Mol Gen Genet (1981) 181:497 504 © Springer-Verlag 1981
Role of the reeF Gene of Eseheriehia eoli K-12 in 2 Recombination Maria-Eugenia Armengod Instituto de Investigaciones Citoldgicas, Amadeo de Saboya 4, Valencia 10', Spain
Summary. When Escherichia coli K12(2) lysogens are infected with heteroimmune 2 phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial reeF gene. It has been shown that in E. coli K12 postconjugational recombination, the ReeF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which 2 replication is blocked, the recombination pathway dependant on the reeF gene is fully active in producing viral recombinants even, if the phage is Red +, in the presence of exonuclease I. In contrast, removal of)~ exonuclease and fl protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the ReeF pathway and that this cooperation involves overcoming the inhibitory effects of exonuclease I. In the absence of 2 exonuclease, /~ protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence offl protein, full efficiency of the ReeF pathway can be obtained either via cooperation with )~ exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of 2 exonnclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that/I exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I. When )~ replication is allowed, the Rec system produces viral recombinants mainly via a reeF-independent mechanism. However, a major contribution of the ReeF pathway to 2 recombination is observed after removal of the Red system and exonuclease I.
In~oduction
General recombination in E. coli is mediated by the Rec system. The performance of this system depends on the product of the recA gene, the RecA protein, which catalyzes initiation of strand exchanges during recombination (McEntee et al. 1979; Shibata et al. 1979). It has been proposed thai: other proteins of the Rec system operate concertedly in two major recA-dependent pathways (for review: Clark 1973, 1974). The RecBC pathway, which seems to account for about 99% of postconjugational recombination in wild-type strains, uses in part the product of the recB and recC cistrons (exonuclease V). The RecF pathway, Offprint request to. M.-E. Armengod, Department of Molecular Biology, University of California, Berkeley, Califl~rnia94720, U.S.A.
* Obra social de la Caja de Ahorros de Valencia (Director: S. Grisolia)
which seems to account for the remaining 1% of recombination, depends on the recF gene, whose product until now has not been identified. This pathway can be as efficient as the RecBC pathway in sbcB mutants that have lost exonuclease I and, accordingly, it has been suggested that exonuclease I interferes with full expression of the RecF pathway by converting a necessary intermediate into an intermediate in the RecBC pathway (Clark 1973; Horii and Clark 1973; Kushner etal. 1971). Although it is not possible as yet to specify with certainty the in vivo role of exonuclease I, the postulation of two recombination pathways appears to be the easiest way to explain the observed relationships among the various genes of E. coli involved in postconjugational recombination (Clark 1973). General recombination in bacteriophage 2~can be mediated by its own recombination system (Red system) and/or by the recombination system of its host, E. coll. Under conditions in which )o DNA replication occurs, the Red system (in recAcrosses) is more efficient in producing viral recombinants than is the Rec system (in red crosses). However, under conditions in which 2 DNA replication is blocked, the efficiency of the Red system decreases drastically to a level close to that of the Rec system, which remains unaffected. When both systems are present, the number of recombinants is always significantly greater than that resulting from the sum of the values obtained when either is present alone, suggesting a cooperation in the production of viral recombinants (Blanco and Armengod 1976; Stahlet al. 1974). Since Rec activity seems to be obligatory for pronounced Red activity when 2 replication is blocked, it has been proposed that the Red system, though ineffective in initiating recombination of unreplicated DNA, operates on recombinational intermediates which require the Rec system for their formation (Stahl et al. 1978). The aim of this work was to investigate to what extent 2 can use the recombination pathways provided by its host bacteria and to determine the genetic bases for cooperation between viral and bacterial systems under conditions of blocked replication. Since the gamma protein encoded by 2 inhibits all activity of the bacterial exonuclease V, thus blocking the RecBC pathway (Enquist and Skalka 1973; Karu et al. 1975; Unger et al. 1972; Wilkins and Mistry 1974), the work reported here concentrated especially on the recombination pathway controlled by the recF gene orE. coli K12. Materials and Methods Bacteriophage Strains
The following strains were obtained from R. Devoret: 2c1857 Oam29, )~imrn21 cI int6 red30am29, )~h clind-, 2imm434, 2imm434 intl02
0026-8925/81/0181/0497/$01.60
498 Table 1. Bacterial strains Basic Genetic B a c k g r o u n d (1)
Genotype
No phage
reeA
reeB
recC
sbcB
recF
Su (2)
+ -
+ +
+ +
+ +
+ +
-
+ +
+ 21
+ +
+ +
+ +
+ +
+ 21
+ +
+ +
+ 128 +
+ + 21
+ + +
+
21
+ +
+ +
+ + + + + + + 56 13
+ + + + + 21 21 + +
G Y 7 4 6 x AB1157
+ 56
G Y 7 4 6 x JC9239
Strains lysogenized by
D e r i v a t i o n or reference (3)
)dmm434
2h c l i n d -
W3110 N100
V3284 V3285
-
-
V5701 IC9
IC3 IC21
-
143 143
-
V5702 IC33
V5800 IC47
-
+ + +
+ + +
+ + +
GY158 GY6128 V3245
V3225 V3227 V3305
-
+
+
143
+
IC35
IC48
-
+ +
+ +
+ +
+ +
ABl157 AB2497
-
V3378
+ + + + + 22 22 + +
+ 15 + + 15 15 15 + +
+ + 143 143 143 + + + +
+ + + + + + ++ + +_
IC59 IC58 JC9239 IC160 IC64 IC37 JC7623 AB2463
IC61 IC60 IC180 IC66 IC49 IC389 -
IC74 IC73 V3376 IC181 IC75 V3375 V3379
B a c h m a n n 1972 T h y m i n e r e q u i r i n g derivative of AB1157 ( B a c h m a n n 1972) His + t r a n s d u c t a n t of AB2497 from d o n o r IC52 d Idem H o r i i a n d C l a r k 1973 Sal + t r a n s d u c t a n t of IC59 f r o m d o n o r V5702 Val ~ t r a n s d u c t a n t o f IC58 f r o m d o n o r V5697 c Sal + t r a n s d u c t a n t of JC8111 ~ from d o n o r V5701 H o r i i a n d C l a r k 1973 Tet 1 t r a n s d u c t a n t of IC61 f r o m d o n o r JC10240 r B a c h m a n n 1972
+ +
+ +
+ +
+ +
-
IC384 -
IC386 IC387
-
G Y 7 4 6 g x A B 1157 ( G a l + Stff selection) Tet" t r a n s d u c t a n t of IC386 f r o m d o n o r JC10240 f
+ + + + +
+ + + + +
+ + + + +
+ + + 15 15
143 + 143 + 143
---
V5653 IC153 IC154 IC170 IC174
V5663 IC157 IC158 IC176 IC183
-
G Y 7 4 6 g x JC9239 ( G a l + Stff selection) Sal + t r a n s d u c t a n t of V5653 f r o m d o n o r V5737 h Idem His + t r a n s d u c t a n t of IC153 f r o m d o n o r IC52d VaP t r a n s d u c t a n t of IC170 f r o m d o n o r V5697 °
G Y 7 4 6 x JC7623
+
21
22
15
+
-
IC391
IC392
-
G Y 7 4 6 ~ x JC7623 ( G a l + Str r selection)
G Y 3 0 6 x JC8111
+
2i
22
15
143
--
V5661
V5662
-
G Y 3 0 6 i x JC8111 e ( G a l Str r selection)
Miscellaneous
+
+
+
+
+
+
C600
-
-
+
+ +
+ +
+ +
+ +
-
CR63 Y10T5
-
-
B a c h m a n n 1972 B a c h m a n n 1972 Eisen et al. 1966
W3110 JGl13
GY158
ABl157
+
-
B a c h m a n n 1972 M. M e s e l s o n a A r m e n g o d a n d Blanco 1978 M. Blanco. IC9 is a Thy + t r a n s d u c t a n t of JG113 from d o n o r AB2470 b A r m e n g o d a n d Blanco 1978 VaP t r a n s d u c t a n t of IC9 from d o n o r V5697 ~ D e v o r e t a n d Blanco 1970 Bailone et al. 1975 Thy + t r a n s d u c t a n t of G Y 1 5 8 f r o m d o n o r AB2470 b Val" t r a n s d u c t a n t o f V3245 from d o n o r V5697 c
(1) JG113 (origin: J. Gross) is a t h y m i n e - r e q u i r i n g derivative of W3110. R e l e v a n t genetic m a r k e r s o f G Y 1 5 8 are: thr-1 leu-6 thi-1 t h y A 6 p y r F l a c Y 1 supE44 t o n a l deoC34. The r e m a i n d e r of the m u t a n t genotype of A B l 1 5 7 is as follows: thr-1 leuB6 thi-1 lacY1 g a l K 2 ara-14 xyl-5 mtl-1 p r o A 2 his-4 argE3 rpsL31 tsx-33 sup-37. The c o m p l e t e genotype of C600 is: thr-i leu-6 thi-1 lacY1 tonA21 supE44. R e l e v a n t genetic m a r k e r s o f C R 6 3 are: F + l a m - C R 6 3 supD. R e l e v a n t genetic m a r k e r s o f Y10T5 are: t h r A l leu-6 thi-1 supE44 (2t5). (2) In this p a p e r it is c o n s i d e r e d t h a t a s t r a i n has Su + p h e n o t y p e when, used as i n d i c a t o r strain, it is able to suppress a m b e r m u t a t i o n s in ) x I 8 7 5 Oam29, 2imm21 c l - int6 r e d 3 0 a m 2 9 or 2 c I bio69 Oam205. These p h a g e m a k e very small p l a q u e s on A B l 1 5 7 , suggesting t h a t in this strain the sup-37 allele is n o t efficient in s u p p r e s s i n g the viral m u t a t i o n s , F o r this r e a s o n is c o n s i d e r e d to AB1157 have Su + phenotype. D e r i v a t i v e strains of A B l 1 5 7 vary in the degree o f s u p p r e s s i o n w h e n used for p l a t i n g the a b o v e m e n t i o n e d phage. Thus, JC9239, AB2497, a n d JC8111 show Su + p h e n o t y p e b u t JC7623 a n d AB2463 show Su + phenotype. (3) P h e n o t y p e a b b r e v i a t i o n s used are the following: Thy, t h y m i n e ; Gal, galactose ; Sal, salicine ; Val, valine; His, histidine ; Str, s t r e p t o m y c i n ; Tet, t e t r a c y c l i n e ; superscript", resistance; superscript + , i n d e p e n d e n c e or utilizing. N100 is M e s e l s o n ' s strain 152 (see G o t t e s m a n a n d Y a r m o l i n s k y 1968) b AB2470 is a recB21 derivative of A B l 1 5 7 (see B a c h m a n n 1972) V5697 is a Val r s p o n t a n e o u s m u t a n t o b t a i n e d f r o m JC9239 (see M i l l e r 1972). The m u t a t i o n responsible for Val r p h e n o t y p e m a p s in the ilv region located at 84 m i n ( B a c h m a n n a n d L o w 1980) a IC52 is a His + t r a n s d u c t a n t of JC7623 f r o m d o n o r V5701 ° JC8111 is a recB21 recC22 sbcB15 recF143 derivative of AB1157 (Horii a n d C l a r k 1973 ; K u s h n e r et al. 1971) f JC10240 is a tetracycline-resistant t r a n s d u c t a n t of JC5088 ( B a c h m a n n 1972) f r o m d o n o r JC10236 ( T e m p l i n et al. 1978) w h i c h carries the srl-3OO::TnlO m u t a t i o n (see C l a r k et al. 1979) g R e l e v a n t genetic m a r k e r s of G Y 7 4 6 (origin: R. Devoret) are: H f r H thi m a l uvrB5 2 P1 r h V5737 is a s p o n t a n e o u s salicine-fermenting m u t a n t o b t a i n e d from a derivative strain of W3110 R e l e v a n t genetic m a r k e r s o f G Y 3 0 6 (origin: R. Devoret) are: H f r H thi
499 red3 and 2imm434 bioll. The strain 2cI- bio69 Oam205 was a gift of R. Thomas. The strain 2imm434 bio69 was isolated from a cross between 2imm434 bioll and 2cI bio69 Oam205 by selecting for turbid plaques on the recA Su- strain N100 (see Zissler et al. 1971). red3
mutation determines inactive 2 exonuclease and /~ protein (Shulman et al. 1970). bio69 and bioll are substitution mutations, bio69 deletes the redX (2 exonuclease) but not the redB gene (/~ protein) and bioll deletes both genes and the gamma gene (y protein) (see Manly et al. 1969; McMitin et al. 1974; Shulman et al. 1970; Zissler et aL 1971). Bacterial Strains
The bacterial strains employed in this study are described in Table 1. The nomenclature is that used by Bachmann and Low (1980). Media
LBT contained 10 g NaC1, 10 g Bacto-Tryptone, 5 g yeast extract and 40 nag thymine/liter of bidistilled water. GT contained 5 g NaCI, 5 g of Bacto-Tryptone, 8 g Peptone and 20 g Difco agar/liter of bidistilled water. This medium was used for plating phage 2 on indicator strains. Phage dilutions were made in 0.01 M MgSO4. Marker Rescue Experiments
Bacteria grown in LBT to about 2 x 108 cells/ml were harvested by centrifugation and resuspended in 0.01 M ]~vIgSO4to 1/3 of the initial volume. They were infected at a multiplicity of 0.05 phage/cell and incubated for 20 rain to allow adsorption of the phage. Samples were diluted 1/50 in prewarmed LBT and incubated with vigorous aeration for 85 rain. Chloroform was then added and, after dilution, aliquots were plated on indicator strains. All experiments were carried out at 37° C. Construction of Special Strains
1. Construction of the reeF143 Sal * strain was as previously described (Armengod and Blanco 1978). 2. To obtain Val r mutants the method used was as described by Miller (1972). 3. Construction of the sbeB mutant strains: Red- phage make small plaques when plated on wild-type strains. However, when an sbcB mutant strain was used for plating, it was observed that Redphage made plaques similar to those formed by Red + phage. The sbcB gene maps at 44 rain (Bachmann and Low 1980) and is cotransducible with the his locus so that this marker is useful in transferring sbcB mutations from one strain to another. Accordingly, the methodology was as follows: P1 grown on an sbcB15 His + strain was used to transduce an sbcB + His- strain. After purification, some His + transductants were used for plating Red- phage. It was assumed that the His + transductants on which Red- phage make "large" plaques (about 50% of those selected) had inherited the sbcB15 allele. The presence of the sbcB15 mutation in the selected clones was later confirmed by the observation that P1 grown on cultures of the new sbcB mutant strains produced ultraviolet light-resistant clones when utilized to transduce a recB21 His to His + (Kushner et al. 1971). Since, in this test, prexisting suppressed Rec* revertants of the recB21 recipient would be preferentially transduced to His +, the inverse test was performed also using a recB21 sbeB15 his.-4 strain as recipient and His + transductants were screened for sensitivity to ultraviolet light. Considering that no clone was found to be sensitive to ultraviolet light in this case, it was concluded that the donor strains used carry the sbcB15 mutation. Results and Discussion 1. Rescue o f the 0 + Gene f r o m the Prophage by the Superinfecting Phage D N A
The experimental system, which has been previously described (Blanco and Armengod 1976), involves superinfection of a strain
lysogenic for 2 by a heteroimmune 2 phage which carries an amber mutation in the O gene. The D N A of this phage replicates only in an Su + host (Ogawa and Tomizawa 1968), and when it infects an Su- lysogenic strain, its replication depends on rescue by the parental D N A of the O + gene from the prophage. In this case, recombination occurs in the absence of viral replication.
a) Recombination in the Absence o f Viral Replication. The results
are shown in Table 2. Since during superinfection of Su- hosts there is no burst of the parental O - phage, the number of recombinants obtained per infected cell is compared in this Table. When the superinfecting phage is Red +, the presence of the r e c F m u t a t i o n in the lysogenic host reduces the number of recombinants per infected cell to about 10% of that obtained with the rec + host. This indicates that recombination between the prophage and the unreplicated D N A of the heteroimmune superinfecting phage depends on the reeF gene. As expected, considering that a rec + strain acquires R e c B - phenotype after infection by gam + phage (Enquist and Skalka 1973; Karu et al. 1975; Unger et al. 1972; Wilkins and Mistry 1974), the production of viral recombinants in the recB mutant is similar to that in the rec + strain. It should be noted that recombination in reeF143 strains is about four times higher than that obtained with recA mutants in which the Rec-dependent recombination is completely abolished (see Clark 1973). This could indicate that a further r e e f (and recBC)-independent mechanism also contributes to 2 recombination. However, considering the striking reduction provoked by the recF mutation, it is clear that in the absence of 2 replication, the Rec system produces viral recombinants mainly through the RecF pathway. It has been shown that during postconjugational recombination in E. coli, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973; Horii and Clark 1973). However, since the reeF mutation reduces the amount of viral recombination in sbcB + strains significantly (see Exo2+/~ + phage, Table 2), it appears that under conditions in which 2 replication is inhibited, the RecF pathway is fully efficient in producing viral recombinants even, if the phage is Red +, in the presence of exonuclease I. This conclusion is further supported by the fact that in sbeB and recB recC sbcB strains, which have lost exonuclease I, no increase in recombination of Red + phage was observed as compared with ree + and recB21 strains. Interestingly, a significant contribution of the RecF pathway to recombination between bacterial plasmids has been demonstrated and in this case also the activity of this pathway is independent of exonuclease I (Willetts 1975). In rec + cells, removal of the Red system drastically reduces the number of recombinants per infected cell (compare data for Exo2+/? + and Exo2-/~- phage, Table 2), suggesting an important role for the Red system, if the Rec system is present, in recombination of unreplicated DNA. Table 2 shows that when both systems are present simultaneously (see data for Exo2+/~ + in rec + strain), the number of recombinants obtained is considerably greater than the simple sum of the values obtained for each system working separately (see data for Exo2+/~ + in the recA mutant and for Exo2-/~- in the rec + strain). This indicates that the Red and Rec systems cooperate in the production of viral recombinants (Blanco and Armengod 1976; Stahl et al. 1974). Furthermore, following the same reasoning but using the data for the recF mutant in place of those for the reeA mutant, it can be concluded that such cooperation takes place mainly through the RecF pathway.
500 Table 2. Rescue of the O + marker under conditions of blocked replication Properties of Su- ;.imm434 lysogen Genetic background
Strain no.
Recombinants[100 infected cells by superinfecting with 20am29 phage Genotype recA
recB
recC
sbcB
reeF
Red + (Exo2 +fl +)
Red (Exo2 fl-)
+ +
+ +
+ +
+ +
14.0 0.3
0.3 0.01
15.0 19.0 1.2 1.9
0.2 0.3 0.05 0.04
13.0 0.2
0.2 0.02
12.5 1.0" 12.5 0.8
0.2 0.04" 1.5 0.07
25.0
5.7
0.8
0.03
W3110
V3284 V3285
+ -
JGI13
IC3 IC21 V5800 IC47
+
+
+
+
+
+
21
+
+
+
+
+
+
+
21
+
+
+ 143 143
IC386 IC387
+
+
+
+
+
56
+
+
+
+
IC157 IC158 IC176 IC183
+ + + +
+ + + +
+ + + +
+ + 15 15
143 + 143
GY746 x JC7623
IC392
+
21
22
I5
+
GY306 x JC8111
V5662
+
2I
22
15
143
GY746 x ABt157 GY746 x JC9239
+
-
Red + and Red- phage were 2c1857 Oam29 and 2imm21 eI int6 r e d 3 0 a m 2 9 , respectively. The recombinants, having the immunity of the superinfecting phage and the O + gene of the prophage, were scored by plating on V3285. The number of infected cells was assumed to be equal to that of the infective centers obtained by titrating the viral suspension used in each experiment on C600 indicator strain. Infective centers were determined after an adsorption period of 20 min at 37° C. The number of infected cells/ml was 2 x 106-5 x 10 6. Since in the course of each experiment, following the phage adsorption period, infected cell cultures were diluted 1/50 to facilitate later (number of recombinants/ml) x 50 manipulations, the following expression was used to calculate the number of recombinants/infected cell: number of infected cell/ml a Similar data were obtained for V5663
Although when t h e superinfecting phage is R e d - the number of recombinants per infected cell in the rec + strain is already low, it is even lower in the r e c F mutant (see Exo2-/~- phage, Table 2). This shows that in the absence of the Red system, the r e e F - d e p e n d e n t pathway also works to produce viral recombinants in the ree + strain. However, in contrast to the Red + case, inactivation of exonuclease I significantly increases recombination of the R e d - phage (compare data for sbcB and reeB r e c C sbcB mutants with those for rec + strains). Since the s b c B - dependent increase is abolished by the r e e F mutation (see sbcB r e c F and recB r e c C sbcB r e e F mutants), it is clear that the extra production of viral recombinants in sbcB15 and recB21 reeC22 sbcB21 strains occurs through the recombination pathway controlled by the r e e F gene. It must be concluded, therefore, that the r e e F - d e p e n d e n t pathway is not fully effective in sbcB + strains when the superinfecting phage is Red . Considering that the R e c F pathway is only insensitive to exonuclease I in the presence of the Red system, it appears that cooperation between the ReeF pathway and the Red system involves overcoming the inhibitory effects of exonuclease I. b) R e c o m b i n a t i o n under Conditions where Viral Replication Occurs. In these experiments, Su + strains lysogenic for 2 i m m 4 3 4 are infected by 2ci857 O a m 2 9 or 2 i m m 2 1 c I - int6 r e d 3 0 a m 2 9 .
Since Su + hosts permit the replication of superinfecting phage D N A , it is possible here to refer the observed number of recombinats both to the number of infected cells and to the total number of phage. The results obtained with 2ci857 O a m 2 9 are shown in Table 3
(see ExoR+/~ + phage). Removal of the bacterial system by the recA mutation, decreases the recombination frequency to about 10% of that observed with the rec + strain, revealing the contribution of the Rec system to recombination between prophage and superinfecting phage D N A under conditions which permit replication of the latter (Blanco and Armengod 1976). The presence of the recB and/or recF mutations in the host strain does not change the values of recombination with respect to those obtained in the rec + strain. The similarity of the recombination values in recB21 and rec + strains can again be explained by the g a m m a - d e p e n d e n t inhibition of the recBC enzyme in the rec + strains. However, the lack of effect of the r e c F mutation contrasts with the results obtained in the absence of replication and suggests that under conditions of permissive replication, the recF+ product is unnecessary for an efficient contribution of the Rec system to ~, recombination, at least in the presence of the Red system. The possibility was also tested that the activity of exonuclease I could be responsible for this apparent ineffectiveness of the r e e F - d e p e n d e n t pathway. However, since experiments using sbcB15 and recB21 reeC22 sbcB15 strains showed that the absence of exonuclease I does not improve the yield of viral recombinants (Table 3, Exo)~ +/3 + phage), this hypothesis was ruled out. Likewise, when the superinfecting phage was R e d - (see results for Exo2-/3- phage, Table 3), the reeF mutation had no noticeable effect on the recombination of replicating D N A , supporting the hypothesis that the Rec system can promote 2 recombination via a r e e F - i n d e p e n d e n t mechanism. However, in experiments using the sbcB15 strain, the number of recombinants obtained was always several times higher than in the rec ÷ strain.
501 Table 3. Rescue of the O + marker under conditions of permissive replication Recombination by superinfecting with 2 0 a m 2 9
Properties of Su* 2imm434 lysogen Genetic background
ABI157
GY158
Strain no.
IC61 IC389 IC180 IC60 IC66 IC49 V3225 V3227 V3305 IC48
Genotype recA
recB
recC
sbcB
recF
+
+ + + + +
+ + + + +
+ + +
+ +
21
128
+ +
56
+ + + + +
22
15 15 15
143 + 143 +
÷ +
+ +
+ +
+ +
21 21
+ ÷
+ +
+ 143
Red + (Exo2+/~ +)
Red (Exo)~ /~-)
45 5 33 30 25 15
(0.4) a (0.09) (0.2) (0.3) (0.2) (0.5)
0.4 0.02 0.2 2.5 0.5 1.3
(0.008)" (0.001) (0.006) (0.04) (0.007) (0.043)
39 3 50 35
(0.5) (0.04) (0.5) (0.5)
0.5 0.05 0.2 0.2
(0.009) (0.002) (0.008) (0.009)
The number in parenthesis is the percentage recombination frequency; the number not in parenthesis is the number of recombinants per 100 infected cells. Red + and Red- phage were as in Table 2. Total progeny phage were measured by plating on C600 and recombinants were scored, having the immunity of the superinfecting phage and the O ÷ gene from the prophage, by plating on V3285. Recombination frequency is defined as the ratio No. recombinants/No, total phage. The number of total phage was 5 x 106 1 × 107 in the superinfections by Red ÷ phage and 2 x 106-6 x 106 in the superinfections by Red phage. Number of recombinants/infected cell was calculated as indicated in the legend to Table 2. The number of infected cells/ml was 3 x 106-5 x 106
Since the increase disappeared in the sbcB r e c F mutant, it can be concluded that in this case the r e c F gene also regulates the extra production of recombinants resulting from the inactivation of exonuclease I. It should be noted that the a m o u n t o f recombination in the s b c B r e c F m u t a n t is similar to that in the rec + and recF143 strains a n d far greater t h a n that obtained for the recA mutant. Therefore, the activity c,f the RecF pathway is only perceptible in the s b c B m u t a n t where, due to elimination of exonuclease I, the RecF pathway is more efficient t h a n is the r e c F - i n d e p e n d e n t mechanism. Why, is the activity of the RecF pathway not observed when the superinfecting phage is Red +, at least in the sbcB15 strain? It has been shown that the Red system is more efficient t h a n is the Rec system in promoting recombination between the prophage a n d the replicating D N A of the superinfecting phage a n d that b o t h systems, when simultaneously present, cooperate in the production of viral recombinants (Blanco a n d A r m e n g o d 1976; see also Table 3). The fact that recombination of Red + phage is decreased by the recA mutation, but not by the r e c F m u t a t i o n indicates that cooperation between the Rec a n d Red systems occurs in r e c F mutants. Perhaps the high efficiency of this r e c F - i n d e p e n d e n t cooperation on replicating D N A conceals the activity of the R e c F pathway, which can be only observed after removal of the Red system and inactivation of exonuclease I. A l t h o u g h the Rec system appears to promote recombination of replicating D N A via a recFindependent mechanism, the possibility that other components from the RecF pathway are required shouid not be excluded.
2. R e s c u e o f the h M a r k e r f r o m the Prophage by the D N A o f a R e d Superinfecting P,~age
To confirm that under conditions in which 2 replication occurs inactivation of exonuclease I facilitates r e c F - d e p e n d e n t recombination of R e d - phage, the rescue of a marker was analyzed that is lying on )~ D N A in a different region from that of the O gene. The experimental system involved, in this case, rescue of the h marker at the J gene from 2cIind h prophage by D N A undergoing replication of 2 i m m 4 3 4 i n t l 0 2 red3 superinfecting phage.
Table 4. Rescue of the h marker by replicating DNA of Exo2-fisuperinfecting phage Strain no.
Genotype recA recB recC sbcB recF
IC74 V3379 IC181 IC73 IC75 V3375
+
+
+
+
+
13
+
+
+
+
+
+
+
+ +
+ + 21
+ + 22
15 15 I5
+ 143 + 143 +
+
Recombinants/Recombination infected cell frequency (x 100) (x 100) 1.5 a 0.2 0.8 b 10.0 0.8 34.4
0.044 0.015 0.04 b
0.3 0.04 1.4
The phage was )oimm434 intl02 red3. Having the immunity of the superinfecting phage and the h marker of the prophage, recombinants were scored by plating on a mixture of one part of Y10T5 and two parts of CR63. Total phage were estimated by plating on C600. The recombination frequency and the number of recombinants per infected cell were calculated as for Tables 2 and 3. In all experiments the number of total phage and the number of infected cells/mI was 3 x 1064 × 106 a Similar data were obtained for V3378 b Similar data were obtained for V3376
The results are shown in Table 4. It can be seen that the recombination frequency in the sbcB m u t a n t is at least seven times higher than in the rec + strain. Moreover, this increment disappears with the addition of the r e c F m u t a t i o n (see sbcB15 recF143 strain) but not of the recB a n d r e c C mutations (see recB21 recC22 sbcB15 strain). As expected when viral replication occurs normally, the values of recombination are similar in the r e c F single m u t a n t a n d in the rec + strain. No increase in recombination was observed in the s b c B mutant with respect to the rec + strain when the superinfecting phage was Red + (data not shown). These results, therefore, support the conclusion that the RecF pathway mediates recombination of replicating D N A but that its activity can be only observed after removal of the Red system and exonuclease I.
502 Table 5. Rescue of the O + marker by Exo2-p + phage under conditions of blocked replication Recombinants infected cell (x t00)
Properties of Su- ,~imm434 lysogen Genetic Strain background no.
Genotype recA recB recC sbcB recF
GY746 xABI157
IC386 IC387
+
+
+
+
+
56
+
+
+
+
GY746 x JC9239
IC157 IC158 IC176 IC183
+ + + +
+ + + +
+ + + +
+ + 15 15
+ 143 + 143
3.2 0.08 2.6 0.5 13.8 0.8
The phage was 2cI- bio69 Oam205. Recombinants were scored as in Table 2. In all experiments the number of infected cells/ml was 2 x 106
3. Effect o f the sbcB Mutation on the Development o f R e d - Phage
An alternative explanation of the sbcB -dependent increment in the number of recombinants observed in superinfections by Red phage is that inactivation of exonuclease I simply facilitates the development of R e d - phage, either recombinants or nonrecombinants, rather than recombination. Although analysis of the total phage produced in superinfections by R e d - phage did not show differences between sbcB + and sbcB15 strains (see legends to Tables 3 and 4) and, therefore, indicated that this possibility was unlikely, the development of R e d - phage was studied in sbcB15 and sbcB + strains. The results (data not shown) revealed no difference in either the burst size or the kinetics of phage growth on either strain, supporting the hypothesis that inactivation of exonuclease I facilitates recombination between the prophage and superinfecting R e d - phage. 4. Cooperation between the ReeF Pathway and the Red System
The results obtained under conditions of blocked replication (Table 2) indicate that the RecF pathway is only sensitive to exonuclease I when the Red system has been eliminated. Since the performace of the Red system depends on 2 exonuclease (Exo)0 and /~ protein (Shulman et al. 1970), an investigation was made into whether the 'protective effect' of the Red system on the ReeF pathway was due to both proteins or to only one of them. In the absence of 2 replication, an analysis was made of the rescue of the O + gene from the prophage by 2ci- bio69 Oam205, which produces/3 protein but not 2 exonuclease (Manly et al. 1969; Shulman et al. 1970). The results are shown in Table 5. The number of recombinants per infected cell was always higher than that obtained when the superinfecting phage was Exo2-/3- (see Table 2). Therefore, it seems that recombination in this case is favoured by the activity of/~ protein. However, even if/3 protein is present, elimination of exonuclease I is still necessary for pronounced activity in the RecF pathway (see data for rec + and sbcB15 strains, Table 5). Although/3 protein increases recombination in reeF and sbcB recF mutants (compare data from Tables 2 and 5), indicating that /3 protein stimulates reeF-independent recombination, it should be noted that when the recF+ gene product and/3 protein are simultaneously present, in either sbcB + or sbeB15 strains (see Table 5), the amount of recombination is significantly
Table 6. Rescue of the O + marker by Exo2-/?+ phage under conditions of permissive replication Strain no.
Genotype recA recB recC sbcB recF
IC61 IC389 IC180 IC60 IC66
+
+
+
+
+
56
+
+
+
+ 143 + 143
+
+
+
+
+ +
+ +
+ +
15 15
Recombinants/ Recombination infected cell frequency (x 100) (x 100) 4.6 1.2 3.1 45.4 3.1
0.08 0.03 0.06 0.5 0.06
The phage was 2ci- bio69 Oarn205. Recombinants and total phage were scored as in Tables 2 and 3. In all experiments the number of total phage and of infected cells/ml was 2 x l0 p Table 7. Rescue of the h marker by replicating DNA of Exo,~-/3 + superinfecting phage Strain no.
Genotype recA recB recC sbcB recF
IC74 + V3379 13 IC181 + IC73 + IC75 +
+
+
+
+
+
+
+
+
+
+
+ +
+ +
15 15
+ 143 + 143
Recombinants/ Recombination infected cell frequency (× 100) (x 100) 4.3 0.4 3.3 46.5 4.4
0.1 0.03 0.1 0.5 0.1
The phage was )dmm434 bio69. Recombinants were scored as in Table 5. In all experiments the number of total phage and of infected cells/ml was 2 x 106 greater than that resulting from the sum of the values obtained when either is present alone (see data for Exo2-/~- in rec + or sbcB15 strains, Table 2, and data for Exo)~-/~ + in recF143 or sbcB15 recF143 strains, Table 5). This suggests that/~ protein also stimulates recombination via the RecF pathway although obviously it does not overcome the inhibitory effects of exonuclease I. Since exonuclease I deficiency also facilitates recF-dependent recombination between the prophage and replicating D N A of the superinfecting R e d - phage (Tables 3 and 4) an additional test was whether the presence of/~ protein made elimination of exonuclease I unnecessary under conditions of permissive replication for a completely efficient RecF pathway. The results obtained (Tables 6 and 7) show that during superinfection by replicating Exo)~ /~+ phage, exonuclease I-deficiency increases the number of recombinants per infected cell (or the recombination frequency) with respect to that in the sbeB ÷ strain (compare data for sbcB15 and the wild-type strains). This indicates that, also in this case, activity of/~ protein does not suffice to prevent the negative effect of exonuclease I on the RecF pathway. On the other hand, comparison of Tables 4 and 7, 3 and 6, shows that /~ protein also stimulates recombination under conditions of permissive replication. Although this protein seems to mediate a Rec-independent recombination (see data for recA mutants) it is clear that it cooperates with the Rec system and that such cooperation is much more efficient when the RecF pathway is fully active, i.e., in the absence of exonuclease I (see data for Exo)~-/~- and Exo2-/? + in the sbcB and sbcB recF mutants). Although the effect of/3 protein appears in the case of the O + rescue more marked than in the h rescue, when the rescue of both markers from ,~h imm434 prophage by 2imm21 cI- int6 red30am29 and 2cI- bio69 Oam205 was analyzed simultaneously (data not shown), it was found that 1~protein favoured
503 the rescue of the h marker as much as the O +. This suggests that the observed effect of/~ protein on recombination is not interval dependent, at least for the intervals containing the O and J genes. If fl protein does not suffice to prevent the negative effect of exonuclease I on the RecF pathway, an alternative possibility is that 2 exonuclease could be the viral protein which cooperates with the RecF pathway and makes it insensitive to exonuclease I. In this context, it is relevant to note, by comparing Tables 2 and 6, that in the rec + strain the amount of recombination for Exo2-fl + (2.6) is smaller than that: obtained for Exo2+fl + phage (12.5), i.e., 2 exonuclease increases recombination of unreplicated D N A in the rec + strain. This increase appears mainly to be due to cooperation between 2 exonuclease and the RecF pathway because the amount of recombination when 2 exonuclease and the recF + product are simultaneously present is greater than that resulting from the sum of the values obtained when either is present alone. Moreover, it should be observed that the increase due to the activity of 2 exonuclease in rec + strains is greater than in recF mutants suggesting that the contribution of 2 exonuclease to the recombination of unreplicated D N A is unimportant when the RecF pathway is blocked. Since the amount of recombination for Exo2+fl + in the rec + strain (12.5) is similar to that obtained for Exo2-fl + phage in the sbcB mutant (13.8), it is concluded that, in the presence of/~ protein, a fully efficient RecF pathway can be obtained either via cooperation with 2 exonuclease (even in sbcB + strains) or, if the viral exonuclease is absent, via inactivation of exonuclease I. What, then, is the role of )~ exonuclease in the recF-mediated recombination? The viral enzyme could operate on a specific intermediate promoted by the RecF pathway and thus mediate in the production of recombinant DNA. This extends the previous hypothesis that in the absence of 2 replication " t h e products of the red genes can operate upon recombinational intermediates which require recA activity for their formation" (Stahl et al. 1978). It has been proposed that in recombination 2 exonuclease catalyzes the strand assimilation step that increases the length of the heteroduplex region. The hypothesis holds that 2 exonuclease promotes assimilation of an invasive strand by starting at a 5' terminus, digesting the strand of the recipient molecule which will be replaced by the invasive strand (see Radding 1978). Our results suggest that in the absence of 2 exonuclease, other protein(s), probably bacterial protein(s), would be able to perform the role of 2 exonuclease in the recF-dependent recombination, but that then this process would be sensitive to exonuclease I. This enzyme of E. coli is specific for singlestranded D N A and acts processively starting at a 3' terminus (Lehman and Nussbaum 1964; Thomas and Olivera 1978). Perhaps in the absence of 2 exonuclease an appropiate substrate for exonuclease I appears in the RecF pathway. In conclusion, it is proposed that 2 exonuclease diverts material from the RecF pathway in a shunt reaction that hinders the production of the intermediate able to be recognised by exonuclease I, but that at the same time allows the completion of recF-initiated recombinational intermediates. Acknowledgements. I am especially grateful to Dr. M. Blanco for stimu-
lating discussions throughout this work and to Drs. A.J. Clark and E. Knecht for critical reading of the manuscript and valuable suggestions. I am also grateful to Dr. F. Thompsom for correction of the several drafts of this paper, to P. Collado, L. Pom6s, and J.E. Rebollo for helpful comments and to S. Tortosa for technical assistance. The generosity of Dr. A.J. Clark in supplying Dr. Blanco's laboratory with JC7623, JC9239, and JCS111 strains is gratefully acknowledged.
References
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