JOURNAL OF BACTERIOLOGY, June 1978, p. 958-9660021-9193/78/0134-0958$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 134, No. 3

Printed in U.S.A.

Recombination Levels of Escherichia coli K-12 MutantsDeficient in Various Replication, Recombination,

or Repair GenesJANINE ZIEG, VALERIE F. MAPLES, AND SIDNEY R. KUSHNER*

Program in Genetics, Department ofBiochemistry, University of Georgia, Athens, Georgia 30602

Received for publication 20 December 1977

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE,lig, polA, dam, or xthA were constructed and tested for conjugation and trans-duction proficiencies and ability to form Lac' recombinants in an assay systemutilizing a nontandem duplication of two partially deleted lactose operons(lacMS286k80dIIlacBKI). lexA and rep mutants were as deficient (20% of wildtype) as recB and recC strains in their ability to produce Lac' progeny. All theother strains exhibited increased frequencies of Lac' recombinant formation,compared with wild type, ranging from 2- to 13-fold. Some strains showedmarkedly increased conjugation proficiency (dam uvrD) compared to wild type,while others appeared deficient (polA107). Some differences in transductionproficiency were also observed. Analysis of the Lac+ recombinants formed by thevarious mutants indicated that they were identical to the recombinants formedby a wild-type strain. The results indicate that genetic recombination in E. coliis a highly regulated process involving multiple gene products.

The study of genetic recombination in Esch-erichia coli K-12 has been based primarily onobserved alterations in either conjugation ortransduction proficiency (6). Since recipientstrains carrying mutations in recA, recB, or recCyield relatively few recombinant progeny afterconjugation (100- to 1,000-fold reduction) (7, 32,35), these gene products likely mediate reactionsessential for recombinant formation. On theother hand, the rep and lexA genes appear to bemore involved in repair or replication processes,since they exert only minor alterations in recom-bination after conjugation (4, 25).The development by Konrad (15) of a strain

of E. coli carrying a specially constructed dupli-cation of the lactose operon (lacMS286480dII-lacBKI) has provided an additional means ofstudying recombination. Since the 480dlI-lacBKI contains a small deletion in the proximalportion of the lacZ gene, and lacMS286 is de-leted in the distal portion of lacZ, Lac' recom-binants occur at low frequencies. Konrad andLehman used this strain to isolate derivatives ofE. coli with increased frequencies ofLac' recom-binant formation ("hyper-Rec") (16, 17). Re-cently Zieg and Kushner (35) have demonstratedthat the increases or decreases in the number ofLac+ recombinants obtained in the presence ofa specific mutation provide a sensitive measureof genetic recombination within the cell. Thisassay therefore appeared useful as a means of

studying the regulation of genetic recombinationin E. coli. Accordingly, a series of isogenic strainscarrying mutations in rep, lexA, xthA, uvrD,uvrE, polA, dam, ig, and uvrA were constructedand tested for conjugation, transduction, and lacgene recombination proficiency. The results pre-sented in this paper suggest that most of theabove gene products are involved in genetic re-combination either directly or indirectly.

MATERIALS AND METHODSMaterials. Reagents were obtained from the fol-

lowing sources: lactose, glucose-free, U.S. BiochemicalCorp.; 2,3,5-triphenyl-2H-tetrazolium chloride, methylmethane sulfonate, and ethyl methane sulfonate, East-man Kodak Co.; streptomycin sulfate and amino acids,Sigma. Spectinomycin sulfate was the generous gift ofThe Upjohn Co. All other chemicals were of reagentgrade.

Bacterial strains and bacteriophages. Bacterialstrains are listed in Table 1. Nomenclature conformsto that of Demerec et al. (9). Gene symbols are thoseused by Bachmann et al. (1). Figure 1 shows thelocations of the various genes and Hfr's described inthe paper. All strains were constructed by either con-jugation or Plvir transduction. Inheritance of uvrD,uvrE, lexA, uvrA, and dam mutations was detectedby sensitivity to UV light using the replica-platingtechniques of Clark and Margulies (7). The presenceof rep alleles was measured by the inability to supportP2vir22. poLA and xthA mutations were analyzed bysensitivity to 0.08% methyl methane sulfonate in Luria

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GENETIC RECOMBINATION IN E. COLI 959

TABLE 1. Bacterial strainsSex arg his thr klu pro thi rpsE" rpsLb sup lac Other markersF- E3 4 1 6F- E3 4 + +F- E3 4 1 6

F- - 4 + +F- E3 4 + +F- E3 4 1 6F- E3 4 1 6F- E3 4 1 6F- + + + +F' + + + +F- + + + +F- + + + +Hfr + + + +F- E3 4 1 6F- E3 4 1 6

F- E3 4 1 6Hfr + + + +

A2 1

A2 I

A2 I

A2 I

A2 -

A2 1

A2 1A2 I

+ +

+ +

+ +

+ -

+ -

A2 1

A2 1

A2 1

+

Source or derivation+ 31 37 Y) A. J. Clark+ + ? - aroD6 A. J. Clark+ 31 37 Y) uvrA6 thyA R. Cole

deoB+ + ? Y) metA28 B. Bachmann+ - ? - ilvpabA1 G. Tritz+ 31 37 Y) xthA1 B. Weiss+ 31 37 Y) AxthA 4ipncA B. Weiss+ 31 37 Y) lexA3 D. Mount+ - ? + trp uvrD3 D. Young+ + ? + F'Iac A. J. Clark+ + 19 - ilvD)88 uvrE4' R. Cole+ + ? + dam-3 M. Marinus+ + + - serA A. J. Clark+ 31 37 Y) cysC43 A. J. Clark+ 31 37 Y) metE46 recB21 A. J. Clark

recC22 sbcB)5+ 31 37 Y) ilvA A. J. Clark+ + + + B. Bachmann

KL209 Hfr + + + + + + + + + + B. BachmannKMBL1789 F- A101 + + + + + + + + + pheA97 bio-87 B. Glickman

endAlO1poLA107

KMBL1493 F- AIOI + + + + + + + - + pheA97 uvrD)O) B. Glickmanbio-87 endAlO)

KS244 Hfr + + + + + I + + + + lig-7(Ts) E. B. KonradKS391 Hfr + + + + + + + + + BKid E. B. KonradPM5 F- + + + + + + + + + + rep-5 R. CalendarRa-2 Hfr + + + + + + + + + + B. BachmannRep-3 F- + + + + + + + + - + rep-3 A. J. ClarkRS9 F- + + + + + + + - ? BK) uvrElOO E. B. KonradRS5033a F- + + + + + + + - + BK) dam-4 E. B. KonradSK212 F- E3 4 + + + 1 + 31 + BK) ilvA JC8403 x KS391

Pro+ conjugantSK217 F- E3 4 + + + I + 31 + BK) uvrE)(X SK212 x ES271Iv+

transductantSK236 F- E3 4 + + + I + 31 + BK) metE46 SK212 x JC4729

llv+ tranaductantSK246 F- E3 4 + + + I + 31 + BK) uvrD3 SK212 x DY81 Ilv+

transductantSK274 F- E3 4 + + A2 1 - + ? - aroD6 RpsE- derivative of

AB1360SK276 F- E3 4 + + + 1 + 31 + BK) poIA107 SK236 x

KMBL1479MetE+ transduc-tant

SK277 F- E3 4 + + + I + 31 + BK) SK236 xKMBL1479MetE+ transduc-tant

SK285 F- E3 4 + + + I + 31 + BK) rep-3 SK212 x Rep-3Ilv+transductant

SK287 F- E3 4 + + + I + 31 + BK) SK212 x Rep-3Ilv+transductant

SK329 F- E3 4 + + + - + - + BK) pabA) AB3292 x KS391Pro+ conjugant

SK336 F- + 4 + + + - + 31 + BK) ilvA metA28 SK212 x AB2569ArgE+ transduc-tant

SK343 F- E3 4 + + + 1 - + + BK) aroD6 SK274 x KS391ProA conjugant

SK366 F- E3 4 + + + 1 + 31 + BK) IexA3 ilvA SK336 x DM49MetA+ transduc-tant

SK368 F- E3 4 + + + 1 + 31 + BK) ilvA SK336 x DM49MetA+ transduc-tant

SK377 F- E3 4 + + + I - + + BK) SK343 x BW9091AroD+ transduc-tant

StrainAB1157AB1360AB2500

AB2569AB3292BW9091BW9101DM49DY81E3ES271GM33JC158JC2915JC4729

JC8403KL96,7 tlW

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960 ZIEG, MAPLES, AND KUSHNER

Table 1-ContinuedStrain Sex arg his thr ku pro thi rpsEa rpaL' sup lac Other markers Source or derivation

SK383 F- E3 4 + + + - + - + BKI dam-4 SK329 x R95033aPabA+ tranaduc-tant

SK385 F- E3 4 + + + - + - + BKI dam-3 SK329 x GM33PabA+ tranaduc-tant

SK395 F- + 4 + + + I + 31 + BKI uvrA6 ilvA SK336 x AB2600MetA+ tranaduc-tant

SK703 F- E3 + + + + I + 31 + BKI metE46 lig- 7(Ts) SK236 x KS244His+ conjugant

SK705 F- E3 + + + + I + 31 + BKI metE46 SK236 x KS244His+ conjugant

SK866 F- E3 4 + + + I + 31 + BKI uvrE4 SK212 x ES271IlvA+ transduc-tant

SK1175 F- E3 4 + + + I + 31 + BKI rep-5 SK212 x PM5 Ilv+tranaductant

SK1314 F- E3 4 + + + I - + + BKI xthAI SK343 x BW9091AroD+ tranaduc-tant

SK1328 F- E3 4 + + + I - + + BKI Axth SK343 x BW9101AroD+ transduc-tant

SK1455 F- E3 4 + + + I + 31 + - Lac- revertant fromLac+ recombi-nant of SK287

SK1466 F- E3 4 + + + I + 31 + - Lac- revertant fromLac+ recombi-nant of SK287

SK1468 F- E3 4 + + + I + 31 + - rep-3 Lac- revertant fiomLac+ recombi-nant of SK256

SK1469 F- E3 4 + + + + + 31 + - uvrD3 Lac- revertant fromLac+ recombi-nant of SK246

a Formerly stA.b ForMerly pc.Formely nwtU4.dBK1 indicates presence of lacMS2864)dIIlacBKI.

agar plates. The duplication of the lactose operon usedin this work was that of Konrad (15) and Konrad andLehman (16) and was detected as described previously(35). For introduction of the lactose operon duplica-tion, 2-h matins were employed.

Bacterial viruses were obtained from the followingsources: 480am2, Ethan Signer, Plvir, A. J. Clark;T4D, T4B22, and T4N82, Bruce Alberta; 80vir, MaxGottesman; and P2vir22, Richard Calendar. The pres-ence of amber suppressors was tested by the ability ofstrain to plate T4B22 and T4N82 amber mutants.

Media. The complete medium used was Luria brothas described previously (19). For solid medium, 2%agar was added. Lactose- tetrazolium agar indicatorplates were prepared as described by Miller (24). Theminimal medium used for plates (M56/2) has beendescribed by Willetts et aL (31). M9 medium (30)supplemented with amino acids to a final concentra-tion of 50 pg/ml was used for liquid cultures. Lactoseminimal agar plates contained lactose as the sole car-

bon source.

Conjugation and transduction. The proceduresuaed for conjugation and transduction were those ofWilletts et aL (31). In testing for the ability of cells totake up an F' plasmid, F'iac was used. Ninety-minute

incubation times were used for these experiments. Fordetermination of quantitative transduction frequen-cies, Plvir grown on JC158 was used as the donor.Trnsduction frequencies are expressed as number oftransductants per 107 plaque-forming units. In deter-mining conjugation frequencies, Hfr donors were cho-sen which would transfer selected markers early butwhich would not transfer the wild-type alleles of themutations being examined within the 60-min matingperiod. Matings were interrupted by mechanical agi-tation after 60 min.

Lactose recombination assay. Five single colo-nies of each strain to be tested were grown individuallyin 7-ml Luria broth cultures to cell densities of ap-proximately 10" cells per ml at 37°C. Viable countswere determined on each isolate using Luria agarplates, and Lac' recombinants were measured on lac-tose minimal agar plates. Between 10i and 107 cells (inLuria broth) were plated on each minimal agar plate.All plates were incubated at 37°C (Luria agar platesfor 24 h, minimal agar.plates for 48 h). Unless other-wise noted, all plates were overlaid with soft agar. Insome cases the cells were washed three times withM56/2 buffer prior to plating on the minimal agar

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proA

FIG. 1. E. coli genetic map. Locations of genes and Hfr's described in text are presented according toBachmann et al. (1).

Other methods. Lac' recombinants were analyzedas described by Zieg and Kushner (35). Respreadingexperiments were carried out as outlined previously(35). Cell growth was followed densitometrically witha Klett-Summerson photometer (no. 42 green filter).For UV sensitivity analysis, plates were exposed toapproximately 1,000 ergs/mm2 from a 15-W GE ger-micidal lamp.

RESULTS

Recombination proficiency of strains de-ficient in UV repair. Mutations inpolA, uvrA,uvrD, uvrE, and lexA result in increased sensi-tivity to UV light, but have not generally beenassociated with genetic recombination (8, 11, 25,26, 28, 29, 34). The uvrA gene product is thoughtto introduce single-strand breaks in the vicinityof pyrimidine photoproducts (3).polA (deficientin DNA polymerase I) or uvrD strains can excisepyrimidine dimers normally or at a reduced rate,but appear deficient in repair synthesis or someother later function (2, 14, 26). uvrD and uvrEmutants exhibit increased spontaneous muta-tion rates as well as enhanced sensitivity to UVlight (28). lexA is thought to be the structuralgene for a repressor protein which controls anumber of cellular repair functions (13).

A series of isogenic strains were constructedcarrying pol107, uvrA6, uvrD3, uvrDlOl,uvrE4, uvrEl(X, or lexA3. A summary of Lac'recombinant formation and conjugation andtransduction proficiencies for these strains isshown in Table 2. With the exception of lexA3,mutations affecting the repair of UV-irradiatedDNA led to a 2- to 10-fold stimulation in theformation of Lac' recombinants over wild-typestrains. In the case of SK246 (uvrD3), SK217(uvrE4), and SK866 (uvrEl(X), conjugation andtransduction proficiencies were also enhanced.After conjugation the extent of increase ap-peared dependent both on the marker selectedand the Hfr donor employed. Ability to take upan F'lac plasmid also increased in the uvrD3mutant. SK246 (uvrD3) exhibited the largestincrease in transduction proficiency ofany straintested. In contrast, SK276 (poL4107) consist-ently demonstrated a 90 to 95% drop in conju-gation proficiency, while its efficiency of trans-duction was not affected appreciably. As shownpreviously, lexA3 strains (SK366) appeared al-most as conjugally proficient as a wild-type con-trol (25). On the other hand, the formation ofLac' recombinants was reduced dramatically, aswas transduction proficiency.

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962 ZIEG, MAPLES, AND KUSHNER

TABLE 2. Recombination proficiency of various strains

Relevant geno- No. of Lac' col- Conjugal deficiency index' F'lac inherit- Transductants/107 PFUbStrain type onies/plate ance defi-ttKL96 Ra-2 KL209 ciency index' Arg' His+ Ilv+

ASK368 Wild type 118 1 1 1 81 1,868SK395 uvrA6 237 0.7 1.2 1 105 2,182SK366 lexX3 24 1.4 1.2 1 18 1,060SK276 polA107 396 20 12 4 292 60SK277 po,A4+ 97 1 1 1 222 30SK287 uvrD' 99 (46)C 1 1 1 323 71SK246 uvrD3 479 (237)c 0.6 0.1 0.4 850 154SK1493 uvrDlOl 446 (286)CSK218 uvrE+ 172 (12) 1 1 1 149 53SK217 uvrE4 421 (154)c 0.7 0.2 1 147 76SK866 uvrElOO 420 (130)c 0.9 0.8 1 224 99

BSK287 rep+ 99 1 1 1 32 22SK285 rep-3 22 3.1 1.8 0.4 50 18SK1175 rep-5 29 1.3 3.0 58 22

CSK387 dam+ 185 (50)c 1 1 1 362 110 4,849SK385 dam-3 633 (190)C 0.33 0.16 1.1 368 141 1,284SK383 dam-4 854 (288)c 0.12 0.05 1 245 121 761

DSK377 xthA+ 103 (56)C 1 1 105 33SK1314 xthAl 1,264 (638) 1 1 103 30SK1328 AxthA 1,341 (743) 2 1 350 60

ESK703 lig-7(Ts) 17 (191)d 1,000 (2)d (1)d (56)d (58)eSK705 lig+ 60 (49)d 1 (1)d (1)d (46)d (50)ea Conjugal deficiency indexes represent wild-type frequencies divided by mutant frequencies for 60-min

matings selecting [His+] [Smr] recombinants with KL96 or [Arg+] [Smr] recombinants with either Ra-2 orKL209. F'lac inheritance was determined by mating E3 with appropriate recipients for 90 min. Deficiency indexis as defined for conjugal crosses. All transductions were carried out with Plvir grown on JC158. Resultsrepresent the average of two determinations.

b PFU, Plaque-forming units.'Numbers in parentheses indicate values obtained by washing cells with M56/2 buffer prior to plating on

lactose minimal agar plates.'Values obtained at 30°C. Although lig-7(Ts) is a conditionally lethal mutation, no loss in viability at 37°C

was detected in these experiments.eMet+ transductants determined at 30°C.

Recombination proficiency of strainswith various defects in DNA metabolism.The rep protein has been shown to be involvedin the replication of certain E. coli bacterio-phages such as P2 and 4X174 (4, 10). Addition-ally, rep strains show slightly enhanced sensitiv-ity to UV light. Both rep alleles tested hereshowed significantly reduced formation of Lac'progeny and small reductions in conjugationproficiency. Transduction efficiencies appearedunaffected (Table 2).

Polynucleotide ligase (lig) is required for cellviability presumably because of the need to gen-erate covalently closed DNA (12, 18). Konradhas shown that ligase-deficient mutants yieldhigher levels of Lac' recombinants (15). Asshown in Table 2, an interesting phenomenonwas observed in the case of the temperature-sensitive allele 1ig- 7(Ts). At 30°C, Lac' progenyformation was stimulated, while conjugation and

transduction proficiencies remained unaltered.However, at 37°C, both Lac' progeny formationand conjugation proficiency dropped dramati-cally (Table 2), although cell viability was notaffected at this temperature.The absence of DNA adenosine methylase in

E. coli leads to an accumulation of single-strandbreaks in the cellular DNA (20). As shown inTable 2, not only was Lac' progeny formationincreased, but conjugation proficiency was alsoincreased dramatically by 3- to 20-fold. Theextent of stimulation appeared to be a functionof the donor Hfr. Transduction proficiency ap-peared to be marker dependent. The number ofllv+ (a gene close to the origin of DNA replica-tion) transductants was reduced in both dam-3(SK385) and dam-4 (SK383) mutants, while thefrequencies ofArg+and His' transductants wereunaffected.Exonuclease III-deficient mutants of E. coli

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GENETIC RECOMBINATION IN E. COLI 963

demonstrate increased sensitivity to methylmethane sulfonate but not to UV light (22, 33).Recently it has been shown that xthA mutantsare also missing endonuclease II (33). Althoughconjugation proficiency was unchanged, Lac'recombinant formation increased over 10-foldwith both the point mutant (SK1314 xthAl) andthe deletion (SK1328 AxthA) (Table 2). Trans-duction proficiency also increased in the strain(SK1328) carrying the deletion (Table 2).Nature ofLac+ recombinants. Konrad (15)

and Zieg and Kushner (35) have shown that inwild-type strains of E. coli, a recombinationalevent takes place such that an intact lactoseoperon is generated on the defective 080 lysogen.A similar analysis of Lac- revertants derivedfrom Lac+ recombinants produced by either a"hyper-Rec" strain (SK246 uvrD3) or a "hypo-Rec" strain (SK285 rep-3) is presented in Table3. Although the absolute number of sponta-neously aring Lac- colonies is related towhether the strain is "hyper-Rec" or "hypo-Rec," the fraction of those Lac- derivatives re-

taiing the 080 prophage is identical within ex-perimental error for all three strains. Those fewLac- revertants that papillated produced Lac'colonies independent of the mutation presentwithin the cell (Table 4) and may have arisenfrom some type of chromosomal rearrangement.

Previous rsults have suggested that in wild-type strains Lac+ recombinants occur as the cellsenter late-log-phase growth (35). As shown inFig. 2, the formation of Lac+ progeny in a "hy-per-Rec" strain (SK246 uvrD3) was similar tothat of a wild-type control (SK287). Addition-ally, the ratio of mutation rates could be calcu-lated from these curves, yielding an average

value of 13.3 ± 4.0 over the first 6-h period. Thisnumber contrasts with the 5.2-fold increase inthe total number of Lac+ recombinants mea-

sured by the standard plate assay (Table 2).

DISCUSSIONFrom the results presented above it appears

that Lac' recombinants arising in "hyper-Rec"or "hypo-Rec" mutants are formed by a mech-anism similar to that which occurs in wild-typestrains. This would account for the absence of

this event in recA strains (35). Additionally,recombinant fonnation in "hyper-Rec" strainsappears to occur when the cells enter late-log-phase growth. Since the assay can measure bothincreases or decreases in recombination profi-ciency, the lacMS28&j80dIIlacBKl duplicationprovides a powerful method for studying recom-bination in E. coli.The data presented in Table 2 and by Zieg

and Kushner (35) represent the analysis of ge-netic recombination employing three independ-ent assays. The differences in the methodsshould be noted. Formation of Lac+ recombi-nants does not require an extraceliular DNAdonor. The information necessary is always pres-ent, but the process may be regulated, since itoccurs at a very low frequency during logarith-mic growth of the cell. On the other hand, bothconjugation and transduction require an exter-nal source of donor DNA. Transfer from stableHfr donors is highly efficient, and recombina-tional proficiencies close to the theoretical limitcan be observed in these cases. In contrast, gen-eralized transducing particles make up a smallpercentage of the total phage population, andmany of these lead to abortive events. As a

result, transduction proficiency is typically quitelow and can be dependent upon the particularmarkers selected.

TABLE 4. Analysis ofpapillating Lac- revertantsobtained from Lac' recombinants'

No. of Lac'Strain Genotype Origin colonies/

plateParental strainsSK287 Wild type 99SK285 rep-3 22SK246 uvrD3 479

Lac- revertantsSK1455 Wild type SK287 6SK1456 Wild type SK287 5SK1458 rep-3 SK285 7SK1459 uvrD3 SK246 16

a Lac' recombinants were isolated from SK287, SK285, andSK246 as described previously (35). Strains SK1455, SK1456,SK1458, and SK1459 are representatives of the class of pap-illating Lac- revertants obtained from Lac+ recombinants (seeTable 3).

TABLE 3. Analysis of spontaneously occurring Lac- revertants arising from Lac' recombinantsa

Avg no. of Lac- col- No. of Lac- colo- Fraction of Lac- Fraction of Lac-Strain Genotype onies/104 Lac+ cells nies tested colonies retaining colonies papillating$OdIIlac

SK246 uvrD3 11.3 382 0.08 0.02SK287 Wild type 1.4 128 0.05 0.02SK285 rep-3 0.5 63 0.10 0.02

'Twenty independently isolated Lac' recombinants were isolated from each strain and grown overnight inL broth. The cells were plated on lactose tetrazolium agar plates. Spontaneously arising Lac- colonies wereanalyzed as described previously (35).

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964 ZIEG, MAPLES, AND KUSHNER

10

0-10 0

(I)>

I-~~ I E-ous

Z

grw inKndurndsrbdntetx.A ie

z

CID~~~ ~ ~ ~ ~~~~I

0

w >W_i02 108

1010 lo?r w 0 m o

M5/ bufr Adiinapae3Jr wahe wit62.0

TIME (hours)

FIG. 2. Growth of total cell mass and Lac' recoi-

binants on lactose minimal agarplates. Strains were

grown in K medium as described in the text At timesindicated, four plates were removed from the 370C

incubator and re-spread with 0.1 ml of prewarmed

M5612 buffer. Additionalplates were wa-shed with 2.0

ml ofM5612 buffer for determination of total celsperpltte. (0) Lac' recombinants obtained with SK246

(uvrD3); (0) Lac' recombinants obtained with SK287

(uvrDm); (0) total SK246 cells on lactose minimalagarplates; (U) total SK287 cells on lactose minimal

agar plates.

Based on these inherent differences, it would

not be surprising if the control of the presynapticsteps for these three recombination systems dif-fered considerably whereas postsynaptic events

(e.g., elongation of the paired regions and gen-

eration of an intact recombinant molecule)

might be more closely related. Accordingly, anal-

ysis of a variety of mutants employing the three

assays might uncover such divergences.As shown in Table 5, the recA gene product is

absolutely required for all forms of homologousrecombination in E. coli. However, the effects of

recB and recC mutations on Lac' progeny for-

mation are much less striking than they are on

transduction and conjugation. It is unlikely that

the decrease observed in the reeB and recC

mutants can be explained on the basis of de-creased cell viability, since the Lac' recombinantassay is independent of the number of cellsplated. Exonuclease V may play only a marginalrole in this type of recombinational event. Onthe other hand, the lethal sectoring observed inrecB and recC strains (23) may be of muchgreater significance when an external DNA do-nor is involved.kexA and rep mutations reduced Lac' progeny

formation to levels similar to recB and recCstrains, but did not appear to appreciably affectrecombinant yields after conjugation or trans-duction. This result is ofparticular interest, sincelexA mutants phenotypically resemble recAstrains in all characteristics except for conjuga-tion proficiency (25). Results presented here sug-gest, however, that the lexA protein is requiredfor some types of genetic recombination. Sup-port for this hypothesis is provided by the factthat strand rejoining during repair of psoralencross-linked DNA is reduced markedly (R. Sin-den and R. Cole, personal communication).With the exception of recF143, mutations in

any of the genes examined led either to a stim-ulation of or a reduction in Lac' progeny for-mation (Table 5). The increases varied from 1.6(xseA7) to 13 (&xthA). As has been pointed outpreviously (16), the formation of single-strandbreaks in the chromosome is apparently a rate-limiting step in the formation of Lac' recombi-nants, since strains which accumulate suchbreaks (polA, lig, dam, and sof [15-17]) showincreased Lac' recombinant formation. Al-though strand breaks may be required for theinitiation ofthese events, inability to repair themmight also dramatically reduce the yield of re-combinants. The results obtained at 370C withthe 1ig-7(Ts) strain (Table 2) seem to supportthis hypothesis.

It is not clear, however, whether strand breaksare rate limiting or even required for conjugationor transduction. With SK703 1ig-7(Ts), conju-gation and transduction were unaffected at30°C. On the other hand, in the polA107 strain(SK276), conjugation was actually reduced 90 to95% while transduction was stimulated slightly.dam mutants showed a dramatic increase inconjugation proficiency, particularly when un-stable Hfr donors (Ra-2) were employed. Mari-nus and Konrad have shown changes in theunselected marker inheritance in such crosses(20). In contrast, the number of transductantsfor a marker very close to the origin of DNAreplication (ilv) was significantly reduced, whilethe fiequencies for other more distant markerswere unaffected.The greatest stimulation in Lac' recombinant

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TABLE 5. Effect of variouw mutations on recombination proficiencyIncrease (decrease)' in recombination proficiency

Relevant genotype Lac+ progeny Conjugation Transduction Sourceformation Ra-2 KL96 Arg+ His+

recA13 (>100) (5,900) (3,000) (100) (32) 35recA142 (47) (5,000) (428) (42) (19) 35recB21 recC22 (5.5) (121) (358) (62) (17) 35recC22 (5.0) (113) (251) (36) (100) 35recB21 (5.0) (96) (176) (548) (365) 35kexA3 (5.0) (1.2) (4.5) This paperrep-3 (4.4) (1.8) (3.1) (1.3) (1.3) This paperrep-5 (3.4) (2.9) (1.2) (1.2) (1.2) This paperrecF143 (1.1) (1.5) 1.1 1.1 (1.8) 35xseA7 1.6 5uvrA6 2.0 1.2 1.3 This papersbcB15 2.4 1.2 2.7 35recL152 2.7 3.1 1.7 1.4 1.8 35poLA480 (poLAexl) 3.1 5recB21 sbcB15 3.5 35dam-3 3.8 6.3 3.3 1.0 1.3 This paperlig-7(Ts) 3.9 (1C) 1.2 1.2 This paperpoLA107 4.1 (12.2) (20) 1.3 2.0 This paperuvrD3 5.2 12.7 1.8 2.6 2.2 This paperdam-4 5.8 20.3 8.6 (1.5) 1.1 This paperuvrDlOl 6.2 This paperuvrE4 10.8 1.2 1.1 1.5 1.8 This paperuvrEl(X 12.0 4.6 1.4 1.7 1.8 This paperxthAl 12.3 1.0 1.0 1.0 This paperAxthA 13.0 (2.0) 3.0 1.8 This paper

a Increases were determined by dividing the mutant frequency by the wild-type frequency. (Decreases)represent the ratio of wild-type to mutant frequency.

formation was observed in strains where accu-mulation of single-strand breaks has not as yetbeen observed (uvrD, uvrE, xthAl, AxthA). Al-though the function of the uvrD and uvrE geneproducts remains to be determined, the sub-strate specificity of exonuclease III (xth.A) iswell characterized (27). Its ability to form single-strand gaps in nicked duplex DNA moleculesmakes it an appealing candidate for an enzymemediating recombination. Although its absencehas little or no effect on conjugation or trans-duction, Lac' recombinant formation is in-creased markedly. Differences in the presynapticevents for the three processes could account forthe observations reported above.From the data shown in Table 5, it is clear

that genetic recombination is a complex processin E. coli. Many gene products may serve tolimit the nature and extent of genetic recombi-nation within the cell presumably by competingfor common intermediates or by preventing theformation ofDNA structures which would facil-itate genetic exchanges. For example, the lactoseduplication system may provide an analog forrecombination events which can take place be-tween daughter strands or multi-chromosomeswithin the cell, such as may be involved in

recombinational repair. Although these eventswould normally go undetected because of a lackof selection method, their occurrence could infact interfere with DNA replication, chromo-some folding, or cell division.

ACKNOWLEDGMENTSWe thank E. B. Konrad and D. Vapnek for many useful

suggestions.This work was supported by Public Health Service grant

GM-21454 and Research Career Development Award GM-00048 to S.R.K., both from the National Institute of GeneralMedical Sciences.

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