Biochimie ( ! 991 ) 73,277-280 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris
277
C h a r a c t e r i z a t i o n o f recF s u p p r e s s o r s in Bacillus subtilis JC Alonso, G Ltider Max-Planck-lnstitut fiir Molekldare Genetik, lhnestrasse 73, D- 1000 Berlin 33, German v (Received 27 November 1990; accepted 13 February 1991)
Summary - - A reeF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNAbinding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response. recF gene / SOS induction / DNA repair / recA / ssb
In an e m e r g e n c y response to severe D N A d a m a g e , m a n y cells have a m e c h a n i s m that enables t h e m to induce D N A repair e n z y m e s [ 1-2]. G e n e s i n d u c e d in this stress response (SOS response), are n o r m a l l y repressed by a repressor, t e r m e d L e x A in Escherichia coli. T h e y are turned on f o l l o w i n g proteolytic inactivation o f the L e x A protein. In vitro both E coli R e c A and Bacillus subtilis R e c E proteins mediate proteolytic c l e a v a g e o f the repressor [1-5]. The B subtilis fetE_ r e e f and ,-~,,-,M gon,~e are "h~ h,,....., . . . . . . . . c E coli recA, recF and recR genes, respectively [6-7]. At the recent International C o n f e r e n c e on the Bacillus subtilis g e n o m e (2-5 S e p t e m b e r 1990, Paris, France) was suggested the recA and recR designations for the genes p r e v i o u s l y referred to as recE and recM in B subtilis. W e will, hereafter, follow their r e c o m m e n dation. In E coli has previously been reported that: a) SOS induction is h a m p e r e d in recF or ssb (ssb-I and ssb1 13) mutant strains [8-9], b) the i m p a i r m e n t in SOS induction could be reversed either by o v e r e x p r e s s i n g the ssb-I gene product from a clone, w h e n the recA441 mutation is placed in the ssb-I 13 strain [9], or w h e n the recA801 mutation is placed in the recF400 genetic b a c k g r o u n d [10], and c) certain recA mutants partially suppressed the recF sensitivity to U V - l i g h t [11-12]. The S O S - r e s p o n s e model, as adapted for B subtilis, postulated that the Ssb, RecF, R e c L and R e c R proteins are involved in R e c A ' a c t i v a t i o n ' [13]. In this report we e x a m i n e s o m e o f the a b o v e prediction [13] b y m e a s u r i n g the i n f l u e n c e o f different recA alleles and heterologous Ssb proteins in the B subtilis recF genetic background. __
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Materials and Methods Bacterial strains, plasmids and phages B subtilis strains YB1015 (recA4), BG115 (recA73), BGi01 (recP149), BGI03 (recR27), BGI07 (recLl6), BGII9 (recH342), BGI25 (addA5), BGI26 (addB72), BGi29 (reeF15). BGI45 (recA4 recFl5) and BGI33 (recA73 recFl5) are isogenic derivatives of YB886 (trpC2 metB5 amyE sigB xin-1 attSP~) 1141. Plasmids pEB4 [i5] and their derivatives bearing either the E coli ssb gene (pBTI86) or 029 ssb (gene 5) gene (pBTI88) were used. The source of the E coli ssb DNA was a 2.2 Kb Kpnl fragment of plasmid pLN84 (a gift of JM Louarn). Gene 5 gene from the B subtilis phage 029 was obtained as a 0.8 kb EcoRI-Rsal fragment from plasmid pGM26 [161. Plasmids pBTI86 and pBTI88 were constructed and kindly provided by J Kupsch and H Leonhardt. The B subtilis bacteriophage ~105 was used 1171. Prophage induction was measured as described previously [14]. Media and transformation B subtilis cells were grown on TY (if not stated otherwise) or minimal medium [181. Competent cells were prepared as previously described [19]. Transformants were selected on TY agar medium containing either phleomycin (100 ng/ml) or neomycin (5 Hg/mi). Chemical treatment Methyl methanesulfonate (MMS) was purchased from Eastman KodaK, USA. Ethyl methanesulfonate (EMS) was obtained from TCI, Japan. 4-nitroquinoline-l-oxide (4NQO) was purchased from Sigma Chemical Co, USA. The chemical treatment of the mutant strains was performed essentially as described in [141.
278
JC Alonso, G Liider
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protein could participate in the recA-dependent postreplication repair processes without the need for RecF protein. To understand such an event, the r e c A 7 3 r e c F l 5 strain was challenged with different D N A damaging agents or tested for its ability in the induction of resident prophages. B subtilis wild type, r e c A 7 3 , r e c F 1 5 and r e c A 7 3 r e c F l 5 strains were exposed to the killing action of 10 m M MMS, 160 m M EMS or 500 ~M 4 N Q O and the rate of survival was recorded. As shown in figure 1, the r e c A 7 3 r e e F 1 5 cells were more resistant to MMS, EMS or 4 N Q O than each of the singlemutant parents. Identical results were obtained when the r e c F null mutant strain ( r e c F 3 1 ) was used (data not shown). Hence, the recA suppressor mutation allows D N A repair to proceed in the absence of a functional RecF protein. Is this 'suppressing' phenotype due to a higher SOS-induction than in the single mutant strains? To quantify the involvement of r e c A mutations in the induction of the SOS response in the r e c F 1 5 strain, the wild-type, recA4, recA73, r e c F l 5 , recA4 r e c F 1 5 and r e c A 7 3 r e c F 1 5 strains were lysogenized with bacteriophage ~ 1 0 5 and then induced by irradiation with UV light. The results of these experiments are shown in table I. The r e c A 4 , r e c A 7 3 and r e c F 1 5
B suhtilis following exposure to 10 mM MMS (A), 160 mM EMS (B), or 500 laM 4NQO (C). Strains tested were rec + (c), recA73 (v), recFl5 (.~) and recFl5 recA73 t~).
Results and Discussion Suppression o f the r e c F l 5 by the recA73 mutation
mutation residers o subtilis cells very sensitive to DNA damaging agents [14]. It has been reported that: a) increased synthesis of RecA protein does not alleviate the r e c F defect [13], and b) the r e c A 7 3 r e c F l 5 strain was less sensitive to methyl methanesulfonate (MMS) than the r e c F 1 5 strain [14]. Hence, we tried to established whether the RecA73 mutant m
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Table I. Maximal UV induction of prophage ~105 Strai~z
YB886 BG115 YB 1015 BG 129 BG 136 BG145
Relevant gellotype
Maximal UV induction of ~105 (fold) a
183 1.8
Rec ÷ recA73 recA4 reeF15 recA73 recA4
277
C h a r a c t e r i z a t i o n o f recF s u p p r e s s o r s in Bacillus subtilis JC Alonso, G Ltider Max-Planck-lnstitut fiir Molekldare Genetik, lhnestrasse 73, D- 1000 Berlin 33, German v (Received 27 November 1990; accepted 13 February 1991)
Summary - - A reeF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNAbinding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response. recF gene / SOS induction / DNA repair / recA / ssb
In an e m e r g e n c y response to severe D N A d a m a g e , m a n y cells have a m e c h a n i s m that enables t h e m to induce D N A repair e n z y m e s [ 1-2]. G e n e s i n d u c e d in this stress response (SOS response), are n o r m a l l y repressed by a repressor, t e r m e d L e x A in Escherichia coli. T h e y are turned on f o l l o w i n g proteolytic inactivation o f the L e x A protein. In vitro both E coli R e c A and Bacillus subtilis R e c E proteins mediate proteolytic c l e a v a g e o f the repressor [1-5]. The B subtilis fetE_ r e e f and ,-~,,-,M gon,~e are "h~ h,,....., . . . . . . . . c E coli recA, recF and recR genes, respectively [6-7]. At the recent International C o n f e r e n c e on the Bacillus subtilis g e n o m e (2-5 S e p t e m b e r 1990, Paris, France) was suggested the recA and recR designations for the genes p r e v i o u s l y referred to as recE and recM in B subtilis. W e will, hereafter, follow their r e c o m m e n dation. In E coli has previously been reported that: a) SOS induction is h a m p e r e d in recF or ssb (ssb-I and ssb1 13) mutant strains [8-9], b) the i m p a i r m e n t in SOS induction could be reversed either by o v e r e x p r e s s i n g the ssb-I gene product from a clone, w h e n the recA441 mutation is placed in the ssb-I 13 strain [9], or w h e n the recA801 mutation is placed in the recF400 genetic b a c k g r o u n d [10], and c) certain recA mutants partially suppressed the recF sensitivity to U V - l i g h t [11-12]. The S O S - r e s p o n s e model, as adapted for B subtilis, postulated that the Ssb, RecF, R e c L and R e c R proteins are involved in R e c A ' a c t i v a t i o n ' [13]. In this report we e x a m i n e s o m e o f the a b o v e prediction [13] b y m e a s u r i n g the i n f l u e n c e o f different recA alleles and heterologous Ssb proteins in the B subtilis recF genetic background. __
.
. . . . . . . . . .
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Materials and Methods Bacterial strains, plasmids and phages B subtilis strains YB1015 (recA4), BG115 (recA73), BGi01 (recP149), BGI03 (recR27), BGI07 (recLl6), BGII9 (recH342), BGI25 (addA5), BGI26 (addB72), BGi29 (reeF15). BGI45 (recA4 recFl5) and BGI33 (recA73 recFl5) are isogenic derivatives of YB886 (trpC2 metB5 amyE sigB xin-1 attSP~) 1141. Plasmids pEB4 [i5] and their derivatives bearing either the E coli ssb gene (pBTI86) or 029 ssb (gene 5) gene (pBTI88) were used. The source of the E coli ssb DNA was a 2.2 Kb Kpnl fragment of plasmid pLN84 (a gift of JM Louarn). Gene 5 gene from the B subtilis phage 029 was obtained as a 0.8 kb EcoRI-Rsal fragment from plasmid pGM26 [161. Plasmids pBTI86 and pBTI88 were constructed and kindly provided by J Kupsch and H Leonhardt. The B subtilis bacteriophage ~105 was used 1171. Prophage induction was measured as described previously [14]. Media and transformation B subtilis cells were grown on TY (if not stated otherwise) or minimal medium [181. Competent cells were prepared as previously described [19]. Transformants were selected on TY agar medium containing either phleomycin (100 ng/ml) or neomycin (5 Hg/mi). Chemical treatment Methyl methanesulfonate (MMS) was purchased from Eastman KodaK, USA. Ethyl methanesulfonate (EMS) was obtained from TCI, Japan. 4-nitroquinoline-l-oxide (4NQO) was purchased from Sigma Chemical Co, USA. The chemical treatment of the mutant strains was performed essentially as described in [141.
278
JC Alonso, G Liider
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Exposuretimelmin) Fig 1. Suwival of recombination-deficient strains of
protein could participate in the recA-dependent postreplication repair processes without the need for RecF protein. To understand such an event, the r e c A 7 3 r e c F l 5 strain was challenged with different D N A damaging agents or tested for its ability in the induction of resident prophages. B subtilis wild type, r e c A 7 3 , r e c F 1 5 and r e c A 7 3 r e c F l 5 strains were exposed to the killing action of 10 m M MMS, 160 m M EMS or 500 ~M 4 N Q O and the rate of survival was recorded. As shown in figure 1, the r e c A 7 3 r e e F 1 5 cells were more resistant to MMS, EMS or 4 N Q O than each of the singlemutant parents. Identical results were obtained when the r e c F null mutant strain ( r e c F 3 1 ) was used (data not shown). Hence, the recA suppressor mutation allows D N A repair to proceed in the absence of a functional RecF protein. Is this 'suppressing' phenotype due to a higher SOS-induction than in the single mutant strains? To quantify the involvement of r e c A mutations in the induction of the SOS response in the r e c F 1 5 strain, the wild-type, recA4, recA73, r e c F l 5 , recA4 r e c F 1 5 and r e c A 7 3 r e c F 1 5 strains were lysogenized with bacteriophage ~ 1 0 5 and then induced by irradiation with UV light. The results of these experiments are shown in table I. The r e c A 4 , r e c A 7 3 and r e c F 1 5
B suhtilis following exposure to 10 mM MMS (A), 160 mM EMS (B), or 500 laM 4NQO (C). Strains tested were rec + (c), recA73 (v), recFl5 (.~) and recFl5 recA73 t~).
Results and Discussion Suppression o f the r e c F l 5 by the recA73 mutation
mutation residers o subtilis cells very sensitive to DNA damaging agents [14]. It has been reported that: a) increased synthesis of RecA protein does not alleviate the r e c F defect [13], and b) the r e c A 7 3 r e c F l 5 strain was less sensitive to methyl methanesulfonate (MMS) than the r e c F 1 5 strain [14]. Hence, we tried to established whether the RecA73 mutant m
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Table I. Maximal UV induction of prophage ~105 Strai~z
YB886 BG115 YB 1015 BG 129 BG 136 BG145
Relevant gellotype
Maximal UV induction of ~105 (fold) a
183 1.8
Rec ÷ recA73 recA4 reeF15 recA73 recA4
