-8) Gm' Hg' Tcr transconjugant ED8764 x A(pro-lac),111 supe spc rpob Rifr derivative of X5119 MW2050 Hfr(RP4Aatt) rpsl supe reca56 (R26-8) Rec- derivative of MW2023 JC6650 StrW host for RP X5119 A(pro-lac).111 supe spc Laboratory stocks ED8764 thr leu rpsl supe (R26-8) N. Willets TC1 Hfr(RP4Aatt) rpsl supe reca56 (R26-8) TC2 rpsl supe reca56 (RP4Aatt) TC3 rpsl supe reca56 (R26-8) Derivatives of MW2050 that have sur- TC4 rpsl supe reca56 (RP4Xatt) vived A infection TC5 rpsl supe reca56 (R26-8) TC6 rpsl supe reca56 (R26-8) Plasmid RP4Aatt Ap' Km' Tcr IncP, sria2-3 12; R. Pastrana, Ph.D. thesis RP1-85 Ap' Tcr Hg' IncP 10 R26-8 Ap' Km' Gmr Hg' Smr Su' IncP 9 TABLE 2. Incompatibility properties of RP4Xatt strainsa Donor strain Donornid pla Recipient strain Selection FOT Score JC6650 RP1-85 MW2006 Hgr 4.0 x /32 KMr (Hfr) Hg' 7.5 x /32 KMr Hgr ED8764 R26-8 MW2006 Gmr 1.2 x /32 Tcr (Hfr) Gmr 4.7 x /32 Tc' Gmr Hgr ED8764 R26-8 TC2 Gm' 1.7 x /16 Tc' TC4 Gmr 6.7 x 1i-5 0/16 Tcr a Matings between donor and recipient strains were either liquid or plate matings carried out as described in the text. Transconjugants were purified on the selection media and then scored separately for the presence of both donor and recipient plasmids. FOT, Frequency of transfer, expressed as transconjugants per donor bacterium. JC6650 was counterselected by streptomycin. ED8764 was counterselected by media lacking threonine and leucine.

3 464 WATSON AND SCAIFE in the parent. Studies on MW2023, an Hfr derivative with R26-8 in the autonomous state, led to the same conclusion (Table 3). The rare occurrence of either Kin transconjugants (with MW2021) or Tc' transconjugants (with MW2023) in these crosses is of considerable interest. It is likely that these are formed by recombination between the two coexisting plasnids (see below). The conclusion that strain MW2021 and MW2023 carry RP4Aatt only in the integrated state is given added weight by the analysis of their cytoplasnic DNA. The parental Hfr strain can be seen to carry RP4Aatt in the autonomous stage (Fig. 1, track 2). MW2021 and MW2023 are characterized by loss of cytoplasmic RP4Aatt and the appearance of RP1-85 and R26-8, respectively (Fig. 1, tracks 4 and 6). The plasmids can be compared with DNA from strains carrying only autonomous RP4att, RP1-85, or R26-8 (Fig. 1, tracks 1, 3, and 5). xis-promoted recovery of integrated RP4Aatt The stable coexistence of an integrated and autonomous plasmid was not an expected result. We have shown above that this lack of incompatibility extends to other IncP plasmids. Does the integrated plamid have a defect in a replication or incompatibility function? We will now show that there is no detectable defect when the plmid is restored to the Donor strain MW2021 MW2023 MW2050 TC1 TC2 TC3 TC4 TC5 TC6 TABLE 3. Transfer properties ofrp4aatt strain' Recipient strain Selection FOT X x 10-4 Km' Tc' 2.5 x 10-5 X x 10-4 Ki' 1.4 x 10- Hg' 2.4 x x 10-5 Tcr Gmr Hg' Aptr Mr Apr jmr Pro' Ap' Km Apr Kin' Ap' Kmn' Apr Km' Apr Kirn autonomous state. The integrated pamnid was recovered from the chromosome by infecting the Hfr with phage A. This provides the Int and Xis proteins necessary for excision of RP4Xatt by site-specific recombination (1). The Hfr strain MW2023, containing autonomous R26-8, was made reca to block any rescue of mutant excised plnids by recombination with the autonomous resident. Phage A was plated for single plaques on strain MW2050. We expected that the turbid centers of these plaques would contain bacteria with RP4Aatt excised. The bacteria were picked, purified, and tested for R26-8. We found (Table 4) that many of them had, in fact, become Gm! Hg, inplying that they had lost R26-8. By contrast, none of the bacteria lost R26-8 after infection with Axi-, which cannot promote the relevant site-specific exchange. We infer that the RP4Xatt is excised when phage infects and causes loss of R26-8 by incompatibility. This inference is confirmed by the following tests. Six isolates were tested for their transfer properties by comparison with the Hfr parent strain, MW2050. Chromosomal transfer was tested by selection for Pro' and drug resistance transfer by selection for Apr and Kmr. The drug-resistant transconjugants could contain either RP4Xatt or R26-8. Two isolates, TC2 and TC4, have RP4Xatt in the autonomous state. They cannot 2.9 x x x x 10- Score 70/94 Hg 0/94 Hg 105/200 Gmr Hg' 0/200 Tc' 16/16 Gmr 16/16 Tcr 16/16 Tc' 16/16 Gmr J. BACTERIOL. a Pro' recombinants or drug-resistant transconjugants were purified on the appropriate selection media and then scored for their drug resistance phenotype. Crosses involving X5119 used spectinomycin as a counterselection. Crosses involving used spectinomycin plus rifampin as a counterselection. FOT, Frequency of transfer, expressed as transconjugants per donor bacterium. -, Not tested.

4 VOL. 142, 1980 FIG. 1. Restriction analysis ofplasmid DNA from derivatives of Hfr(RP4Aatt). Track 1, MW2006- (RP4Aatt); track 2, (RP4Aatt); track 3, JC6650(RP1-85); track 4, MW2021(RPI-85); track 5, ED8764(R2&8); track 6, MW2050(R2&8); track 7, TC2(RPAatt); track 8, TC4(RP4Aatt). Plasmid DNA was isolated and digested by EcoRI restriction endonuclease, then analyzed by electrophoresis on 0.7% horizontal agarose gels (5, 6). In tracks 1, 2, 7, and 8, the larger band is RP4 whereas the smaller band is the Aatt fragment. In tracks 3 and 4, the two largest bands (almost a doublet) are from RP1 whereas the smaller bands are from Tn501. In tracks 5 and 6, the precise allocation ofbands is unknown; nevertheless, R26-8 is clearly distinguishable from RP4Aatt and RP1-85. TABLE 4. Excision of integrated RP4Aatt by phage A infection- Infecting phage No. of isolates having lost R26-8 No. tested Aint' xis8 Aint+ xis 'Bacteria from A-infected MW2050 were picked from the turbid centers of plaques and purified on L agar. Isolates were then tested for their drug resistance phenotype. Those that were sensitive to gentamicin and mercuric chloride but remained resistant to tetracycline were considered to have lost R26-8. transfer Pro' at high frequency but can transfer the drug resistance markers of RP4Xatt (Table 3). Three others, TC3, TC5, and TC6, could not transfer. They have lost the integrated RP4Xatt but still contain R26-8. The remaining isolate, TC1, was unchanged by A infection. It continued to transfer at high frequency, and it still had R26-8. Proof that RP4Xatt does INTEGRATIVE COMPATIBILITY OF PLASMID RP4 465 return to the autonomous state after A infection is shown by analysis of the plasmid DNA of isolates TC2 and TC4. They (Fig. 1, tracks 7 and 8, respectively) show the restriction pattem of RP4Xatt (Fig. 1, track 1) and not that of R26-8 (Fig. 1, track 5). Thus, in all cases where RP4Aatt has demonstrably returned to the autonomous state, R26-8 is lost. We conclude that this event regenerates its incompatibility property. This is confirmed by transferring R26-8 to TC2 and TC4 by selection for gentamicin-resistant transconjugants. Analysis of these transconjugants shows that all (16 out of 16) had lost RP4Aatt (Table 2). The results presented above show that a chromosomally integrated P group plasmid (RP4Xatt) is able to coexist stably with other P group plasmids (RP4Xatt, RP1-85, and R26-8) in the autonomous state. This appears to be a fundamental property of RP4, as no mutation is required in replication or incompatibility functions. We call this property integrative compatibility. The replicon hypothesis can explain integrative compatibility (3). If integrated RP4Xatt becomes part of the chromosomal replicon and is replicated passively, it could vacate a specific membrane attachment site necessary for replication. This site would thus become available for an autonomous plasmid. Alternative repressor dilution hypotheses for the control of replication (7, 11) can also be adapted to explain integrative compatibility. The level of a plaidspecific repressor determines the plasmid copy number. Repressor is made by the plasmid immediately after replication and prevents further replication initiation events. As the bacterium grows and divides, the concentration ofrepressor falls, allowing replication to proceed once more. RP4Aatt integrated at a site replicated late may have an effective copy number lower than that normally found for autonomous IncP plasmids. The level of repressor maintained by the integrated plasmid could therefore be insufficient to prevent replication of an autonomous plasmid. Alternatively, integration of the plasmid into the chromosome may block repressor synthesis. It would be of great interest to discover whether integrative compatibility depends on the position at which RP4Aatt is integrated in the chromosome. Elucidation of the mechanism of integrative compatibility could greatly increase our understanding of plasmid replication control. It would be very interesting to know whether this new property is a general feature of certain plasmid groups.

5 466 WATSON AND SCAIFE ACKNOWLEDGMENTIS We would like to thank J. Watson, N. Willets, and S. LeGrice for invaluable discusions during this work; N. Willets for plasmid R26-8; B. Fischer for XcI857 xi8-i; and R. Somerville and G. Warren for reading the manuscript of this paper. This work was supported by Medical Research Council progrsm grant G974/828. LITERATURE CITED 1. Enquist, L W., and R. A. Weisberg The red plaque test: a rapid method for identification of excision defective variants of bacteriophage lambda. Virology 72: Hedges, R. W., and N. Datta Plasmids determining I pili constitute a compatibility complex. J. Gen. Microbiol. 77: Jacob, F., S. Brenner, and F. Cuzin On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28: Kaiser, A. D., and T. Masuda Evidence for a prophage excision gene in A. J. Mol. Biol. 47: McDonell, M. W., ML N. Simon, and F. W. Studier Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis J. BACTERIOL. in neutral and alkaline gels. J. Mol. Biol. 110: Murray, N. E., W. J. Brammar, and K. Murray Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150: Pritchard, R H., P. T. Barth, and J. CoUlins Control of DNA synthesis in bacteria. Symp. Soc. Gen. Microbiol. 19: Scaife, J. G., and J. D. Gross Inhibition of multiplication of an F-lac factor in Hfr cells of E. coli K12. Biochem. Biophys. Res. Commun. 7: Stanisich, V. A., and P. MW Bennett Isolation and characterisation of deletion mutants involving the transfer genes of P-group plasmids in Pseudomonas aeruginosa. Mol. Gen. Genet. 149: Stanisich, V. A., P. M. Bennett, and M. H. Richmond Characterisation of a transocation unit according resistance to mercuric ions that occurs on a nonconjugative plasmid in Pseudomonas aeruginosa. J. Bacteriol. 129: Warren, G., and D. Sherratt Incompatibility and transforming efficiency of Col El and related plasmids. Mol. Gen. Genet. 161: Watson, M. D., and J. G. Scaife Chromosomal transfer promoted by the promiscuous plamid RP4. Plasmid 1: Downloaded from on April 11, 2019 by guest