PLASMID

27,93- 104 ( 1992)

RecP, a New Minor Pathway of General Recombination in Escherichia co/i Encoded by Plasmid Ri drd-19 LEONID

S. CHERNIN,’ MICHAIL A. TEFWNTYEV, AND MARIANNA I. OVADIS

Institute of Chemical Physics, USSR Academy of Sciences, II 7334 Moscow, USSR Received December 28, 1990; revised August I, I99 1 Plasmid Rldrd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K 12 as measured by Hfr crosses and increases their resistance to uv inactivation. The effect correlates with the production of an ATPdependent ds DNA exonuclease in recB/R Idrd-19 cells. This paper further investigates the suppressive effect of plasmid R 1drd- 19 on the recB mutation of E. co/i. The gene(s) responsible for the effect was localized to the 13. I-kb EcoRI-C fragment of the resistance transfer factor (RTF) portion of Rldrd- 19. The plasmid-encoded activity does not merely replace the RecBCD enzyme failure but differs in several significant ways. It promotes a hyper-recombinogenic phenotype, as judged by the phenomenon of supcroligomerization of the tester pACYC 184 plasmid in recB/R 1drd- 19 cells and two inter- and intramolecular plasmid recombination test systems. It is probably not inhibited by X Gam protein and does not restrict plating of T4gp2 mutant. No significant homology between the E. co/i chromosomal fragment carrying recBrecCrecD genes and the EcoRI-C fragment of Rldrd-19 was observed. It is suggested that the plasmid-encoded recombination activity is involved in a new minor recombination pathway (designated RecP, for Plasmid). RecP resembles in some traits the RecBCD-independent pathways RecE and RecF but differs in activity and perhaps substrate specificity from the main RecBCD pathway. Q 1992 Academic

The general recombination

processes in

mids from different incompatibility groups, may be strain-specific, and is not associated with the protective or mutagenic effect of plasmids (for review, see Chemin and Mikoyan, 198 1). The Hr+ phenotype has been studied in more detail in plasmid Rldrd- 19 (IncFII). It was found that in recB or recBrecC mutants R 1drd- 19 shows a complementation effect seen as a significant increase in postconjugational recombination and resistance to uvcaused lethality as well as the appearance of an exonuclease activity specific to doublestranded DNA. Like exonuclease V, which belongs to the RecBCD complex (Smith, 1988), this exonuclease is also ATP-dependent (Chemin and Ovadis, 1980). In the present work we continued the study’ of the recombinogenic properties of Rldrd19. Our results suggest that R 1drd- 19 codes for a recombination activity whose in vivo properties are in some respects different both from those of the RecBCD nuclease and,

Escherichia coli are controlled by about 20 genes localized in the chromosome whose activity is associated with the major, RecBCD, and the additional, RecF and RccE, recombination pathways, as well as with the DNA processing that accompanies recombination (for reviews, see Clark and Low, 1988; Mahajan, 1988; Smith, 1988). Despite this highly polygenic chromosomal control of general recombination, some plasmids can also contribute to the process (Oliver and Stacey, 1977; Chemin and Ovadis, 1980; Pembroke and Stevens, 1984; Syvanen et ai., 1986). It was found that some plasmids can affect the level of postconjugational recombination in the host strain when the latter is being crossed with a Hfr donor. This property, designated Hr+ phenotype (host recombination) (Chernin and Ovadis, 1980), is possessed by plas’ To whom correspondence

should be addressed. 93

0147-619X/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction m any form reserved.

94

CHERNIN, TERENTYEV,

AND OVADIS

TABLE 1 ESCHERICHIACOLISTUAINSANDF'LASMIDS Strain or plasmid

Relevant characteristics

Size W

-

JC411

-

JC4535 JC1553 JC8679 HfrC

recB60 recA 1 recB2 1recC22sbcA23 metB

RI&d-19 RThw pSM185

Ap’ Cm’ Km’ Sm’ AP’

94.2 65.0 24.5

pSM187

Ad

24.5

pSM187::TnS

Ap’ Km’

30.3

pACYC184 pPE230 pFS1 l-04

Cm’ Tc’ Ap’ recB+ Ap’ recBCD+

4.0 22.3 23.7

pSF117 pSF119 pAL210

Apr gam S+ Ap’ gam S201 AP’

4.8 4.8 7.4

pAL23 1

AP’

4.4

pAL242

AP’

4.0

Description

Source

k-6 lacy 1 gal6 his-l malA1 xyl7 mtl-2 argG6 m&B1 str-104 sup58 Derivative of JC4 11 Derivative of JC4 11 Derivative of AB1157 -

A. J. Clark

Derivative of R 1drd- 19 pRSF2 124 + EcoRI-C fragment of RI&d-19 pRSF2 124 + EcoRI-C fragment of R 1drd- 19 in opposite orientation compared with pSM185 Derivative of pSM 187 with insertion of Tn5 into the EcoRI-C fragment of R ldrd- 19 PAT 153 + gene recB of E. coli pBR322 + genes recB, recC and recD of E. coli pCQV2 + gene gamS of phage X Mutant of pSF 117 Derivative of pBR322 carrying duplication of gene tet, with deletion in tet-1 gene and insertion in tet-2 Derivative of pBR322 with deletion of BamHI site in tet gene Derivative of pACYCl84 with deletion of Sal1 site in tet gene

K. Nordstrom This work K. Nordstrom

A. J. Clark A. J. Clark A. J. Clark W. Hayes

K. Nordstrom

This work A. Stepanov I. Hickson M. Anai J. Hays J. Hays A. Cohen

A. Cohen A. Cohen

Media, conjugation, uv irradiation, and probably, from the exonuclease VIII, the product of the recE gene which can compen- ATP-dependent exonuclease assay. These sate for the absence of RecBCD activity have been described earlier (Chernin and (Clark and Low, 1988). The plasmid-coded Ovadis, 1980). Transformation of E. coli cells, isolation of activity may be responsible for a new supplementary recombinational pathway that oper- plasmid DNA by the alkali or boiling techates in RecBCD-deficient mutants and may nique, electrophoresis of plasmid DNA, rebe designated the RecP (for Plasmid) striction analysis of plasmids, electroelution from agarose gels, and Southern blotting. All pathway. procedures were performed essentially as deMATERIALS AND METHODS scribed by Maniatis et al. (1982) with minor Bacterial strains and plasmids. The E. coli modification. Hybridization was performed strains and plasmids are listed in Table 1. under mild-stringency conditions. Filters

PLASMID-ENCODED

were incubated with a radioactive probe at 37°C in the presence of 50% formamide and washing was performed at 7 1“C in 2X SSC + 0.2% SDS. Plasrnid oligomerization test. The procedure used was a modification of one described elsewhere (James et al., 1982) but the amplification step was omitted. The strain to be tested was transformed with electrophoretically purified monomeric plasmid pACYC184. The transformation mixture was plated onto LB2 agar (LBA) plates containing Tc (12.5 pg/ml) and the plates were incubated at 37°C until the colonies reached l-2 mm in diameter. Such colonies were transferred into tubes containing 5 ml of L broth with Tc. The cultures were grown with aeration at 37°C to the optical density of 0.6 at 590 nm. Plasmid DNA then was isolated and subjected to electrophoresis in a horizontal 0.5% agarose gel. For an assay of GamS dependency of oligomerization, tester plasmids pSF117 (GamS) and pSF119 (GamS201) were used. The strains carrying these plasmids were grown overnight at 3O”C, diluted 50- to loo-fold into fresh LB (+Ap, 100 pg/ ml), and divided into two portions, one of which was grown at 30°C and the other at 39°C with aeration to an optical density of 0.6 at 590 nm. Plasmid DNA was then isolated and studied as described above. Electron microscopy. The DNA solution was gamma-irradiated to induce relaxation of supercoiled structures. The samples for electron microscopy were prepared by a standard technique (Davis et al., 197I).

Isolation of a RTF derivative ofRldrd-19. JC4535/R 1drd- 19 cells were inoculated into LB (pH 7.6) containing 40 pg/ml ethidium bromide (Sigma) at ca. 500 cells/ml. The culture was grown without aeration overnight at 37°C diluted, and plated on LBA without antibiotics. The colonies were replicated onto Ap (100 pg/ml) plates. The colonies on the ’ Abbreviations used: LB and LBA, Luria broth and LB agar, respectively; Ap, ampicilline; Cm, chloramphenicol; Tc, tetracycline; Km, kanamycin; Sm, streptomycin; r, resistant.

RECOMBINATION

95

initial plates that failed to replicate were tested for resistance to Ap, Cm (25 &ml), and Km (25 pg/ml). About 0.2% of the cells lost the resistance to all three drugs together. One clone surprisingly still possesseda plasmid which, as shown by EcoRI restriction analysis, contained only sequences of the whole RTF portion of R ldrd- 19 plasmid. This plasmid was designated RTFR,-19. Assay of sensitivity to mitomycin C. This assay was performed as described by Sasaki and co-workers (1982). Frequency of intra- and intermolecular recombination. The frequency was determined essentially according to Laban and Cohen (1981). The strains were transformed with plasmid pAL210 (Laban and Cohen, 1981) or a mixture of pAL23 1 and pAL242 (Laban et al., 1984).

Effect of GamS protein on postconjugational recombination. Recipient cultures were grown overnight at 30°C in L broth or in LB with 100 pg/ml Ap for strains with plasmid pSFl19 or pSF117, respectively. In the morning the incubation temperature was increased to 42°C for 2.5 to 3 h. To 1.6-ml aliquots, 0.4 ml of fresh HfrC donor culture was added, and the crossing mixture was incubated for 1 h at 37°C washed, resuspended in 0.89% NaCl, and plated from a dilution onto selective agar to select Leu+Str+ recombinants. For pSFl17 or pSFll9 the agar also contained Ap. The plates were incubated for 48 h at 37°C. Efect of GamS protein on uv sensitivity of strains. The cultures were grown at 30°C as described above and then diluted in 0.89% NaCl depending on the strain uv sensitivity predetermined by the droplet method and plated on 1.5% LBA containing Ap for pSF1 I7 or pSFl19. The plates were then incubated at 39°C for 18 h. Eficiency of T4g2-phage propagation.The cultures were grown overnight at 30°C in LB (+Ap for strains with pSFl17 or pSFl19) mixed with the phage and plated in the upper 0.6% agar layer, and the plates were incubated at 39°C.

96

CHERNIN, TERENTYEV,

AND OVADIS

TABLE 2 INFUJENCE OF PLASMID Rl drd- 19 AND ITS DERIVATIVES ON MUTANT PHENOTYPE JC4535(~~cB60) LINE

Plasmid

Rl drd-19 pSM185 pSM187 pSM 187::TnS

Recombinants Leu+Str+ (%)’

Activity of ATP-dependent exonuclease’

Mitomycin C

UV

0.1 (15) 12.8 (8) 0.1 (6) 28.8 (12) 0.1 (4)

0.01 (12) 1.1 (5) 0.01 (5) 5.7 (8) 0.01 (2)

0.001 (3) 18.8 (2) ND 91.0 (5) ND

0.01 (14) 1.3 (4) 0.01 (3) 2.0 (5) 0.01 (3)

Sensitivity (W)’

Note. The number of experiments is indicated in parentheses. ND, not determined. ’ 100%for crossesJC411 X HfrC (4.8 X lo-‘/lo0 cells Hfr donor). b Units/mg of protein, for ret+ cells (JC411) 0.8 U/mg. ’ Survival of JC4 11 cells under this condition (0.1% mitomycin C) is 82%; survival after uv irradiation ( 15 J/m’) is 9%.

ties (Table 2). A possible explanation may be the opposite orientation of the fragment in Identification of the region of Rldrd-19 the two plasmids relative to the vector, estabthat determines its recombinogenic proper- lished by restriction analysis (Fig. 1). Eflect of Rldrd-19 on oligomerization of ties. Plasmid R 1drd- 19 and eight of the PSM series hybrid plasmids carrying different pACYC184. The strains JC411 and JC4535 EcoRI fragments of RI drd- 19 RTF region transformed by electrophoretically pure (Molin et al., 1979) were transferred by conju- monomers carry both the monomeric and gation or transformation, respectively, to the the dimeric forms of pACYC 184, although in JC4535(recB60) strain. Only one ofthese hy- the latter strain it is less apparent (data not brid plasmids pSM 187 carrying the EcoRI-C shown). A similar observation was made on fragment ( 13.1 kb) possessedseveral proper- AB1157 and its recB mutant (James et al., ties characteristic of RI drd-19; that is, it in- 1982). The presence of Rldrd- 19 in JC4 11 creased the recombinant frequency in the did not change the oligomerization pattern JC4535 strain, as well as the mutant’s resis- (data not shown). However, when pAtance to uv and mitomycin C, and deter- CYC184 monomers were transferred to the mined the synthesis of ATP-dependent exo- recB derivative carrying Rldrd- 19 (or nuclease activity instead of defective Exo V pSMl87), the oligomerization was much more profound (Figs. 2 and 3). In contrast, (Table 2). The conclusion that the gene(s) determin- the introduction of pPE230 or pFS1 l-04 ing the protective and recombinogenic effects carrying recB or recBCD genes, respectively of R 1drd- 19 is located on the EcoRI-C frag- (Hickson and Emmerson, 1981; Umeno et ment (which is carried by pSM187) is con- al., 1983) into JC4535 (recB), did not change firmed by an insertion mutagenesis experi- the pattern of pACYC 184 oligomerization ment with Tn5. The mutant derivative (Fig. 3). The plasmids that lost the ability to pSM187::Tn5 in which the Tn5 insertion determine the ATP-dependent exonuclease was mapped inside the EcoRI-C fragment together with the protective and recombino(unpublished data of A. Myashkauskas in our genie activities of Rldrd- 19 (because of an lab) lost all the above-mentioned effects of incorrect orientation of the EcoRI-C fragpSM 187. However, another hybrid plasmid, ment or a transposon-induced mutation) pSM 185, which also carried the same frag- were also unable to induce pACYCl84 sument of R 1drd- 19, did not show these activi- peroligomerization (Fig. 3). RESULTS

PLASMID-ENCODED

97

RECOMBINATION

Bamtl

I

FIG. 1. Orientation of the Rldrd-19 EcoR1-C fragment into the pSM187 and pSM185 plasmids. The thin line depicts the pRSF2 124 vector sequences.The EcoRI, BarnHI, and SalI sites, replication origin (ori), colicin El (c&l), and &lactamase @la) genes (shown by shaded boxes) and direction of their transcription (shown by thin arrows) are indicated (Dougan et al., 1978; Casabadan ez al., 1981). The sequencesof the EcoRI-C fragment inserted into the pRSF2 124 vector in pSM 187 or pSM 18.5are shown by internal and external thick lines, respectively. Orientation of EcoRI-C fragment into the RI&d-19, pSMl85, and pSM187 plasmids was established by BumHI + S&I hydrolysis and from data on the asymmetric location of the SalI site in the EcoRI-C fragment (Clerget et al., 198I). The direction of transcription of the tru operon in the EcoRI-C fragments (thick arrows) is indicated and is based on the relationship between plasmids F and R 1 and data obtained for F (Willetts and Skurray, 1987) and for R 1 (Koronakis et al., 198.5).

It is known that oligomerization of plas- 2%, respectively. About 10%of the molecules mid DNA can decrease its stability in host were in the shape of an 8; these could be incells (Summers and Sherratt, 1985). A muta- termediate recombination products or catention in the recB gene was found to have no ants (Potter and Dressier, 1979; James et al., effect on the stability of ColE 1-type plasmids 1982). In contrast, the examination of 140 (Bassetand Kushner, 1984). We have made a molecules of pACYC184 isolated from the similar observation with pACYC184 (data original recB host revealed a ratio of 79: 18:3 not shown). However, the presenceof residen- for monomers, dimers, and all other structial R 1drd- 19 plasmid causesthe fast elimina- tures in total, respectively. tion of superinfected tester plasmid from Efect ofRldrd-19 on intra- and intermolecrecl3 cells grown for 50 generations without ular recombination of tester plasmids. One of special selection, while Rldrd- 19 itself is the molecular substrates of recombination stably maintained even after 100 generations used in the present study was plasmid (data not shown). pAL2 10, which has an inner repeat of the tetThe conformation of pACYC184 oli- racycline resistance gene (tet) and two mutagomers in recB/R 1drd- 19 cells was examined tions at different sites in the two copies (Fig. by electron microscopy of plasmid DNA iso- 4). Intramolecular recombination between lated from the cells after transformation with the defective copies of the tet gene may result pACYC 184 monomers and grown for 40 gen- in the recovery of the gene function and erations in the presence of Tc. Among the hence in the appearance of Tc-resistant colo120 molecules examined were circular mono- nies (Laban and Cohen, 1981). mers, dimers, trimers, tetramers, and higher The other tester system is composed of the order oligomers constituted 22, 55, 7, 5, and pAL231 and pAL242 plasmids, which carry

98

CHERNIN, TERENTYEV,

11

9 7* lo-

-

z 4 3 2

,l

AND OVADIS

RTF,,-,, cells, or plasmidless JC411 (ret+) and JC4535 (recB60) strains with plasmids pAL231 and pAL242 or pAL210, we determined the frequency ofintra- and intermolecular recombination. The results summarized in Table 3 suggest that recombination between the two defective copies of the tet gene in plasmid pAL2 10 (intramolecular recombination) and interplasmidic exchanges is not significantly reduced in the recB mutant compared with the ret+ strain. This is in agreement with the observation that plasmid recombination is recBCD independent (Fishe1et al., 1981;James et al., 1982). The RTF derivative of Rldrd-19 stimulated the frequency of interplasmidic recombination in recB cells (Table 3).

FIG. 2. Superoligomerization of pACYC184 monomers in recB- cells carrying the residental pSM 187(II) or R ldrd- I9 (III) plasmid. Oligomers of pACYC 184 plasmid in initially plasmid-free recB- cells (I). (1) Monomers, (2-9) pACYC184 oligomers; (10) pSM187; (11) Rldrd-19.

different mutations in the tet gene (Table 1). The intermolecular recombination in the region between the two deletions in these plasmids following co-transformation could result in the recovery of the tet gene function (Laban et al., 1984). This system of substrates for studying intra- and intermolecular recombination thus employs ampicilline resistance as a selective marker. For this reason, it cannot be used to study the effects of R 1drd- 19 because Ap resistance is one of markers of this plasmid. To overcome this difficulty, we used a derivative of the plasmid lacking the r-det region of R 1drd- 19 (seeMaterials and Methods). Cells of JC4535 (recB60) carrying RTFrom19 lost the resistance to Ap, Cm, and Km but retained their auxotrophic markers, high activity in the pACYC 184 monomer oligomerization test (data not shown), uv resistance, and the ability to correct the recombinational deficiency of the recB mutant (see Table 4 and Fig. 6). Following transformation of JC4535/

67-

FIG. 3. Specificity of the effect of plasmid Rldrd-19 and pSM 187 on oligomerization of pACYC 184 monomers in recB- cells. Oligomerization of pACYC184 monomers in the initially plasmid-free recB strain (I) and its derivatives with plasmids pFS1 l-04 (II), pPE230 (III), Rldrd-19 (IV), pSM187 (V), pSM185 (VI), pSM187::TnS (VII). (1) Monomers, (2-5) oligomers of pACYC184; (6) pFSll-04; (7) pPE230; (8 and 9) pSM187 and pSM185; (10) pSM187::Tn5; (11) RI&d19.

PLASMID-ENCODED

99

RECOMBINATION

FIG. 4. Generation of the main viable products by intraplasmidic recombination of two mutated tet genes in pAL210 plasmid. Solid thick line-insertion of the X DNA; solid triangle-site of deletions; shaded boxes-short repeated sequences;and open boxes-long repeated sequences.The cross symbols designate possible modes of recombinational interactions. Arrows with numbers indicate the corresponding recombination products. The map of pAL210 was borrowed from Laban and Cohen (198 1).

The results of intraplasmidic recombination proved to be rather complicated. We undertook clonal analysis of the frequency of intramolecular recombination in recB/ RTF,,-i9 cells. Sixty transformant clones grown on a solid medium with Ap, cultured in LB with Ap, and then grown on a solid medium with Tc were studied. These clones can be divided into four groups. Group I clones (29 of those studied) did not produce colonies on medium containing Tc. Group II (11) and III (16) clones formed colonies in the presence of Tc with frequencies of l-3 X

lop3 and l-2 X 10m2,respectively. Finally, four of the transformants classified as group IV produced colonies in the presence of Tc at the highest frequency (2-5 x 10-i). We isolated DNA from transformants of each of the groups, subjected it to electrophoresis, and found that each of the groups had a characteristic plasmid profile (Fig. 5). All group I transformants contained monomers and oligomers of a plasmid with a monomer size of 3.7 kb. In group II and III transformants, both monomers and dimers of pAL2 10 were present; additionally, dimers predominated

TABLE 3 INTRA- AND INTERPLASMIDIC RECOMBINATION IN RECB- CELLS CARRYING RTFx,+, PLASMID Frequency of interplasmidic recombination pAL23 1 X pAL242 Strain

Frequency of intraplasmidic recombination pAL2 10

Mean

Relative

Mean

1.0 0.9 3.5

3.6 x 1O-4(12) 1.5 x 10-4 (7) Pronounced clonal heterogeneity (60)

JC4 11 (ret+)

1.6 x 1O-4(21)

JC4535(recB60)

1.5 x lo-4(17)

JC4535/RTF,,vu,

5.6 X 1O-4(21)

Note. The number of experiments is indicated in parentheses.

Relative 1.0

0.4

100

CHERNIN, TERENTYEV.

AND OVADIS

Sau3A, and Tag1 enzymes has shown that the plasmid is a deletion derivative of pBR322. The deletion is about 630 bp in size and corresponds to the one in the tet-1 gene of pAL210 (Fig. 4). No other differences be-1 tween pBR322 and the 3.7-kb plasmid were -12 found. GamS dependency of the recombinogenic activity of Rldrd-19. The GamS protein of X phage is known to inhibit the activity of the =37 llE. coli RecBCD enzyme in vitro (Kant et al., -8 lo1975) and in vivo (Friedman and Hays, Q1986). We used a hybrid plasmid, pSF117, carrying a cloned sequence of the gums gene -4 which is expressed from a thermoinducible 8promoter controlled by the X cIts857 repressor and mutant of the plasmid pSFl19 FIG. 5. Electrophoretic analysis of the oligomeric (gamS20 1). As a control of the GamS protein forms of pAL2 10 plasmid and its recombinational prod- activity we employed ret’ strain JC411. ucts. (1) RTFR,.,P; (2) pAL210 monomer; (3) pAL210 dimer; (4) monomer; (5-7) pBR322 oligomers; (8) mono- Strains JC4 11/pSF 117 and JC4 11/pSF 119 mer; (9-l 1) 3.7-kb plasmid oligomers; and (12) frag- were compared with regard to their profiments of chromosomal DNA. (I-IV) Groups of clones ciency of postconjugational recombination, (see text). ability to maintain the growth of phage T4g2, and sensitivity to uv. It was found that when the two plasmids in group II and monomers predominated in (Rldrd-19 or RTF,,-,9 and pSF117) were group III. In group IV transformants the prod- present in recB cells, thermoinduction of uct of intramolecular interaction between the GamS protein did reduce the yield of recomsmall homology regions of pAL2 10 (Fig. 4) binants (two- to fourfold) but it remained at a can be seen to be the monomer of plasmid level much higher than that in recB cells without R 1drd- 19 (Table 4). The conclusion pBR322 and its oligomers. It is worth mentioning that Laban and Co- that the effects of Rldrd- 19 in the recB muhen (198 l), who studied the recombination tant are almost insensitive (or at least mildly products of pAL2 10 in ret” and some ret sensitive) to the GamS protein is even more strains, did not refer to a 3.7-kb plasmid. Our obvious from our uv sensitivity data (Fig. 6): own data show that the transformants of Expression of GamS protein at 39°C markJC411 (ret’), JC4535 (recB60), JC1553 edly decreases the uv resistance of JC41 I/ (recAl), and JC8679 (recBCsbcA) that re- pSFl17 (Fig. 6A), but does not influence the ceived pAL2 10 and were then grown with Ap protective activity of Rldrd-19 or RTFRlT19 contained only monomers or oligomers of in the recB background (Fig. 6B). It is worth the plasmid. In addition, a clonal analysis of noting that the temperature induction of the JC8679/pAL210 transformants did not re- gums+ gene did not cause a further increase veal heterogeneity in their growth pattern in in uv sensitivity or recombination deficiency the presenceof Tc. All 20 transformants stud- of the JC4535/pSF117 strain (data not ied formed Tc-resistant colonies at an aver- shown). We have also investigated the GamS deage frequency of l-2 X 1Oe2,which is close to pendency of superoligomerization of tester that of our group III transformants. Restriction analysis of the 3.7-kb plasmid plasmids caused by R 1drd- 19 recombination using CfrI, CfrlO, EcoRI, HueIII, MspI, activity. The tester plasmids used were

PLASMID-ENCODED TABLE 4 EFFECTOFGAMS PROTEINONPOSTCONJUGATIONAL

strain

Plasmid

JC41 l(rec+) JC411 JC411 JC4535 (recB60) JC4535 JC4535 JC4535 JC4535 JC4535

pSFll7(gumS+) pSFl19(gumS201) Rl drd-19 RTFRI-IP Rldrd-19, pSFl17 RTFa,+,, pSFll7 RTFa,.,g, pSFll9

Recombinants Leu+Str+ (%) 100.0” (8) 15.6 (4) 67.7 (3) 0.5 (4) 14.7 (3) 22.8 (4) 5.1 (4) 9.4 (4) 23.5 (2)

101

RECOMBINATION

pSM 187 and used as the labeled DNA probe and the plasmids pSF1 l-04 and pPE230 carrying the recBrecC and only the recB genes,respectively (Hickson and Emmerson, 1981; Umeno et al., 1983). No specific hybridization was found. A similar negative result was obtained in the cross-hybridization experiment using the recBC-carrying fragment of pFS1 l-04 as the DNA probe (data not shown). DISCUSSION

In this study we continued the investigation of the recombinogenic activity of plasn The level of recombination in JC4 11 X HfiC crosses; mid R 1drd- 19 discovered earlier in E. coli see footnotes to Table 2. The number of experiments is K12 cells defective in the RecBCD enzyme indicated in parentheses. (Chernin and Ovadis, 1980). We found that the ability of this plasmid to compensate partially for the recB defects in postconjugapSFl17 and pSFl19. Regardlessof the culti- tional recombination and DNA repair as well vation temperature, both plasmids were as to determine the synthesis of ATP-depenstrongly oligomerized in the presence of dent exonuclease is encoded in the EcoRI-C fragment of its RTF part. A complete genetic RTF,,-,, (data not shown). A comparison of T4g2- propagation in map of R 1drd- 19 is not yet available. HowJC4 11, JC4535 (recB), and the derivatives of ever, because its RTF portion has strong hothe latter carrying Rldrd-19 or RTFRI+, gave mology with tra operons of related plasmids a rather unexpected result. The recB mutant, F and RlOO (Willetts and Skurray, 1987) it with or without the two plasmids, showed normal plaques, whereas its parent (JC411) produced very small plagues, if any. As is known, T4g2- mutants fail to grow on recB+ recC+ cells becausethey lack the Gp2 protein needed to protect double-stranded ends of T, DNA from exonuclease V (Lipinska et al., 1989). It appears, therefore, that the ATP-dependent exonuclease activities encoded by R 1drd- 19 and the recBCD genesare not quite identical. Because Rldrd-19 did not affect the propagation of T4g2- phage in recB cells, we could not use this model system to study the effectsof the GamS protein on the recom10 20 @30 10 20 30 J/m2 bination activity encoded by R 1drd- 19.

.,I\ i\.I *

A study of homology between Rldrd-I9 and chromosomeregions codingfor plasmid recombination activity and the RecBCD enzyme, respectively.This study was performed by Southern hybridization between the EcoRI-C fragment of R 1drd- 19 isolated from

FIG. 6. The influence ofGamS inhibitor on uv sensitivity of JC41 l(rec+) and JC4535(recB60) cells and their derivatives carrying Rldrd-19 or RTF,,-rS plasmid. (0) JC411; (@) JC411/pSF117; (0) JC4ll/pSF119; (A) JC4535; (A) JC4535/RTF,,-,,; (0) JC4535/Rldrd-19; (A) JC4535/RTFa,.,9, pSFl19; (m) JC4535/Rldrd-19, pSF117; (A) JC4535/RTF,,-,,, pSFll7.

102

CHERNIN,

TERENTYEV.

can be inferred that the &RI-C fragment covers the central portion of the tra operon where genesinvolved in sex-pili assembly are located. The fragment probably does not carry the leading region of the R 1drd- 19 plasmid and therefore does not include the ssb gene for single-strand DNA-binding protein, which is also involved in recombination processesin E. coli (Golub and Low, 1985). The gene(s) responsible for R 1drd- 19 recombinogenie properties is most probably not functionally connected with the transfer properties. Yet this gene (or genes)seemsto be transcribed as part of the giant polycistronic mRNA from the tra operon promoter because its expression in hybrid plasmids pSM 187 and pSM185 is dependent on the orientation of the EcoRI-C fragment and could be achieved in the former case from strong promoters of the pRSF2 124 vector (Fig. 1). The same follows from our data that various TnlO and Tn5 insertions in Rl drd19 or pSM187 may have a polar effect on their recombinogenic activity but did not change the conjugativity of the former plasmid (L. Chernin, M. Ovadis, and A. Myashkauskas, unpublished). A comparison of the recombination yield in crosseswith HfrC and of the uv and mitomycin C sensitivities in a ret+ strain (JC4 11) and its recB60 mutant (JC4535) carrying Rldrd-19 or pSM187 (Table 2) suggested that the plasmid-determined recombination activity at least partially compensates for the defect of RecBCD nuclease. Although the exact nature of the recB60 allele (Clark, 1967) is still unknown, the suppressive effect of R 1drd- 19 is not allele-specific because it was earlier observed in another genetic background, e.g., recB and recBrecC mutants of ABl157 (supE44) strain (Chernin and Ovadis, 1980) and even in the recB deletion mutant V360 isolated in the G. R. Smith laboratory (data not shown). We have shown that the presenceof R 1drd19 or pSM 187 in a recB mutant induces a marked oligomerization of pACYC 184 plasmid monomers (Fig. 2), whereas in the reef strains the oligomerization was much lessap-

AND

OVADIS

parent (Fishel et al., 1981; James et al., 1982). The oligomerization level in the former caseis similar to the pattern observed in a recBrecCsbcA mutant, where it is due to ATP-independent exonuclease VIII (James et al., 1982). We therefore cannot exclude the possibility that although the plasmid-coded exonuclease is ATPdependent, it can somehow activate the RecF pathway. It could be accomplished, for example, by inducing SOS functions, as the RecBCD enzyme can do (Chaudhury and Smith, 1985), or by the appearance of an ATP-dependent helicase that prepares substrate DNA for some ATP-independent single-strand exonuclease in extracts of recB cells carrying R 1drd- 19. Thus, the superoligomerization of pACYC184 monomers in recB cells carrying Rl drd- 19 may occur when either the plasmid-encoded recombination activity ensures an unusually high level of tester plasmid recombination or it somehow interferes with the replication of tester plasmid DNA. At any rate, the observed effect is not merely a replacement of the missing RecBCD activity since plasmids pPE230 and pFS 1l-04, which carry cloned recB or recBrecC genes, fail to enhance tester plasmid oligomerization in a recB mutant and do not oligomerize themselves despite the increased level of RecBCD enzyme expression (compared with ret+ strain which they induce) (Hickson and Emmerson, 1981; Umeno et al., 1983). The difference between the RecBCD enzyme and the R 1drd- 19 recombination activity is also apparent from a comparison of inter- and intraplasmidic recombination frequencies (Table 3) and from clonal analysis of intramolecular recombination frequency and its products in the recB/RTF,,-,, strain transformed with pAL210. The transformants showed marked differences in the ability to form colonies on a Tc-containing medium and in the fate of pAL2 10 DNA in them (Fig. 5). About one-half of the transformants selected for Ap resistance gave little if any tet-resistant subclones. In all these cases pAL2 10 could not be found; instead there was a 3.7-kb monomer and its oligomers. A

PLASMID-ENCODED

restriction analysis suggested that this plasmid was similar or identical to pALl99, which was one of the two components used to construct pAL2 10 in vitro (Laban and Cohen, 1981). In explaining why the 3.7-kb product formed at such a high frequency, it should be noted that the pAL210 plasmid carries two pairs of repeats differing in length. Recombination between short repeats (270 bp) leads to restoration of functional tet gene, while recombination between long repeats (about 1 kb) results in formation of plasmid with the defective tet- 1 gene (Fig. 4). This may explain the absence of Tc’ clones in progeny of cells harboring the 3.7-kb plasmid. The circumstances may be decisive if it is assumed that the R 1drd- 19 recombination activity strongly and nonspecifically stimulates homologous recombination processes. Differences between the RI drd- 19 recombination activity and the RecBCD enzyme have also been revealed in our tests on the sensitivity of phage X to the GamS protein. Several phenomena resulting from inhibition of the RecBCD enzyme by this protein have been reported (Friedman and Hays, 1986; Cohen and Clark, 1986; Berger and Cohen, 1989). Our evidence suggeststhat the stimulation of postconjugational recombination and tester plasmid oligomerization in a RecBCD host as well as its protection against uv caused by R 1drd- 19 is essentially GamS-independent (Table 4, Fig. 6). Another difference between the two activities is that the plasmid-encoded activity doesnot attack double-stranded ends of T, DNA, whereasexonucleaseV does. Finally, our hybridization tests failed to reveal any homology between the EcoRI-C fragment of Rldrd- 19, which is responsible for its recombinogenic activity, and the cloned chromosome regions containing recBrecCrecD or only recB genes. Taken together with our previous study (Chernin and Ovadis, 1980), the present results make it possible to conclude that plasmid R 1drd- 19determines a novel recombinational pathway in E. co/i cells. We propose that it be designated the RecP (for Plasmid)

RECOMBINATION

103

pathway. This is a minor pathway that has been revealed in cells defective in the RecBCD enzyme. Some processesof general recombination on the RecP pathway, such as intra- and intermolecular recombination of plasmids and plasmid oligomerization, may occur with even higher efficiency than those in ret+ cells. It is possible, therefore, that the recombinogenic determinant of R 1drd- I9 (gene recP?) can make its own contribution to recombinational events in the host cell, despite the fact the latter has more than a dozen genes controlling its recombinational potential. Further investigations of recombinogenie activity of Rldrd-19 are under way in our laboratory. ACKNOWLEDGMENTS We are grateful to Alvin J. Clark, Kurt Nordstrom, Am&am Cohen, J. Hays, I. Hickson, and T. Anai for strains and plasmids. We also sincerely thank A. J. Clark, A. Cohen, S. Levy, and R. Novick for helpful discussions and comments.

REFERENCES BASSET,C. L., AND KUSHNER,S. R. (1984). ExonucleasesI, III, and V are required for stability of ColE 1-related plasmids in Escherichia coli. J Bacterial. 157, 121-127. BERGER,I., AND COHEN,A. (1989). Suppression of recA deficiency in plasmid recombination by bacteriophage X protein in recBCD- ExoII Escherichia coli cells. J. Bacterial. 171, 3523-3529. CASABADAN,M. J., CHOU,J., LEMAUX, P., Tu, C.-P. D., AND COHENS. N. (198 1). Tn3 transposition and control. Cold Spring Harbor. Symp. Quant. Biol. 45,269273. CHAUDHURY,A. M., AND SMITH, G. R. (1985). Role of Escherichia coli RecBC enzyme in SOS induction. Mol. Gen. Genet. 201, 525-528. CHERNIN, L. S., AND MIKOYAN, V. S. ( 198 1). Effects of plasmids on chromosome metabolism in bacteria. Plasmid 6, 119-I 40. CHERNIN,L. S., AND OVADIS,M. I. ( 1980).Plasmid control of recombination in E. coli K12. Mol. Gen. Genet. 179,399-407.

CLARK, A. J. (1967). The beginning of a genetic analysis of recombination proficiency. J. Cell Physiol. 70, 121-127. CLARK,A. J., AND Low, K. B. (1988). Systemsand pathways of homologous recombination in Escherichia coli. In “The Recombination of Genetic Material”

104

CHERNIN, TERENTYEV,

(K. B. Low, Ed.), pp. 155-215. Academic Press,New York. CLERGET,M., CHANDLER,M., AND CARO, L. (1981). The structure of R l&d-l 9: A revised physical map of the plasmid. Mol. Gen. Genet. 181, 183-19 1. COHEN,A., AND CLARK, A. J. (1986). Synthesis of linear plasmid multimers in Escherichia coli K12. J. Bacteriol. 167, 327-335. DAVIS, R. W., SIMON, M., AND DAVIDSON,N. (197 1). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (L. Grossmann and K. Moldave, Eds.), Vol. 21D, pp. 4 13-428. Academic Press,New York. DOHERTY,M. J., MORRISON,P. T., AND KOLODNER,R. (1983). Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli. J. Mol. Biol. 167, 539-560. DOUGAN,G., SAUL, M., WARREN,G., AND SHERRAT~, D. ( 1978). A functional map of plasmid ColE 1. Mol. Gen. Genet. 158,325-327. FISHEL,R. A., JAMES,A. A., AND KOMDNER, R. (198 1). RecA-independent general genetic recombination of plasmids. Nature (London) 294, 184-l 86. FRIEDMAN,S. A., AND HAYS,J. B. (1986). Selective inhibition of Escherichia coli RecBC activities by plasmidencoded GamS function of phage A. t. Bacterial. 171, 3523-3529. GOLUB, E., AND Low, K. B. (1985). Conjugative plasmids of enteric bacteria from many different incompatibility groups have similar genes for single-strand DNA binding proteins. J. Bacterial. 162,235-24 1. HICKSON,I. D., AND EMMERSON,P. T. (1981). Identification of E. coli recB and recC gene products. Nature (London) 294,578-580. JAMES,A. A., MORRISON,P. T., AND KOMDNER, R. (1982). Genetic recombination of bacterial plasmid DNA. Analysis of the effect of recombination deficient mutations on plasmid recombination. J. Mol. Biol. 160,41 l-430. KARU, A. E., SASAKI, J., ECHOLS,H., AND LINN, S. (1975). The protein specified by bacteriophage X: structure and inhibitory activity for the RecBC enzyme of Escherichia coli. J. Biol. Chem. 250, 73777387. KORONAKIS,V. E., BAUER,E., HOGENAUER,G. (1985). The traM gene of the resistanceplasmid R 1: comparison with the corresponding sequence of the Escherichia coli F factor. Gene 36,79-86. LABAN, A., AND COHEN,A. (198 1). Interplasmidic and intraplasmidic recombination in Escherichia coli K-12. Mol. Gen. Genet. 184,2OO-207. LABAN, A., SILBERSTEIN, Z., ANDCOHEN,A. ( 1984).The effect of nonhomologous DNA sequences on interplasmidic recombination. Genetics 108, 39-52. LIPINSKA, B., RAO, A. S. M. K., BOLTEN, B. M., BA-

AND OVADIS

LAKRISHAN,R., AND GOLDBERG,E. B. (1989). Cloning and identification ofbacteriophage T4 gene 2 product gp2 and action of gp2 on infecting DNA in vivo. J. Bacterial. 171,488-497. MAHAJAN,S. K. ( 1988).Pathways of homologous recombination in Escherichia coli. In “Genetic Recombination” (R. Kuscherlapati and G. R. Smith, Eds.), pp. 87-139. American Sot. Microbial., Washington, DC. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK,J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MOLIN, S., STOUGAARD,P., UHLIN, B. E., GUSTAFSSON, P., AND NORDSTROM,K. (1979). Clustering of genes involved in replication control, incompatibility, and stable maintenance of the resistance plasmid R 1drd19. J. Bacterial. 138, 70-79. OLIVER, P., AND STACEY,K. A. (1977). The effect of a drug-resistance factor on recombination and repair of DNA in Escherichia coli K 12. J. Gen. Microbial. 101, 93-98. PEMBROKE,J. T., AND STEVENS,E. (1984). The effect of plasmid R39 1 and other IncJ plasmids on the survival of Escherichia coli after UV-irradiation. J. Gen. Microbiol. 130, 1839- 1844. POTTER,H., ANDDRESSLER, D. ( 1979).DNA recombination: In vivo and in vitro studies. Cold Spring Harbor Symp. Quant. Biol. 43,969-985. SASAKI,M., FIJIYOSHI,T., SHIMADA,K., AND TAKAGI, J. ( 1982).Fine structure of recB and reck gene of Escherichia coli. Biochem. Biophys. Res. Commun. 109, 414-422. SMITH, R. S. (1988). Homologous recombination in procaryotes. Microbial. Rev. 52, l-28. So, M., GILL, R., AND FALKOW,S. (1979). The generation of ColE 1-Ap’ cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet. 142,239-249. SUMMERS,D. K., AND SHERRATT,D. J. (1985). Bacterial plasmid stability. Bioessays 2,209-2 11. SUTCLIFFE,J. G. (1979). Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol. 43, 77-90. SWANEN, M., HOPKINS,J. D., GRIFFIN, T. J., IPPENIHLER,K., AND KOLODNER,R. (1986). Stimulation of precise excision and recombination by conjugal proficient P plasmids. Mol. Gen. Genet. 203, l-7. UMENO, M., SASAKI, M., ANAI, M., AND TAKAGI, Y. (1983). Properties of the recB and recC gene products of Escherichia coli. Biochem. Biophys. Res. Commun. 116, 1144-l 150. UNGER, R. C., AND CLARK, A. J. (1973). Interaction of recombination pathways of bacteriophage X and its host E. coli K12: Effect on exonuclease V activity. J. Mol. Biol. 70, 539-548. WILLEX-~S,N., AND SKURRAY,R. (1987). Structure and function of the F factor and mechanism of conjugation. In “Escherichia coli and Salmonella typhimurium” (F. C. Neidhardt, Ed.), Vol. 2, pp. 1110-I 134. Am. Sot. Microbial., Washington DC.

RecP, a new minor pathway of general recombination in Escherichia coli encoded by plasmid R1drd-19.

Plasmid R1drd-19 markedly improves the recombination deficiency of recB and recBrecC mutants of Escherichia coli K12 as measured by Hfr crosses and in...
2MB Sizes 0 Downloads 0 Views