Res. Mierobiol.

(~) INSTITDT PASTEuR/ELsEVIEr. Paris 1991

1991, 142,381-387

Incompatibility between pSCIO1 and Xdv replicons N. Ekaterinaki and P. Prentki ('1

Department of Molecular Biology, University of Geneva, 30, quai Ernest-Ansermet, 1211 Geneva 4 (Switzerland)

SUMMARY

Escherichia coli cells are unable to co-maintain a Xdv and a minimal pSCl01 replicon. Selection for both plasmtds results in a much reduced growth rate of the cells, whereas selection for one plasmid results in a rapid loss of the other. A chromosomal mutant has been isolated, which restores the normal growth rate of cells harbourlng both plasmids, but does not alleviate the incompatibility. The nature of this new case of incompatibility Is discussed.

Key-words : Plasmid, Incompatibility, Replicon; pSC101, ~,dv, Escherichia coli, Bacte. ri~ ~ ~.~w,~L5.

I NTRODUC'TION The ~dv plasmids a=e deletion derivatives of phage X which replicate autonomously in Escherichia coil They contain 5-10% of the ~. genome and have in common a 2-3-kb region, the "'core" region, which comprises the ~ origin of replication, ori~) the right promoter and operator sequences (PR and OR), the regulatory gene cro, a transcription termination signal tR1 and the replication genes O and P of X (Matsubara and Kaiser, 1968; Berg, 1974; Matsuhara, 1981). The naturally occurring plasmid pSC101 is 9.2-kb long and confers tetracycline resistance to its host (for a review, see Manen and Caro, 1991). It replicates in the absence of DNA polymerase I (Cabetlo et al., 1976), but requires, among other host proteins, the chromosome initiation function DnaA (Fray et al., Submitted October 10, 1990, accepted November 17, 1990. (*)Correspondiag author.

1979) and the integration host factor (1HF) (Gamas et al.. 1986); it also needs the plasmid-encoded RepA protein (Linder et al., 1983). All the plasmid sequences essential for pSC101 replication are carried on a 2.2-kb fragment, consisting of three main regions: par, a locus involved in plasmid partitioning; ori, the locus where replication starts; and repA, coding for the RepA proton. Within ori is a shorter segment which, when cloned into a high copy number vector, inhibits replication of a pSC101 plasmid in trans, thus defining an incompatibility locus, inc (Linder et al., 1983; Vocke and Bastia, 1983; Yamaguehi and Yamaguchi, 1984). The ~dv and pSC101 replicons do not share any known regulatory replication functions and should, therefore, be able to be maintained in the same host. We find, however, that the two ptasmids behave as an

N. EKATERINAtfl AND P. PRENTKI

382

incompatible pair. Moreover, cells forced to carry both replieons grow slowly, but can return to normal growth rates as a result of a mutation which does not affect the apparent incompatibility of the plasmids.

MATERIALS

AND

RESULTS

METHODS

Bacterial strains and plasrnids C600 (F- thiA thrA teuB6 lacYl tonA21 supE44; Appleyard, 1954), ~vas the E. coil KL2 host used thoughout this study. Plasmid pCBI04 is a Oerivative of the hdv plagmid pCBl01 (Boyd and Sherratt, 1986) carrying a truncated segment of the cl gene and a Cm' gone. Piasrnid pBRflE (Prentki et al., 1986) is a derivative of pBR322 with the Str'/Spc' ~ fragment (Prentki and Krisch, 1984), cloned into the EcoRl site. pGB2 (Churchward ¢! al., 1984) is a 4.2-kb plasmid carrying the minimal replication region of p S C I 0 1 linked to the Str'/Spc' gene. DNA manipulation and characterization A modified Birmboin and Doly (1979) technique was used for large scale DNA preparations. Rapid DNA minipreps were made according to Holmes and Quigley (198l). Methods for transformation and gel electrophoresis are described in Maniatis et aL (1982). Media and antibiotic selections

Liquid cattures were in L-broth (Maniatis et ¢1., 1982). Antibiotic concentrations used were chioramphenico] (Crn), 25 #g/ml, spectinornycin (Spc), 100 ~g/ml and tetracycline t~Tc), 10/xg/ml,

Segregation experiments Cultures of C600 harbouring the plasmid pairs pSC101-pCBI0~, pOB2-pCBll)4 or pBR~EpCBI04 were grown overnight in L-broth containing T¢+ Cm, Sp=+ Cm and Spe÷ Cm, respectively, to select for the presence of both plasmids. The following day, dilutions of the cultures were plated on LA (L-Agar; Maniatis et at., 1982), LA+Cm, LA+Tc and LA+Cm+Tc or LA, L A + C r n , LA+Spc and L A + C m + S p c , as

Cm = ¢hloxaJnphcnic.oL kb = kilobasepair. r (superscript) = resistance.

required by the genotypes of the plasmid pairs. At the same time, a 1/10~ dilution of the culture was grown overnight in the absence of antibiotics. These operations were repeated for 5 days thus covering a total growth of about 100 generations,

1) Incompatibility between pSCIOI and Xdv replicons We have tried to construct a fusion between two plasmids: pCBl04, a ~,dv plasmid carrying the chloramphenicol resistance gene from TILO, and pGB2 (Churchward et aL, 1984), a plasmid containing the minimal replication region of pSC101 and a gene s p e d f y i n g Slacr and Str r. In spite of repeated attempts, this construct was never obtained; rare recombinants arising from such a ligation were deleted for one of the two origins, This prompted us to determine whether the two replicons could be maintained in the same cell, Plasmids pSCI01 (or pGB2) and pCBI04 were transformed into the E. colt strain C600, either sequentially or simultaneously ("Materials and Methods"). In parallel, pBRf/E, a derivative of pBR322 carrying the same StrVSpc' gene as pGB2, was eotransformed with pCBI04 into C600 to test for potential effects of the StrVSpc' gene on the co-resident hdv plasmid. The ~,dv-pBRgE double transformants formed normal-size colonies 16 h. after plating of the transformation mixture, while the pCB104-pSC101 transformants produced very small colonies 16 h. after plating, and colonies carrying pCBI04-pGB2 became visible only 24 h. after plating, To verify this observation, the growth rates of cells harbouring both pCBI04 and pGB2, or pSC101, and that of all the

Str - streptomyci~L Spc = spcctinomycin, T¢ = tetracycline.

383

INCOMPATIBILITY BETWEEN pSCIOI AND Xdv REPLICONS

appropriate controls were measured in liquid cultures. Table I shows that each plasmid by itself has slight effects on the doubling time, whether or not the selective antibiotic is present, while the doubling time of cells carrying both pGB2 (or pSCl01) and pCB104 is considerably reduced when selection is applied for both plasmids. No difference is observed in the ease of pCBI04-pBRflE double transformants. Data in table I also show that pCBI04-pSCI01 transformants have a doubling time intermediate between that of the pCBI04-pGB2 and that of the pCBI04-pBRflE transformants, in agreement with the phenotypes observed on plates. These results suggested an incompatibility between Xdv and pSCI01.

2) Stability and segregation of pSCIO1 and hdv in E. coli C600 We examined the maintenance of each of the plasmids individually in E. coli C600. pCBl04 is unstable in E . coli C600 (fig. 1), a behaviour shared by many derivatives of ),dv (Matsubara, 1981). In contrast, plasmids pSCIOI (Meacock and Cohen, 1980) and pGB2 (Churchward et al., 1984) are k n o w n to

be stably maintained in E. coli; this was verified by our results (fig. 2). Segregation experiments were carried out for a p p r o x i m a t e l y 100 g e n e r a t i o n s o n pCBl04-pSCl01, pCB104-pGB2 and

pCBI04

OI -I ~ -2

_a

-3

-5 i

i

I

I

I

2

3

4

Subcultures

Fig. 1. Stability of pCBI04 (Xdv) in C600. The experiment was performed as in "Materials and Methods". The horizontal axis representsthe number of subculturesand the vertical the log of the fraction of the cells which carry pCBI04.

Table I, Generation times of the various strains used in this study, grown in liquid cultures.

Timc

Strain

Plasmid

Antibiotic

(min)

C600 C600 C600 C600 C600 C600 C600 C600 C600 C600 C600 C600 C600 C600 C600"

-pCBI04 pCB104 pSCIOI pSC l01 pGB2 pGB2 pBRt3E pBRflE pCB 104-pS101 pCB 104-pSC 101 pCB 104-pGB2 pCB 104-pGB2 pCB 104-pBRflE --

--Cm -Tc -5v~ -Spc -Cm + Tc -Cm + Spc Cm + SDC --

30 36 36 33 34 3I 30 31 31 30 53 36 70 37 30

384

N, E K A T E R I N A K ! A N D P, P R E N T K I

A

B

pCBIOt,pSCIOI

C pCBIO4-pBR~E

pCBL04pGB2

0 -I

~-Z o_ 3

-5 1

t

I

I

i

[

2

3

4

I 2 3 Subcultures

,

i

|

i

d

I

I

4

I

2

3

4

Fig. 2. Segregation experiments in C600,

The pairs of plasmids analysed were (A} pCB104-pSCI01; (B) pCBI04-pGB2; (C) pCBI04-pBRf,~E. The symbols used were the following: squares: Cm r, triangles: Tc r (in panel A) or Spe r (in panels B and C); circles: CmrTc r (in panel A) or CmrSpcr (in panels B arid

C).

pCB104-pBRfiE double transformants. The results are shown in figure 2. In the absence of selective pressure, the Xdv replicon segregates out of the cell preferentially, leaving pSC101 (or pGB2) as the sole resident of the E. colt host. Given the instability of the kdv replicon, this result is not surprising. But, as observed above, the pCBt04-pGB2 double transformants were found to have very low plating efficiency. As can be seen in figure 2b (t =0), onty about I% of the celts grown in liquid in the presence of both chloramphenieot and spectinomycin formed colonies when plated on Cm/Spc, further underlining the deleterious effect for the celt of having to maintain both plasmids. In order to show more directly that pCBI04 is incompatible with pSC101 or pGB2, we repeated the segregation experiment, this time applying selective pressure for the maintenance of the unstable pCB104. The results (fig. 3), show that selecting for the

presence of the kdv replicon results in a dramatic loss of pGB2 and, to a lesser extent, of pSCI01.

3) Isolation and properties o[ C601~* During the segregation experiments, we observed that CmrSpc r colonies from day 4 of the assay grew normally, in contrast with the slow growth phenotype of colonies from day 1. This was taken as an indication for a possible mutation either on the plasmid or on the chromosome. Starting from such a normally growing colony, we isolated a clone which had lost both resistances, i.e. was plasmid-free ("Materials and Methods"), which we named C600". This strain had the same doubling time as C600 (table I). Moreover, in contrast to what was observed with C600, double transformants of pCBI04 and pGB2 into C600" have doubling times comparable to that of C600' itself, showing

I N C O M P A T I B I L I T Y B E T W E E N pSCIOI A N D hdv R E P L I C O N S

A

B

pCBI04-pSCI01

_ pBRQE C. oCBtO/~

pCBl04pGB2

..------El

I"1

El

385

[]

-I ~,.. - ' )

-'~-3 -5 I

I

i

t

2

3

L

I

I

I

I

I .....

I

I

I

2

3

~

i

2

3

I

Subcultures (.Crn). Fig. 3. Segregation in C600 with selection fc! pCBI04. All subcultures were performed in the presence of Cm. Symb¢ls are the same as in figure 2.

that in this strain the simultaneous presence of both plasmids no longer interferes with cell growth. Another striking characteristic o f C600" is that the hdv-based plasmid pCBI04 has now become very stable (fig. 4). In spite of that, segregation experiments performed on C600" (pCBIt.d-pGB2) cultures clearly show that the two plasmids continue to behave as an incompatible pair (fig. 5).

DISCUSSION

The results presented here show that small plasmids carrying the replication origins o f r , o ~ x v ~ ,a,u ,,u, cannc~t be stably maintained together in wild-type E . c o l i ceils (C600) and that they inhibit cell growth. The two effects are not necessarily linked, as shown by the isolation o f a mutant host (C600") in which the two plasmids, while remaining incompatible, do not confer a slow growth phenotype. AS this mutation renders the ~,dv replicon

more stable, we have also examined the plating efficiency o f phage k in this mutant strain. N o difference between C600 and C600" was observed. The elucidation o f the molecular determinants o f this incompatibility will require further studies. Plasmid incompatibility is generally due to the sharing of one or more elements o f the rep[ieation or partition systems between the two plasmids (Novick, 198"/). Since the t ~ o plasmids are unrelated, it is unlikely that the two replicons compete for a replication factor specified by one o f them. The incompatibility of the two replicons could be an indirect effect due to competition be~'ween the ~wc, c,~igir.s for a host factor. Thus, G e r m a n and Syvanen (1982) have reported an incompatibility between hdv plasmids and the sex factor F. They conclude that it is due to an interaction between the P protein o f X and the DnaB protein o f the host. We have found no evidence of such an interaction here. Plasmids pSFC7, overproducing

386

N. EKATERINAKI AND P. PRENTKI

difference in the growth rates of the resulting transformants was observed.

pCB 106" Ol t-~---a -

Our work has also demonstrated a difference between the behaviour of pSC101 and pGB2. The incompatibi]ity was much stronger t o w a r d s p G B 2 , which carries o n l y the minimal replication region of pSC101. The reason for this difference is not clear. It is not due to selection (To for pSCIOI, Spc for pGB2) as demonstrated by the nearly identical values of the doubling times (table I). It could be due to a site or a factor that is missing in pGB2 and that is present in pSC101.

-I

0 ~-3 -,t.

-S

I I

2

3

t.,

Subcultures

Acknowledgements Fig. 4. Stability of pCBI04 (Xdv) in C600'.

pCBt0~.-pGB2*

pCB 10/,,-t~SB 2'*"

:l Z.

Incornpatibilil~ entre

-~-3

les r6plicons pSCIOI et kdv

-g _ _1

i

I 2 2, Subeu['lures

A

We wish to thank Dave Sherratt and Chris Boyd for provision of pCB104, Costa Georgopoulos for pSFC7, Andrea Boffini, Lueien Caro, Miek Chandler, Michel Clerget, Henry Krisch and Danielle Marten for helpful discussions, and Otto Jenni for drawing the figures. This work was supported by grants from the Swiss National Science Foundation.

i

/.

Subcuttures I+Cml

B

Fig. 5. Segregation of pCB104-pGB2 in C600" without (A) and with (B) selection for pCBI04. The symbols used were: squares: Cmr; triangles: Spd; circles: Cm%pcr.

DnaB (gift from C. Georgopoulos) was transformed into C600 and (2600* harbouring the two small replicons. No effect on growth rates was observed. We have also examined the role of the E. coil D n a A protein by transf o r m i n g a D n a A o v e r p r o d u c i n g plasmid (Churchward et al., 1983) into (2600 and C600" carrying both plasm[ds. Again, no

Le plasmide )~dv ne peat pas ~tre maintenu de mani6re stable duns Escherichia coil en compasnie d'un p]asmide portant la rggion minimale de r~plication de pSC101. La s~lection pour les deux plasmides duns une seule cellule r6sulte en une forte r6duction du taux de croissance, alors que la selection pour un seal plasmide conduit /t la perte de l'autre. Un mutant chromosomique qui r~tablit le taux de croissanee des cellules h6bergeant les deux plasmides sans abolir l'ineompatibilit~ a ~t6 isol6. La nature de ee nouvcl exemple d'ineompatibilit~ est discutge. Mots-clds : Plasmide, Replieon, Incompatibilitg; Escherichia coil, pSC101, Xdv, Croissanee bact6denne.

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I N C O M P A T I B I L I T Y B E T W E E N pSCIOI A N D ~dv R E P L I C O N S

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Linder, P., Churchward. G. & Carp, L. (1983), Plasmid pSCtOl replication mutants generated by insertion of the transposon Tnl000. J. reel. BioL, 170,

287-303. Marten, D. & Carp, L. (1990), Mol. Microbiol (in press). Maniatis, T., Fritsch, E.F. & $ambrook, J. (1982), Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York. Matsubara, K. & Kaiser, A. (1963), 5.dr: an autonomously replicating DNA fragment. Cold Spr. Harb. Syrup. quant. Biol., 33, 769-775. Matsubara, K. (1981), Replication control system in lambda dr. Plesmid 5, 32-52. Meacock, P.A. & Cohen, S.N. (1980), Partitioning of bacterial plasmids during cell division: a cts-acting locus that accomplishes stable plasmid inheritance. Cell, 20, 529-542. Novick, R.P. (1987), Plasmid incompatibility. Microbiol. Rev., 51, 3gl-395. Prontki, P. & Krisch, H. (1984), in vilro insertiona) matagenesis with a selectable DNA fragment. Gone, 29) 303-313. Prentki, P., Toter, B., Chandler, M. & Galas, D.J. (1986), Functional promoters created by the insertion of transposable element ISI. Z reel. Biol., 191, 383-393. Vocke, C. & Bastia, D. (1983), Primary structure of the essential repiieon of the plasmid pSCI01. Prec. nat., Acad. Sci. (Wash.), 80, 6557-6561. Yamaguchi, K. & Yamaguchi, M. (1984), The replication origin of pSC101 : the nucleotide sequence and replication functions of the ori region. Gone, 29, 211 219.