Vol. 163, No. 3

Genetic Recombination of Bacterial Plasmid DNA: Effect of RecFPathway Mutations on Plasmid Recombination in Escherichia coli

RICHARD KOLODNER,* RICHARD A. FISHEL, AND MARTHA HOWARDLaboratory of Molecular Genetics, Dana-Farber Cancer Institute, and Department ofBiological Chemistry, Harvard

Medical School, Boston, Massachusetts 02115

Received 4 March 1985/Accepted 10 June 1985

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designatedrecO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening TnSinsertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency ofrecombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation andmitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UVirradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathwaymutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present inotherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3,recAo281, recN, and ruv mutations had no effect on plasmid recombination. TnS insertion mutations in the lexAgene increased the frequency of plasmid recombination. These data indicate that plasmid recombination eventsin wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecFrecombination pathway and that some component of this pathway besides the recA gene product is regulatedby the lexA gene product.

Genetic analysis has demonstrated that there are multiplepathways or mechanisms for homologous recombination inEscherichia coli. The isolation of mutations that decreasethe frequency of recombination events after mating in wild-type E. coli strains has led to the identification of threegenes, recA, recB, and recC, that define the RecBC recom-bination pathway (6, 11, 42). The isolation of two mutations,sbcA and sbcB, that suppress the recombination defectpresent in E. coli strains containing both the recB and recCmutations led to the discovery of alternate pathways forrecombination (4, 39). Genetic analysis of conjugation-mediated recombination events in recB recC sbcB E. colistrains has demonstrated that recombination in these strainsrequires the products of the recA, recF, recJ, recN, recQ,and ruv genes (5, 16, 26, 27, 29, 36). Mutations in the recF,recJ, recN, recQ, and ruv genes, when present in otherwisewild-type strains, have no effect on conjugation-mediatedrecombination, but do, with the exception of recJ and recQ,have an effect on repair (5, 7, 16, 26, 27, 29, 36). Thusrecombination in recB recC sbcB E. coli strains occurs by apathway that is different from the RecBC pathway; thispathway has been called the RecF pathway. Several genes inthis pathway are regulated as part of the recA/lexcA regulon(5, 27, 29). Conjugation-mediated recombination events inrecB recC sbcA E. coli strains require at least the products ofthe recA, recF, and recJ genes and, in addition, require theproduct of the recE gene (7, 15, 28). These results have beeninterpreted to mean that conjugation-mediated recombina-tion in recB recC sbcB E. coli strains and recB recC sbcA E.coli strains occurs by the RecF recombination pathway, butin each case, activation of the RecF pathway occurs by adifferent mechanism (19, 20; J. W. Joseph, Ph.D. thesis,Harvard University, Cambridge, Mass, 1983). In the formercase, activation is caused by the inactivation of the sbcB(xonA) gene product exonuclease I; in the latter case,activation occurs by induction of the synthesis of the recE

* Corresponding author.

gene product, exonuclease VIII, by sbcA mutations (22-24).These experimants suggest that there are at least two path-ways, the RecBC pathway and the RecF pathway, forconjugation-mediated recombination in E. coli.

Genetic analysis of plasmid recombination has demon-strated that the frequency of plasmid recombination eventsin wild-type E. coli strains is decreased by recA, recF, andtopA mutations (8, 12, 13, 17, 25). recB and recC mutationsstimulate plasmid recombination and alter the distribution ofrecombination products obtained (17). The available datasuggest that plasmid recombination in wild-type E. colistrains appears to be catalyzed by a pathway that is similarto the RecF pathway, even though the RecF pathway onlyinfrequently (<1%) catalyzes conjugation-mediated recom-bination events in wild-type E. coli strains (5, 16). In recBrecC sbcA strains, the frequency of plasmid recombinationevents is 20 times higher than in wild-type E. coli strains andis decreased by recE mutations, whereas recA and recFmutations have no effect (8, 12, 17, 25; Joseph, Ph.D. thesis).Similar results have been obtained when recombination ofbacteriophage A red gam was studied in recB recC sbcA E.coli strains (14). This third recombination pathway, whichhas been designated the RecE recombination pathway, ap-pears to catalyze recombination events beween plasmidDNAs or between phage DNAs more efficiently than doesthe RecF-like pathway. Recently, we demonstrated that thisrecombination pathway catalyzes intramolecular recombina-tion of linearized plasmid dimers 20 to 50 times morefrequently than recombination of circular dimers (L. S.Symington, P. T. Morrison, and R. Kolodner, J. Mol. Biol.,in press). In contrast to this, the RecE pathway appears tocatalyze conjugation-mediated recombination events lessefficiently than does the RecF pathway, if it does so at all.

Although the observation that recF mutations decreasethe frequency of plasmid recombination events in otherwiseRec+ E. coli strains suggests that plasmid recombinationevents are catalyzed by the RecF recombination pathway,other RecF pathway mutations must be tested to substanti-

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GENETIC RECOMBINATION OF BACTERIAL PLASMID DNA

ate this conclusion. In this communication we demonstratethat only a subset of the RecF pathway genes is required forplasmid recombination and that plasmid recombination isregulated as part of the recA/lexA regulon. In addition, wedescribe the isolation of a TnS insertion mutation in whatappears to be a new RecF pathway gene.

MATERIALS AND METHODS

Bacterial strains. The bacterial strains used in this study andtheir derivations and relevant genotypes are listed in Table 1.Note that strain AB1157 contains the kdgKSJ mutation,which is not generally listed as being present in the strain (32).The phenotypic abbreviations used are as follows: Leu,leucine; Thr, threonine; Phe, phenylalanine; Mal, maltose;Srl, sorbitol; Kdg and Eda, glucuronate utilization; Nad,NAD; Pur, purine; Pyr, pyrimidine; Rec, recombination;mit-C, mitomycin C; UV, UV irradiation; Ap, ampicillin; Tc,tetracycline; Km, kanamycin; Sm, streptomycin. Thesuperscripts used with the phenotypic abbreviations are thefollowing: +, independent when used with the abbreviationof an amino acid, utilizing when used with the abbreviationof a sugar, and proficient when used with Rec; -, requiring,nonutilizing, or deficient, respectively; r, resistant; s,susceptible. The presence of mutations that affect recombi-nation and repair was confirmed by performing backcrossesto appropriate recipient strains. P1 vir was used in all P1transductions. X419 b221 cI857 Pam80 rex: :Tn5 was obtainedfrom M. Syvanen and used in TnS mutagenesis experiments.

Media. L broth was used routinely to grow bacterialcultures. Platings were made on T broth, L broth, or VBminimal plates (40) containing 1.5% agar. Media were sup-plemented with amino acids and vitamins at a final concen-tration of 50 ,ug/ml and with sugars at a final concentration of0.2% as required. Mitomycin C, tetracycline, kanamycin,ampicillin, and streptomycin were added to media at finalconcentrations of 1, 15, 30, 50, and 100 ,ug/ml, respectively.Dilutions of phage lysates and bacterial cell suspensionswere made in 0.15 M NaCl.

Genetic methods. Replica plate tests for recombinationproficiency and UV sensitivity were carried out as describedpreviously (6). The frequency of plasmid recombination withpRDK41 as the substrate DNA was determined as describedpreviously (12, 17). P1 transductions were carried out ex-actly as described by Miller (33) except that a multiplicity ofinfection of 0.1 to 0.01 was generally used. Bacterial matingin liquid media was carried out as described by Miller (33)with a donor/recipient ratio of 1:10 with a recipient celldensity of 2 x 108 cells per ml. Matings with Hfr strains werecarried out for 20 min, and matings with F'-containingstrains were carried out for 60 min before interrupting andplating. UV survival assays with bacterial cells grown inliquid media were also carried out as described by Miller(33). TnS mutagenesis was carried out by infecting JC11450with X419 at a multiplicity of 0.2. After absorption at 30°C for20 min, the suspension of infected cells was diluted 1:4 withL broth and incubated at 37°C with shaking for 30 min. Then,appropriate dilutions were plated on L broth plates contain-ing kanamycin; after incubation at 30°C for 24 h, 10,000 Kmrcolonies were pooled and used for further experiments asdescribed below.

RESULTS

Isolation and mapping of recN and recO insertion muta-tions. P1 phage were propagated on a pool of cells containing

10,000 independent TnS insertion mutations, and thesephage were used to transduce E. coli RDK1486 recB21recC22 sbcBJS tyrA4 to Tyr'. A total of 600 Tyr' Kmrtransductants were then screened for their ability to serve asrecipients in Hfr crosses and for their susceptibility to killingby mitomycin C and UV irradiation. After retesting, fourRec- UV' mit-Cs transductants were obtained; one,RDK1563, was strikingly more Rec- UV' mit-Cs than therest. P1 phage were propagated on these four E. coli strainsand used to backcross their respective TnS insertion muta-tions into E. coli RDK1486. The transduction data showedthat the three mutations having the least stringent Rec- UVsmit-Cs phenotype cotransduced with tyrA with a frequencyof 59%o and were tentatively called recN1501, recN1502, andrecN1503. The fourth mutation cotransduced with tyrA at afrequency of 28% and was tentatively called recO1504.Two mutations, recN1502 and recO1504, were selected for

further study. P1 lysates prepared with E. coli strainscontaining these mutations were used to cross these muta-tions into derivatives of E. coli AB1157 that were also eithertyrA4, pheAJ8::TnJO, or srl-300::TnlO. All (>98%) of theKmr transductants obtained with P1 phage grown on therecN1502 mutant had a Rec+ UVr mit_Cs phenotype. Anal-ysis of the linkage data obtained in these experimentsindicated that this mutation mapped in the interval of thestandard E. coli genetic map that lies between srl andpheAltyrA (Table 2). The linkage data obtained withrecNS502::TnS and the effect of recNS502::TnS recombina-tion, UV sensitivity, and mitomycin C susceptibility corre-spond closely to the results obtained with previously isolatedrecN mutations. This suggests that this mutation and theother two mutations discussed above (recN501::TnS andrecNS503: :TnS) are a result of insertion of TnS into the recNgene (27). In contrast to this, all of the Kmr transductants ofAB1157 obtained with P1 phage grown on the recO1504mutant had a Rec+ UV, mit-Cr phenotype. Analysis of thelinkage data obtained in this experiment suggested thatrecOIS04::Tn5 did not map between srl and tyrAlpheA, butrather mapped in an interval located counterclockwise oftyrAlpheA, on the standard E. coli genetic map. The differ-ences in the phenotype caused by recO1504::Tn5 and itsdifferent apparent map location compared with the recNmutations suggests that this mutation is a result of insertionof Tn5 into a different RecF pathway gene.To more accurately determine the map location of the

recO1504::Tn5 mutation, two different three-factor crossesbetween a purI66 nadB4 recipient and P1 phage grown on arecO1504::Tn5 donor strain were carried out (Table 3).These results are most consistent with a gene order of purIrecO nadB, because this gene order explains the observedresults with the least number of abberant recombinationevents (2% quadruple crossovers) compared to any otherpossible gene order (7% quadruple crossovers for recO purInadB and 60% quadruple crossovers for purI nadB recO).The data presented in Table 3 have been analyzed by themethod of Wu (43) to determine the map distances betweenrecO and purI, between recO and nadB, and between nadBand purL. This analysis indicates that recO is located 0.2 minfrom purI and 0.3 min from nadB. These map distances areconsistent with the proposed gene order and are inconsistentwith the other gene orders discussed above. These dataindicate that recO1504::Tn5 maps at 54.4 min on the stan-dard E. coli genetic map (Fig. 1). This location has beenconfirmed by studies with a cloned recO gene (unpublishedresults of L. Gilson and R. Kolodner).

Effect of recN and recO mutations on recombination and

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1062 KOLODNER, FISHEL, AND HOWARD

TABLE 1. E. coli strains

Relevant genotype Additonal mutations present Source

Rec+

HfrH(PO1)tyrA4zib-205::TnlO pyrElexA3sulA

lexA71::TnS

recA13recB21 recC22 sbcB15recF143A(srlR-recA)304Rec+recAo281recJ153ruvB9F' 104(PO1)

ssb-113srl-300: :TnlOpheA18: :TnJOpurI66 nadB4

ssb-113lexA71::Tn5lexA71::Tn5 recAJ3lexA3lexA71::TnS recF143recAo281recB21 recC22 sbcB15 pheA18::TnlOrecB21 recC22 sbcBJ5 tyrA4srl-300::TnlOpheA18::TnlOtyrA4purI66 nadB4recN1502: :TnSrecO1504: :TnSRec+recN1502::Tn5recO1504::Tn5ruvB9recN1501::TnSrecN1502: :Tn5recN1503: :TnSrecO1504::Tn5recN1501: :TnSrecN1503::TnSmalE::TnlO

argE3 his4 leu-6 proA2 thr-l thi-l rpsL31 gaIK2lacYl ara-14 xyl-5 mtl-l supE44 kdgK51

thi-J rel-Ithi-l relAl spoTIgltq7lO metB thi-l lac rpsLAsor AB1157As for AB1157, except also met and His' Arg+Lac'

recA441 sulAIl AlacUl69 thr-l leu-6 his4 argE3ilv(Ts) galK2 rpsL31

As for AB1157As for AB1157As for AB1157As for AB1157As for AB1157, except Sup'As for AB1157As for AB1157As for AB1157, except also zla-3::TnJO eda-50thr-l leuB6 proA2 his4 recA13 argE3 thi-1 ara-14

lacYl galK2 xyl-7 mtl-l rpsL31 tsx33 supE44melA thyA rhaHfr (PO of KL16) thi-l relAlHfr (P01) A(gpt-lac)5 relAl spoTI thi-)argHI thi-l ara-13 lacYl gal-6 xyl-7 mtl-2 rpsL9tonA2 Ar supE44

As for AB1157As for DM1202As for DM1202As for AB1157As for DM1202As for AB1157As for JC7623As for JC7623As for AB1157As for AB1157As for AB1157As for AB1157As for JC7623As for JC7623As for AB1157, except KdgK+As for AB1157As for AB1157As for RDK1534As for JC7623As for JC7623As for JC7623As for JC7623As for JC7623As for JC7623araDI39 AlacU169 rpsL relA fibB deoC

C. C. Richardson (1, 32)

Brooks LowB. BachmannB. BachmannA. J. Clark (35)D. Mount (34)

G. Walker (21)

A. J. Clark (6)A. J. Clark (16)A. J. Clark (16)A. J. Clark (9)A. J. ClarkA. J. Clark (41)A. J. Clark (16, 29)S. Lovett (37)B. Bachmann (30)

C. C. Richardson (18)M. SyvanenN. KlecknerB. Bachmann

Original mutant, this studyOriginal mutant, this studyOriginal mutant, this studyOriginal mutant, this study

T. Silhavya AB1157 was transduced to MalP Tcr with P1 grown on TST1 to yield RDK1304. This strain was then transduced to Mall Tcs UVS with P1 grown on KLC484

to yield RDK1309.b DM1202 was transduced to Mal Tcr with P1 grown on TST1 to yield RDK1393. This strain was then transduced to Mal Tcs Kmr with P1 grown on GW2730

to yield RDK1397. The presence of lexA71::Tn5 in all strains was confirmed by backcrossing it into GW1040 dinDI::Mu(ap lac) and testing for induction of -

galactosidase (21).c RDK1397 was transduced to SrlP Tcr with P1 grown on MS398 to yield RDK1404. This strain was then transduced to Srl+ Tcs UVS with P1 grown on JC2926 to

yield RDK1416.d Mal' Tcs UV' transductant of RDK1304 from P1 grown on JC11457. See footnote a for derivation of RDK1304.' DM1202 was transduced to zib-205::TnlO pyrE with P1 grown on BW229 to yield RDK1422. This strain was transduced to Pyr+ Tcs UV' (recFI43) with P1

grown on JC9239 to yield RDK1424. This was then successively transduced to Mal- Tcr (RDK1427) and then to Mal' Tcs Kmr (RDK1428) as described in footnoteb.f Srl+ Tcs UV' transductant of RDK1489 from P1 grown on JC11457.8 Phe- Tcr transductant of JC7623 from P1 grown on NK6204.Phe+ Tcs Tyr- transductant of RDK1481 from P1 grown on AT2471.Srl- Tcr transductant of AB1157 from P1 grown on MS368.Phe- Tcr transductant of AB1157 from P1 grown on NK6024.

k Tyr- Phe+ Tcs transductant of RDK1490 form P1 grown on AT2471.'Tyr' Pur- Nad- transductant of RDK1498 from P1 grown on PA3306.m Tyr' Kmr Rec- UV' mit-Cs transductant of RDK1486. P1 was grown on RDK1560 for RDK1564, RDK1561 for RDK1530, RDK1562 for RDK1566, and

RDK1563 for RDK1531.n KdgK+ transductant of AB1157 from P1 grown on AB259.Tyr' Kmr mit-Cs transductant of RDK1498 from P1 grown on RDK1560.

P Tyr' Km' UVS transductant of RDK1498 from P1 grown on RDK1563.q RDK1534 was transduced to UV' Eda- Tcr with P1 grown on JC14297 to yield RDK1539. This strain was then transduced to Eda+ Tcs UV' with P1 grown on

AB1157 to yield RDK1542.

Strain

AB1157

AB259AT2471BW229DM49DM1202

GW2730

JC2926JC7623JC9239JC10287JC11450JC11457JC13031JC14297KL723

KLC484MS368NK6024PA3306

RDK1309aRDK1397bRDK1416CRDK1417dRDK1428eRDK1429VRDK14819RDK1486RDK1489RDK1490'RDK1498kRDK1521'RDK1530mRDK1531mRDK1534nRDK15400RDK1541PRDK1542qRDK1560RDK1561RDK1562RDK1563RDK1564RDK1566TST1

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GENETIC RECOMBINATION OF BACTERIAL PLASMID DNA

TABLE 2. Percent cotransduction of pheA srl and tyrA to recNand recO% Cotransduction with selected markerb

Unselected markererecN1502::TnS recO1S04::Tn5

PheA+ 37 8TyrA+ 33 7Srl+ 4 <1C

a Kms recipient strains RDK1490, RDK1498, and RDK1489 were used.They carried, respectively, pheA18::TnlO, tyrA4, and srl-300::TnlO,

b In each case 100 Km' trarnsductants were tested for prototrophy orsorbitol utilization by replica plating.

c No Srl+ transdiictants were found.

repair. The effect of both recN1502: :TnS and recO1504: :TnSon the ability of E. coli JC7623 recB21 recC22 sbcBJS and E.coli AB1157 to serve as a recipient during a standard Hfrcross with HfrH was tested (Table 4). When present inJC7623, the recN mutation reduced the formation of Leu+recombinants 50-fold, whereas the recO mutation reducedthe formation of Leu+ recombinants 1,000-fold. The recOmutation, when present in JC7623, decreased the yield ofLeu+ transconjugants 10-fold in crosses with E. coli F' 104,suggesting that recO mutations might only decrease thefrequency of recombination 100-fold in this genetic back-ground. A similar effect has been observed with ruv muta-tions (26). Neither mutation appeared to have any effect onthe formation of Leu+ recombinants when present in E. coliAB1157. These results confirm previous results with recNmutations and indicate that the recO mutation affects recom-bination during Hfr-mediated crosses like other RecF recom-bination pathway mutations.The effect of both the recN and recO mutations on killing

by both UV irradiation and mitomycin C in both recB recCsbcB and Rec+ backgrounds was determined (Fig. 2, Table5). The recN mutations studied conferred susceptibility tokilling by mitomycin C in both Rec+ and recB recC sbcBbackgrounds and only conferred UV sensitivity in the recBrecC sbcB background. In contrast to this, the recO muta-tion only conferred mitomycin C susceptibility in the recBrecC sbcB background and conferred UV sensitivity in boththe Rec+ and recB recC sbcB backgrounds. In all experi-ments where the recO mutation had an effect on repair, itseffect was much greater than the corresponding effect of therecN mutation tested. These data indicate that recN andrecO mutations have very different effects on DNA repair.

Effect of RecF recombination pathway mutations on plas-mid recombination. We have determined the effect of avariety of RecF recombination pathway mutations on theability of the plasmnid pRDK41 to form Tcr recombinants

TABLE 3. Cotransduction of purI and nadB to recO

Selected Classes of transductants obtainedbmarkera

recOQ504::TnS 20.3% PurI+ NadB+, 60.1% PurI+ nadB4, 7.0opurI66 NadB+, 12.6% purI66 nadB4

PurI+ 12% NadB+ recO1504::Tn5, 2% NadB+ RecO+,42% nadB4 recO1504::TnS, 44% nadB4 RecO+

The Kms recipient strain was RDK1521 that carried purI66 and nadB4.The donor strain was RDK1541 that carried recO1504::TnS.

b In the first experiment, 143 Kmr transductants were tested for prototro-phy or UV sensitivity by replica plating. In the second experiment 200 Pur+transductants were tested for kanamycin resistance, UV sensitivity, orprototrophy by replica plating.

55 56

I I,p'J ruco ridB

5857Jk I I

pheA twA rwNF

FIG. 1. Relative position of recO15(4::Tn5 on the standard E.coli linkage map. The map coordinates were those of Bachmann (2).

during propagation in E. coli AB1157 derivatives containingindividual RecF pathway mutations. The results (Table 6)demonstrated that, in addition to the previously tested recAand recF mutations, the recJ153, recO1504: :TnS, andssb-113 mutations all decreased the frequency of plasmidrecombination substantially. The 10,000-fold decrease ob-served with recJ153 is the largest effect on plasmid recom-bination observed with any mutation. In contrast, the lexA3,recN1502::Tn5, and ruvB4 mutations had no effect on plas-mid recombination.Because of the apparent interrelationship between plasmid

recombination and the RecF pathway for Hfr-mediatedrecombination and the observation that the RecF pathway isregulated by lexA, we have also investigated the effect ofmutations that alter the expression of the recA/lexA regulonon plasmid recombination (Table 6). The lexA71::Tn5 muta-tion that derepresses the recAllexA regulon increased thefrequency of plasmid recombination 10-fold. A 10- to 30-foldstimulation was also observed in the recA441 (tif-1) lexA3lexASI (sprSl) strain DM1187 (34), in which the recAllexAregulon is also derepressed (data not shown). The recAo281mutation, which only derepresses the synthesis of recAprotein, had no effect on plasmid recombination, indicatingthat increased synthesis of recA protein was insufficient tostimulate plasmid recombination. The increased frequencyof plasmid recombination induced by the lexA71::TnS muta-tion was reduced by recA and recF mutations by an extentthat was similar to the effect of these mutations on the levelof plasmid recombination observed in LexA+ strains.

DISCUSSION

To determine the relationship between plasmid recombi-nation events in wild-type E. coli strains and the RecFrecombination pathway, we have tested the effect of avariety of RecF pathway mutations on plasmid recombina-tion. During the isolation of TnS insertion mutations in therecN gene for use in this study we isolated a single TnSinsertion mutation mapping at 55.4 min on the standard E.

TABLE 4. Effect of recN and recO on Hfr-mediatedrecombination

Relative yield of transconjugants'

Mutation tested RecB+ RecC+ recB21 recC22SbcB+b sbcBJ5c

recN recO F' Hfr F' Hfr

+ + 1.0 1.0 1.0 1.01502::TnS + 0.78 1.74 1.49 0.024

+ 1504::Tn5 0.87 0.88 0.11 0,001a Leu+ (Smr) transconjugants per viable recipient were selected. In all cases

more than 10%o of recipient cells were viable. Observed yields for the RecN+RecO+ recipients were as follows from left to right: 1.2 x 10-4, 4.3 x 10-3,1.7 x 10-3 and 1.5 x 10-2. The F' and Hfr strains were, respectively, KL723(F104) and AB259 (PO1).

b Recipient strains were AB1157, RDK1540, and RDK1541.c Recipient strains were JC7623, RDK1530, and RDK1531.

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TABLE 5. Effect of mitomycin C on plating efficiency

Mutation tested Fraction survival on 1 ,ug ofmitomycin C per ml'

RecB+ recB21recN recO RecC+ recC22

SbcB+b sbcB15J

+ + 8.0x 10-' 5.2x 10'1502::Tn5 + 3.5 x 10-3 2.4 x 20-4d

+ 1504::Tn5 5.0 x 10-l 4.1 x 10-8a Cells were grown in L broth at 37°C with aeration until they reached a

density of 5 x 10i viable cells per ml. Culture dilutions were plated on L agarwith or without 1 jig of mitomycin C per ml. The titer determined on theformer plates divided by that determined on the latter is reported as fractionsurvival.

bStrains used were AB1157, RDK1540, and RDK1541.c Strains used were JC7623, RDK1530 and RDK1531.d Similar results were obtained with RDK1564 and RDK1566 carrying

recN1501::TnS and recNIS03::TnS, respectively.

coli genetic map that appears to be located in a previouslyunidentified RecF recombination pathway gene. This genehas tentatively been called recO. Like other RecF pathwaymutations, the recO1504::TnS mutation decreased the fre-quency of Hfr-mediated recombination 1,000-fold when pre-sent in a recB recC sbcB background, but had no effect whenpresent in an otherwise Rec+ background. TherecOJSO4::TnS mutation decreased the recovery of F' trans-conjugants 10-fold in a recB recC sbcB recipient, an effectthat could be due to either an inability to maintain an F'factor or a decreased ability to participate in conjugation.Thus, after correction for a possible defect in conjugation,the recO mutation decreased Hfr-mediated recombination atleast 100-fold in a recB recC sbcB background.The recO1504::TnS mutation also had an effect on DNA

repair that was typical of other RecF pathway mutations. Itrendered recB recC sbcB strains extremely susceptible tokilling by UV irradiation and mitomycin C, but only ren-dered otherwise Rec+ strains moderately sensitive to killingby UV irradiation. One of the striking features of the RecFpathway mutations is the broad variety of effects that theyhave on DNA repair when present in Rec' backgrounds

100.0

h1.0

0.1

0.01 . . , . . . . .0 10 20 30 40 50 10 20 30 40 50

UV Irradiation (J/Mt)FIG. 2. Effect of recN and recO mutations on UV sensitivity. (A)

0, AB1157; *, RDK1540 recN1502::Tn5; A, RDK1541recOJ5O4::TnS. (B) 0, JC7623 recB21 recC22 sbcB15; 0, RDK1530recB2J recC22 sbcB15 recN1502::TnS; A, RDK1531 recB21 recC22sbcBIS recOl504::Tn5.

while having no effect on Hfr-mediated recombination.These effects range from no effect for recJ and recQ muta-tions (29, 36) to causing either sensitivity to UV irradiation inthe case of recO mutations (Table 6), susceptibility tomitomycin C for recN mutations (27), or susceptibility tokilling by both agents in the case of recF and ruv mutations(7, 16, 26) (Table 6). The nature of the relationship betweenthe repair of these different types of DNA damage andHfr-mediated recombination and plasmid recombination isunclear at present.The observation that the frequency of plasmid recombina-

tion in wild-type E. coli strains is decreased by recF singlemutations suggested that plasmid recombination is catalyzedby the RecF recombination pathway. In this study, we havedemonstrated that some RecF pathway mutations, recJ andrecO, decrease the frequency of plasmid recombination,whereas other RecF pathway mutations, lexA3, recN, andruv, had no effect. The failure of the IexA3 mutation to affect

TABLE 6. Effect of RecF pathway mutations on plasmid recombinationLexA+ background lexA 71: :TnS background

Mutation mutationa % Tc' Relative % Tcr Relativecolonies frequency colonies frequency

None 0.091 100 0.76b 100

recA13 S 0.0028 3.1 0.014 1.8A(recA-srlR)304 S 0.0051 5.6 ND'recF143 S 0.00033 0.36 0.038 0.5recJ153 S 0.000022 0.024 NDrecO1504::Tn5 S 0.0019 2.1 NDssb-113 S 0.00056 0.62 ND

recAo281 NS 0.042 46.1 NDrecN1502::TnS NS 0.093 102 NDlexA3 NS 0.083 91.2 NDruvB9 NS d 87.8d ND

a S5 Significant effect; NS, no significant effect.b DM1202, the parent of RDK1397 lexA71::TnS, yielded 0.097% Tcr colonies.c ND, Not determined.d ruvB9 is in a KdgK+ background. Since AB1157 is a kdgK mutant we first made a KdgK+ derivative. This strain, RDK1534, showed twofold more Tcr

recombinants (0.18%) than AB1157. The ruvB9 derivative, RDK1542, showed 0.16%.

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GENETIC RECOMBINATION OF BACTERIAL PLASMID DNA

plasmid recombination is not surprising, since it is thought toblock the induction of the RecF pathway during conjugation,and no inducing treatment was used during the measurementof the frequency of plasmid recombination (27, 28). Theresults obtained with the lexA3 mutation suggest that theeffect of the ssb-113 mutation on plasmid recombination is aresult of some direct involvement of single-stranded DNA-binding protein in plasmid recombination rather than someindirect involvement by blocking the induction of therecAllexA regulon (3). Lloyd and his co-workers have sug-gested that the recN and ruv gene products are involved inthe recombination repair of double strand breaks in DNA(26, 27, 38). If this is the case, then it is not surprising thatrecN and ruv rmutations had no effect on plasmid recombi-nation, because we have shown that the presence of double-strand breaks in plasmids does not appear to affect theirfrequency of recombination (Symington et al., in press).Although the results presented here indicate a relationshipbetween the RecF pathway for Hfr-mediated recombinationand plasmid recombination in wild-type E. coli strains, thelack of a one-to-one correspondence between the geneproducts involved suggests that the two processes mayproceed by different mechanisms. The finding of such differ-ences is not surprising, given the differences in the DNAmolecules that are undergoing recombination: duplex circu-lar DNA in the case of plasmid recombination and a single-stranded donor and a duplex recipient in the case of theRecF pathway for Hfr-mediated recombination (31). How-ever, the proposed mechanisms for these two recombinationreactions have some similarities, in that they both involvethe formation of long regions of heteroduplex DNA (10, 31).The observation that mutations that derepress the

recAllexA regulon also stimulate plasmid recombination isconsistent with the relationship between the RecF recombi-nation pathway and plasmid recombination discussed above(27, 28). Repression by lexA and the absence of an inducercould explain why the frequency of plasmid recombinationin wild-type E. coli strains is so low compared with Hfr-mediated recombination events catalyzed by the RecF path-way (27, 28). The stimulation of plasmid recombination bythe lexA71 mutation and by the recA441(tifl) lexA3lexA5J(sprSl) mutation combination is not just due to in-creased expression of the recA gene, because recA operatormutations that only overproduce the recA protein did notstimulate plasmid recombination. The recN and ruv genesare the only other RecF pathway genes that have beenshown to be regulated by lexA to date (26, 27). This suggestseither that the recN and ruv gene products are only requiredfor plasmid recombination when recombination is dere-pressed or that some other gene involved in plasmid recom-bination is regulated by lexA. The recO gene or otherunidentified genes are possible candidates for this regulatedgene. We are presently attempting to identify this gene(s).

ACKNOWLEDGMENTS

We thank A. J. Clark, B. Bachmann, B. Low, N. Kleckner, M.Syvanen, T. Silhavy, C. C. Richardson, S. Lovett, D. Mount, and G.Walker for the strains they provided. We would also like to thank B.Bachmann, A. J. Clark, S. Lovett, and B. Low for helpful advice anddiscussions.

This work was supported by Public Health Service grantGM26017 from the National Institutes of Health and ACS FacultyResearch Award FRA-271 to R.D.K. and by Public Health Servicepostdoctoral traineeship CA09361 from the National Institutes ofHealth to R.A.F.

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