JOURNAL OF BACrERIOLOGY, Jan. 1975, p. 36-43 Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 1 Printed in U.S.A.

Recombination and the Escherichia coli K-12 Sex Factor F N. S. WILLETTS

MRC Molecular Genetics Unit, Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland Received for publication 6 August 1974

Recombination between two Flac tra- elements to give Flac tra+ recombinants measured in Rec+ and Rec- strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra+ recombinants among the parental Flac tra- elements transferred by complementation. The kinetics, measured in a rec+ strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA- strain, reduced by two-thirds in a recF- strain, and unaffected in recB- and recC- strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB+ recFstrain, and was reduced only fourfold in a recB- redF+ strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surwas

face exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the recA + product or the RecBC and RecF pathways.

To date there have been relatively few studies of recombination between homologous deoxyribonucleic acid (DNA) sequences of bacterial plasmids with regard to the roles of the host recombination systems, or of any plasmiddetermined system. This is due in part to the lack of suitable markers, although recombination has been demonstrated between chloramphenicol-sensitive (10, 12) and tetracycline-sensitive (13) mutants of R factors, and between transfer-deficient mutants of Flac (2, 24). Recombination between chloramphenicol-sensitive R factor mutants required the host recA+ product (10), as did recombination between F DNA segments (21). Two major recA-dependent pathways for recombination between homologous chromosomal DNA sequences have been discovered in Escherichia coli: the RecBC pathway and the RecF pathway (see reference 5 for a recent review). The RecF pathway is about 100 times less active than the RecBC pathway for post-conjugational recombination in wild-type strains, but about equally active in sbcB- mutants which have lost exonuclease I.

Our objectives in the present study were to confirm that the recA+ product is required for recombination between homologous F DNA segments, to determine whether the RecBC and/or RecF pathways are involved, and to investigate whether F itself determines any further pathway. Recombination between two tra- mutations carried by Flac elements was measured; one or both of these mutations was polar, reducing complementation and allowing relatively easy detection of tra+ recombinants. Furthermore, the contributions of these pathways to F' mobilization of the host chromosome and of any F-determined recombination system to chromosomal recombination after conjugation, were evaluated. MATERIALS AND METHODS Bacterial strains. Bacterial strains used are described in Table 1. F recombination was studied primarily in derivatives of JC3272, but part way through the study it was discovered that JC3272 is not only XR but also A immune, due to the presence of a defective X prophage (see reference 3). Some of the experiments were therefore repeated using derivatives of the A- strain ED2024. 36

RECOMBINATION AND THE F FACTOR

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37

TABLE 1. Bacterial strains Strain

JC3272 JC6310 ED91 ED92 ED2123 ED2124 ED2126 ED2130 ED2024 ED2057 ED2058 ED2132 ED2134

Phenotype

rec genotype

rec+ recA56

recB21 recA56 recB21 recB21 sbcB15 recB21 sbcB15 recF143 sbcB15 recF143

recF143 rec+

recB21 recC22 recF143 recB21 recF143

JC5465 ED6 ED94 ED382 ED383

rec+

ED390 ED3539

recA56

b, His- Malb, His- Malb, His- Malb, His- Malb, Malb b b, Hisc c c c c

d d d d, StrR

recA56

recB21 recA56 recA56

Lac-x74 Trp- StrR Su,+

Derivationa

Reference 1 JC5088 x JC3272 Thy P1 AB2470 + JC3272 ThyJC5412 x JC6310 Thy P1 JC7644 + ED91 JC9248 x ED2123 P1 JC3272 + ED2124 ThyJC9248 x JC3272 T6RStrR from ED2020 JC5412 x EC2024 ThyJC5426 x ED2024 ThyJC9248 x ED2024 MalP1 AB2470 + ED2132 Thy Reference 24 JC5088 x JC5465 ThyJC5412 x JC5465 ThyJC5088 x JC3054 PJR ThyJC5088 x JC5458 Thy-

(XdeC)

Lac&,124 T6R SpCR A-

b, His- Mal- Leu- SpcR

rec+

SpcR from ED369 Leu- Spc R from JC3272

a Matings with JC5088, JC5412, and JC5426 (25) were mechanically interrupted after 25 min. References for other parental strains are as follows: AB2470 (26); JC7644 (19); JC9248 (Hfr P4x metB recF143, A. J. Clark, personal communication); JC3054 and JC5458 (1); ED369 (21); ED2020 (2). Lac -x , Gal- Trp- Lys- T6R StrR Su- (Ader). c (Lac-Proax111 His- Trp- T6R StrR Su-)-. d LacAX74 His- Trp- T6R SpcR Su-

(Xd.r).

The required derivatives of these two Rec+ starting strains were isolated by screening Thy+ (for recA56, recB21, or recC22), His+ (for sbcB15), or Mal+ (for recF143) progeny from the appropriate conjugational or P1 transductional crosses (see Table 1) for clones with altered ultraviolet (UV) sensitivity and/or recombination ability. Quantitative measurements of these properties, which confirm their genotypes, are shown in Table 2. Flac elements. The mutant Flac tra- elements JCFL4, JCFL90, and those listed in Table 3 were described previously by Achtman, Willetts, and Clark (1, 2) and Willetts and Achtman (24). EDFL33 is a recently isolated Tra+ surface exclusion-deficient Flac element (N. S. Willetts and M. Achtman, unpublished data). Media. The media used were described previously (9), except that Oxoid nutrient plates were used. Measurement of F recombination. A 0.1-ml sample of an exponential culture of ED383 (RecA- Sul+ T6' Spcs) carrying a suppressible Flac tra- mutant was mixed with 0.1 ml of a similar culture of the "intermediate" strain (Su- T6R Spc) carrying the second Flac tra- element JCFL4. After incubation at 37 C for 45 min, this mating was interrupted by adding 0.2 ml of T6 (1011 particles per ml, treated with 3,000 ergs of UV light per mm2), and incubating for a further 20 min. A 0.6-ml volume of broth was added, followed, after 60 min of incubation to allow recombination in the intermediate strain, by 1.0 ml of an

exponential culture of the recipient strain (RecA- SuT6R SpcR Str'). Ed6 was used as recipient when the intermediate strain was a derivative of JC3272, and ED390 (A) was used when it was a derivative of ED2024. After mating for 60 min, dilutions were plated on medium selective for Lac+ [SpcR] progeny. These carried either Flac tra- elements transferred by complementation, or Flac tra+ recombinants. To determine the proportion of the latter, the Lac+ [SpcRJ progeny were patched on the same selective medium and incubated overnight. After replication to nutrient plates and growth for 6 to 8 h, their transfer abilities were ascertained by replica plate-mating with ED382, selecting Lac+ [StrR ] progeny. This proportion was corrected to take into account the background frequency of transfer from the T6-treated ED383 donor strain. This was measured using the corresponding F- strain as intermediate. The correction was fairly large for some of the derivatives of JC3272 because their efficiences as intermediates were low, due to their reduced growth rates and donor and recipient abilities. These efficiencies were measured separately using a donor strain carrying wild-type Flac (Table 2), and satisfactorily account for the decreased numbers of Lac+ progeny observed in the recombination experiments. In parallel control experiments, using F- donor or intermediate strains, very occasional spontaneous tra+ revertants of the Flac tra- elements were seen, and the results given are corrected for these.

38

WILLETTS

J. BACTERIOL.

TABLE 2. Properties of intermediate strains

Eie

Deficiency indicesb

Strain

rec genotype

KL98

JC3272 JC6310d ED91d ED92d. e ED2123e ED2124e ED2126 ED2130

rec+ recA recBrecA - recBrecB- sbcBrecB- sbcB- recFrecF- sbcBrecF-

ED2024 ED2057 ED2058 ED2132' ED2134

rec+

recBrecCrecFrecB- recF-

Efficiency

UV survivala

53 4 x 10- 4 0.4 7 x 10-5 9 3 x 10-s 0.4 0.7 43 0.2 1 0.5 6 x1O-3

ED945

AB257

as intermediatec

1 1 1 9 x 10. 260 790 2,400 7 x 10, 9 37 3 x 103 1 x 104 1 2 1 2

100 7 5 5

1 580 22 2 9 x 103

100

1 6 x 103 58 2 8 x 104

4 3 24 60

45 53 96 27

aPercentage of survival after treating exponential-phase cells with 200 ergs of UV light per mm2. b Measured in 60-min 1:10 matings with the Hfr strains shown. The recombinants selected were His+ [Stral with Hfr KL98, Trp+ [Met+StrR] with ED945 (MetHfrB10), and Lac+ [Met+StrR] with AB257 (Met-Hfr Cavalli). cMeasured as described in Materials and Methods for measurement of F recombination, except that the donor strain carried wild-type Flac and the intermediate strain was F-. The number of Lac+ [SpcR] progeny obtained are expressed as a percentage of the number obtained using the corresponding Rec+ intermediate strain. d Presence of the appropriate rec mutations was confirmed in complementation experiments with the Hfr strains JC5029 (rec+), JC5412 (recB21), JC5426 (recC22), and JC5088 (recA56) (25). e The presence of recB21 was confirmed by growing P1 on the strain, transducing the rec+ thy- strain JC5422, and showing that 30 to 40% of the Thy+ transductants were UVS Rec- (26). ' Induction of the A lysogen of this strain with 300 ergs of UV light per mm2 gave lysates containing 2 x 109 plaque-forming units per ml, compared to 3 x 1010 from the A lysogen of ED2024. This decrease is similar to that observed by A. J. Clark (personal communication) with other recF- strains. From crosses between JCFL80 and JCFL4 in several intermediate strains (JC3272, ED91, ED2123, ED2124, ED2132, and ED2134), two Tra+ recombinant clones were purified. These had quantitative donor abilities similar to that of wild-type Flac both in the RecA- host or after transfer to a Rec+ strain. The progeny of the Rec+ strain were all Tra+; Trasegregants were never found. These results indicate that in all cases the Tra+ progeny carried true Flac tra+ recombinants.

RESULTS Measurement of F recombination. The experimental technique described above for measurement of recombination between two Flac tra- elements is similar to that previously used in complementation tests (2). In such complementation tests, the majority of the Lac+ progeny carried parental Flac tra- elements transferred by complementation and only about 1% carried Flac tra+ recombinants. In the experiments to be described, the Flac traelement carried by the intermediate strain was always JCFL4; this mutant carries the strong polar mutation traK4 which greatly reduces complementation with traB, traC, traF, traH,

and traG mutants (2). As a result, the proportion of tra+ recombinants among the Lac+ progeny was increased. Furthermore, cells carrying JCFL4 show a very low level of surface exclusion, probably because traK4 is also polar on the surface exclusion gene(s) traS (23, 24). This gave the additional advantage that recombination between the Flac tra- elements could be studied in exponentially growing intermediate cells, rather than stationary-phase "Fphenocopy" cultures. In preliminary experiments, the time required for F recombination in a Rec+ intermediate strain was measured. After formation of JCFL80/JCFL4 heterozygous cells in the initial mating, the culture was incubated for different periods before the second mating with the recipient strain; the proportion of the Lac+ progeny which were Tra+ was then determined (Fig. 1). An incubation period of 60 min gave near maximal numbers of recombinants, and was therefore chosen for routine use. Although the precise times of formation of the heterozygous cells during the initial mating period, and of the recombination event preceding transfer of the Flac tra+ element during the second mating,

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RECOMBINATION AND THE F FACTOR

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not known, the period from 1 t( o 2 h after the initiation of mating during which t he proportion of tra+ recombinants increased wa:s very similar to the periods during which reconnbination occurred within the phoA (16) and l ac (4) genes. Several Flac tra- elements were then tested in crosses with JCFL4 so that the c mne giving the highest proportion of recombinan ts among the Lac+ progeny could be chosen (I 'able 3). It is clear that, although the proporltion of Lac+ progeny which carried recombina nt Flac tra+ elements depended partially on the distance between the tra- mutation ancI traK4, the degree of polarity of traK4 on the 1gene in which the other Flac element was mutan: t, and of both

tra mutations

on genes distal to both, was more important. This is because such polarity effects reduce the level of complementation but not of recombination, and the proportion of Flac tra+ recombinants is therefore increased. The degree of polarity of a tra mutation can be roughly gauged by its effect on traS, easily measured as a surface exclusion index (Table 3, column 3); wild-type Flac has an index of about 400, and this is reduced, for example, by the strong polar mutation traK4 to about 5. Thus, although traK4 and traB16 are relatively close, both mutations are strongly polar; complementation was therefore poor, and the proportion of recombinants was correspondingly high. In contrast, although traK4 and traD83 are relatively distant, traK4 is not very polar on traD, and the proportion of recombinants was low. As a further illustration, the polar traG79 mutation gave a much higher proportion of recombinants with traK4 than did the nonpolar traG81 mutation. Although JCFL16 gave the highest proportion of recombinants with JCFL4, the frequency with which these were obtained was rather low, presumably because of the proximity of the mutations. This was undesirable because of the proportionately increased correction for the background level of transfer from the T6-treated donor strain. Therefore, JCFL80 was chosen for routine use since it gave a high proportion of recombinants at a relatively high frequency. 45 60 75 90 Recombination in RecA- strains. The relime (min) sults shown in Table 4 demonstrate that the FIG. 1. Kinetics of recombination. A culture of presence of a recA- mutation in the intermediheterozygous cells carrying JCFL80 aCnd JCFL4 was ate strain prevented recombination between incubated, and at the times shown samples were JCFL4 and JCFL80. Recombination between withdrawn to assay the number of tral+ recombinants homologous F DNA sequences was therefore per milliliter. dependent upon the host recA+ product.

are

E

0

TABLE 3. Recombination between JCFL4 and different Flac tra- elements

Flac tra- element in donor

JCFL16 JCFL5 JCFL13 JCFL80 JCFL81 JCFL79 JCFL83

tra

mutationa

traB16 traC5 traF13 traH80 traG81 traG79 traD83

Surface

exclusion index'

4 290 410 60 300 55 560

No. of Lac+

Corrected pro-

progeny

portion of tra+

tested

recombinantsc

recombinants

400 500 400 600 500 400 400

192/315 63/466 121/390 145/581 27/495 70/394 8/400

61 13.5 31 25 5.5 18 2

Frequency of recombinantsd 1.4 x 8.9 X 5.2 x 1.2 x 1.2 x 6.6 x 8.7 x

10-2 10-2 10-2

10-l 10-1 10-2 10-2

The order of the tra genes is traK traB traC traF traH traG traS traD (15, 23). b The surface exclusion index is defined as the ratio of the number of His+ recombinants obtained with the F strain JC3272 to the number obtained with the Flac tra- derivative, in crosses with Hfr KL98 (1). cTotal number of Tra+ clones divided by number of Lac+ progeny tested minus the number due to the background level of transfer by the T6-treated donor strain. d Tra+Lac+ [SpcR] progeny per 100 donor cells. a

WILLETTS

40

TABLE 4. Recombination between JCFL4 and JCFL80 in recA - strains Genotype of intermediate straina

Geno ype

rec+ rec+ recA recA -

rec+ recA rec+ recA -

strainpt

Corrected proportion % recomprogeny of tra+ re- binants tested combinantsc No. of Lac+

650 600 600 500

188/642 146/581 0/178 0/151

29 25

Recombination and the Escherichia coli K-12 sex factor F.

Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia col...
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