Dec 21, 1987 - alkA gene or the basal level of RecA protein, but rather required the ada gene. ... involves the products of at least four genes, ada, alkA, alkB,.
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BACTERIOLOGY, Mar. 1988, p. 1354-1359
Vol. 170, No. 3
0021-9193/88/031354-06$02.00/0 Copyright 0 1988, American Society for Microbiology
Inhibition of the SOS Response of Escherichia coli by the Ada Protein J.-ALBERT VERICAT,t RICARDO GUERRERO, AND JORDI BARBE* Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain Received 8 April 1987/Accepted 21 December 1987
Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecFmutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene. The treatment of bacterial cells with nonlethal doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) causes the induction of a set of enzymes to repair the lesions caused in the DNA by this alkylating compound (23, 39). This inducible DNA-repair network, called the adaptive response, involves the products of at least four genes, ada, alkA, alkB, and aidB (17, 22, 24, 50), and it is positively regulated by the ada gene (32, 33). The ada gene is also the structural gene for the 06-methylguanine-DNA methyltransferase enzyme, which corrects the major mutagenic DNA lesion 06-methylguanine by transferring the methyl group to a specific residue close' to the C terminus of the Ada protein (16, 42). Adaptive response is triggered in Escherichia coli by the self-methylation of the Ada protein by transfer of a methyl group from a phosphotriester in alkylated DNA to a cysteine residue in the N-terminal half of Ada (36, 43). Methylation of the Ada protein converts to it in an activator of the transcription of the four adaptive genes (36, 43). The Ada protein is processed by a cellular protease (42) which produces two domains that retain the ability to repair 06-methylguanine and phosphotriesters, although the regulatory activity of the Ada protein is destroyed (43).
cleave or promote the self-cleavage of the LexA repressor (13, 27, 28), resulting in the expression of the SOS genes. The adaptive response and SOS system have separate networks, although some relationships seem to exist between them. For example, MNNG does not induce some SOS functions at either adaptive (15) or high (2) doses; MNNG can also avert the induction of filamentous cell growth or prophage lytic growth triggered by other SOSinducing agents (46). The interference between these responses at the cellular level has already been reported (14, 23). For all these reasons, and to determine the pathway by which the adaptive response affects the induction of the SOS genes, we have studied the expression of recA, sfiA, and umuDC genes, induced by UV radiation, in several DNArepair E. coli mutants preadapted with low doses of MNNG.
The behavior of the Ada protein in the adaptive response strongly resembles that of the RecA regulatory protein in the SOS repair system (29, 51). In this latter repair system, a set of genes is coordinately expressed when the cells are exposed to agents that damage DNA or block DNA replication. Biochemical and genetic data have demonstrated that the SOS response is under the control of the recA and lexA proteins. LexA protein is the common repressor to the SOS genes. The basal RecA protein is reversibly activated to a protease by an inducing signal after DNA damage. This signal is increased by the exonuclease activity of the RecBC enzyme (1, 37). It has also been proposed that RecF protein optimizes the use by RecA protein of the SOS-inducing signal to become a protease (44). Once activated, and with the help of the ssb protein (26, 29, 52), RecA protease can
vir.
MATERIALS AND METHODS Bacterial strains, bacteriophage, and growth conditions. Bacteria used in this work are listed in Table 1. All strains are E. coli K-12 derivatives. The phage used was X imm434
Cultures were grown at 30 or 37°C with shaking in AB liquid minimal medium (11) supplemented with thiamine (10 ,ug mI-'), glucose (0.2% [wt/vol]), and Casein Hydrolysate Acid (0.4% [wt/vol]). When necessary, LB (32) was used as a rich medium. Vitamins and mineral salts were obtained from Merck, and Casein Hydrolysate Acid was from Oxoid. Ampicillin, kanamycin, and streptomycin were a generous gift from Antibi6ticos, S.A. (Leon, Spain). IPTG (isopropylP-D-thio-galactopyranoside) was from Sigma, and o-nitrophenyl-p-D-galactopyranoside and X-Gal (5-bromo-4-chloro3-indolyl-i-D-galactopyranoside) were from Fluka. Lysogenization of bacterial strains. To lysogenize with defective X d (recA::lacZ) and A d (sfiA::lacZ) phages, late-exponential-phase cells grown in LB containing 0.4% maltose were centrifuged, suspended in 10-2 M MgSO4, and infected at a multiplicity of infection of 0.01 with a stock containing a mixture of active and defective A phages of different immunity. After 15 min of adsorption, cells were plated in LB-ampicillin or X-Gal petri dishes. Apr or blue
* Corresponding author. t Present address: Frederick Cancer Research Facility, National Cancer Institute, Frederick, MD 21701.
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TABLE 1. E. coli strains used in this work Strain
Genotype
Source or reference
AB1157 GC2375 GY4786 GW1000
argE3 his4 thr-J proA2 leuB6 thi-I supE44 strA As AB1157, but harboring X d(recA::lacZ) As AB1157, but harboring X d(sfiA::lacZ) lacAUl69 recA441 sfiAll, harboring pSE140 (umuDC::lacZ) As GW1000, but malE::Tn5 umuDC::Mu Amp lac As GW1104, but harboring pYN3059 As AB1157, but harboring pSE140 (umuDC::lacZ) As AB1157, but recAo98 As DM2211, but harboring A d(recA::lacZ) As AB1157, but recA441 recA453 As GY2798, but harboring X d(recA::lacZ) As AB1157, but recA441 As JM12, but harboring X d(recA::lacZ) As AB1157, but harboring plasmid pYN3059 As UA4302, but harboring A d(recA::IacZ) As AB1157, but recB21 recC22 As IC53, but harboring X d(recA::lacZ) thyA recB1009 argA::Tn1O strA As IC1050, but harboring X d(recA::lacZ) As AB1157, but recF143 As JC9239, but harboring X d(recA::lacZ) As AB1157, but ada-i As PJ1, but harboring X d(recA::lacZ) As PJ1, but harboring pYN3059 As UA4303, but harboring X d(recA::IacZ) As AB1157, but ada-5 As BS24, but harboring X d(recA::lacZ) As AB1157, but his' alkA As MS23, but harboring pYN3059 As UA4304, but harboring X d(recA::lacZ)
20 8 21 G. C. Walker G. C. Walker This work This work 18 This work 4 This work 9 This work This work This work 1 1 10 This work 19 This work 22 47 This work This work B. Sedgwick This work 21 This work This work
GW1104 UA4441 UA4318 DM2211 UA4187 GY2798 UA4188 JM12 UA4298 UA4302 UA4362 IC53 UA4110 IC1050 UA4305 JC9239 UA4142 PJ1 UA4186 UA4303 UA4374 BS24 UA4442 MS23 UA4304 UA4363
clones were tested for their immunity and for the presence of either recA::lacZ or sfiA::lacZ fusions. DNA techniques. Plasmid DNA was isolated by the alkaline extraction procedure of Birnboim and Doly (3). This procedure was always followed by CsCl-ethidium bromide isopycnic centrifugation as described by Maniatis et al. (30). When necessary, transformation was performed according to the procedure of Cohen et al. (12). Adaptive treatment. Adaptive treatment was performed according to the method of Jeggo et al. (23). The adaptive procedure always involved a preadaptive treatment of 120 min with 0.05 ,ug of MNNG ml-'. It was followed by adaptive treatment with MNNG at a final concentration of 0.5 ,g ml-' for 90 min. Both treatments were performed at 30 or 37°C with shaking. The procedure to determine the MNNG-adaptive concentration useful to adapt the RecBC- and RecF- strains was that of Jeggo et al. (23). UV irradiation. Cultures were grown to 2 x 108 cells mlin supplemented AB minimal medium. Cells were centrifuged, suspended in 0.01 M MgSO4, and then irradiated in a glass petri dish (10-cm diameter) with a General Electric GY1578 germicidal lamp at a fluence of 0.5 J m-2 s-1 (determined with a Latarijet dosimeter). After irradiation, the cells were centrifuged and suspended in the same volume of supplemented AB minimal medium. To study the behavior of the RecA441 mutant, cells were grown in supplemented AB minimal medium at 30°C. For the temperature-mediated induction of the SOS response, cells were incubated at 42°C in the presence of adenine at 300 jig ml-'. 13-Galactosidase assay. The ,B-galactosidase assay was per-
formed as described by Miller (32). Particular conditions were as previously reported (1). RESULTS Influence of the recA alleles and recBC and recF genes in the induction of SOS genes in MNNG-preadapted cells. The MNNG-adaptive pretreatment produced a decrease of about 40% in the UV-mediated expression of both recA and sfiA genes (Fig. 1). Nevertheless, the induction of the umuDC gene was unaffected by the adaptive pretreatment, in agreement with previous results on the level of mutagenesis (15). This inhibition of expression of SOS genes in the UVirradiated cells was independent of the level of RecA protein because MNNG adaptation also produced a decrease in the expression of the recA::lacZ gene fusion both in a recAo98 strain (with constitutive synthesis of RecA protein) and in the double mutant recA441 recA453 (harboring a promoterdown mutation in the recA gene which blocks the amplification of RecA protein) (Fig. 2). On the other hand, the temperature-mediated expression of the recA gene was not affected by adaptive treatment with MNNG in the RecA441 mutant growing at 42°C (Fig. 2), suggesting that the inhibition of the SOS gene expression by adaptive pretreatment must be attributed to some kind of relationship between the molecular mechanisms of the adaptive response and those molecular events that lead to the activation of the RecA protease. One of the most important and incompletely understood events related to the induction of the SOS response is the molecular signal that activates the system. It has been indicated that the activity of RecBC or RecF proteins or both
1356
VERICAT ET AL.
J. BACTERIOL.
C
0
U
4) 10.~~~~~~ c~~~~~~~~
._O0 10
0
rr
2.
0
L
20
.1
a
40
60
Time
80 100
(min)
FIG. 1. Relative extents of recA (O, *), sfiA (E, A), and umuDC (O, *) gene induction, measured as 0-galactosidase synthesis, in unadapted (open symbols) or adapted (closed symbols) cultures of GC2375, GY4786, and UA4318 strains of E. coli after UV irradiation with 20;Jm-2 . The data are referred to the level of specific units of P-galactosidase presented by each strain without any treatment.
(41, 44) is responsible for the activation of the RecA prote-
ase. For this reason, we studied the effect of MNNG-related induction of the adaptive response on the UV-mediated induction of recA gene in mutants completely lacking exo-
nuclease V activity (recB21 recC22), lacking only the degradative function of exonuclease V (recB1009) (10), or lacking
RecF activity (19). recA gene expression was not affected by the adaptive response either in recB21 recC22 or in recB1009 mutants, whereas there was a clear decrease in recA gene induction in the adapted recF143 mutant (Fig. 3). Importance of the ada and alk genes. The inhibitory effect of the adaptive response on induction of the SOS system is dependent on the Ada protein but not on the Alk protein because the level of recA gene induction was not affected in adapted ada-i mutants whereas the behavior of the alk mutant was very similar to that of the wild-type cells (Fig. 4). The ada-S mutant further showed the same behavior as the ada-i strain (data not shown). The Ada protein inhibits the SOS response either before or after its methylation originated to remove the methylations from DNA. To determine which one of these two possibilities was correct, we studied the induction of the recA gene in a wild-type E. coli strain harboring the plasmid pYN3059 after UV irradiation. The pYN3059 plasmid is a pUC9 derivative in which the ada gene is under the control of the lac promoter (34, 35), and its copy number can be increased by the effect of high temperature (34). Thus, by a combination of high temperature and addition of IPTG, it is possible to increase the intracellular level of the Ada protein by severalfold (40). In the nonadapted UA4362 strain growing in the presence of IPTG, UV-mediated induction of the recA gene was lower at 42°C than at 37 or 30°C (Fig. 5), although Ada protein seemed to be more active in the inhibition of SOS response when the cells were adapted previously to MNNG (Fig. 5). The UV-promoted expression of the umuDC gene was the same in both GW1104 and GW1104(pYN3059) strains growing at 30°C in the presence of IPTG (data not shown). Thus, the inhibitory effect of the Ada protein on the induction of some SOS genes is not due to any indirect effect such as the reduction of the actual dose to cells. Furthermore, the expression of the recA gene in both UV-irradiated ada-i and alkA mutants, harboring the
c 0
C
.Q 0
-
10 U' .4-
c
a)
c
4,0
0 in c do -
K -, U,
>
x
-_.
112
0
20
40 60 80 Time (min)
100
FIG. 2. Relative extents of recA gene induction, measured as ,-galactosidase synthesis, in unadapted (open symbols) or adapted (closed symbols) cultures of both recAo98 (A, A) and recA441 recA453 (0, 0) mutants after UV irradiation with 20 J m2, and of the recA441 mutant growing at 420C (O, *) in the presence of adenine at 300 ,ug ml-'. Data were treated as described for Fig. 1.
.
U,_0
a {s
Time (min)
FIG. 3. Relative extents of recA gene induction, measured as
0-galactosidase synthesis, in unadapted (open symbols) or adapted
(closed symbols) cultures of (A) recB21 recC22 (0, 0) and recBiOO9 (A, A) and (B) recF143 (O, U) mutants after UV irradiation with 20 J m-2. recB and recC mutants were treated in the presence of adenine at 300 pLg ml-'. Data were treated as described for Fig. 1.
EXPRESSION OF SOS GENES IN ADAPTED E. COLI CELLS
VOL. 170, 1988
.2_ c c
10 5 0
Cr10
/7 4
20
Time
60
8
10
( min )
FIG. 4. Relative extents of recA gene induction, measured as
0-galactosidase synthesis, in unadapted (open symbols) or adapted
(closed symbols) cultures of the ada-l (O, *) and alkA (E, A)
mutants after UV irradiation at 20 J m-2 . Data were treated as described for Fig. 1.
pYN3059 plasmid and growing at 42°C in the presence of IPTsG, was the same as that in the UA4362 strain under the same conditions (data not shown). On the other hand, the presence of the pYN3059 plasmid did not produce any decrease in the temperature-mediated induction of the recA gene in a recA441 mutant growing at 42°C in the presence of IPTG (data not shown).
It has been proposed that single-stranded DNA, regions produced when DNA replication is blocked by DNA damage, could be an activating signal for the RecA protein after enhancement of the extension of single-stranded DNA regions by the activity of the RecBC exonuclease (37). Furthermore, recent work (1) has shown that in RecBC- mutants, recA and umuDC genes and lambda prophage are induced by UV irradiation at lower levels and delayed with respect to the wild-type strain. This fact seems to indicate that other DNA nucleases, besides exonuclease V, may also produce a molecular signal that is involved in RecA activation but is not as active as the signal mediated by the RecBC nuclease. Similarly, Salles and Defais (38) have shown that the activity of the UvrABC complex on UV-irradiated cells produced DNA gaps that activated the expression of the SOS functions in nonreplicating DNA. Our data indicate that the inhibitory effect of adaptive pretreatment on UV-mediated induction of the recA gene requires DNA degradation by the RecBC exonuclease (Fig. 3). The Ada protein is able to react with DNA, as shown by the fact that this protein and its C-terminal fragment have both been purified by binding to single-stranded DNA (16, 31). For this reason, it is conceivable that in a cell which is amplifying the concentration of the Ada protein, large quantities of this protein can bind to the single-stranded DNA gaps produced by recBC activity, avoiding direct activation of RecA protein. Similarly, it has been shown that the presence of large quantities of Ssb protein may inhibit the induction of the SOS system (33, 52). It has been proposed that RecF protein combines with RecA protein (48, 49) to obtain optimal SOS-inducing signal usage (45). Nevertheless, the UV-induced recF143 mutant C 0
.2_ 0~~~~~~~~
/
ct Go
DISCUSSION Results obtained in this work showed that the increase in the cellular level of Ada protein produced an inhibition of the UV-dependent expression of recA and sfiA genes. This phenomenon is independent of previous DNA methylations. In the pathway of the SOS response, two steps may be described. First, the SOS-inducing signal is generated after either DNA damage or the detention of the DNA replication fork (5, 41), and RecA protein is activated by interaction with this SOS-inducing signal. Next, the LexA repressor is hydrolyzed and SOS genes are expressed (28). The inhibitory effect of the adaptive treatment must be found in.one of two processes: activation of RecA protein or LexA repressor hydrolysis. The results reported here, showing that the Ada protein does not affect the temperaturemediated induction of the recA gene in a RecA441 mutant, lead us to conclude that the process of LexA cleavage is not inhibited or modified by the expression of the adaptive response. Furthermore, the fact that the intracellular level of RecA protein (Fig. 2) did not affect the inhibitory effect caused by the MNNG-adaptive treatment suggests that the process of amplification of RecA protein is not the step affected by the increase of the Ada protein, or its activity, during the adaptive pretreatment. Thus, the activation of the RecA protein to a protease must be the step disturbed by the high levels of Ada protein after MNNG-mediated adaptation.
1357
C 5H 0
a'10
4, K
44
0
20
40
Time
60
80 100
(min)
FIG. 5. Relative extents of recA gene induction, measured as P-galactosidase synthesis, in unadapted (open symbols) and adapted (closed symbols) cultures of E. coli GC2375(pYN3059) growing in the presence of IPTG (10 mM) at 42°C (O, *), 37°C (V), or 30°C (A), after UV irradiation with 20 J m-2. Relative rates of recA gene induction in strain GC2375 without pYN3059 and after UV irradiation at 20 J m2 are shown as a control (0, 0). Cells were grown in minimal AB supplemented medium with thiamine, Casamino Acids (as described in Materials and Methods), and glycerol (0.2% [vol/vol]). IPTG was added 10 min before UV treatment. Data were treated as described for Fig. 1.
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VERICAT ET AL.
showed the same behavior as the wild-type strain when cells were preadapted (Fig. 3), indicating that the hypothetical RecF-RecA protein complex is not the target of the adaptive response-mediated inhibition of SOS gene transcription. Finally, the results reported in this paper also suggest that some differences must exist in the regulation of the umuDC genes in comparison with other SOS genes, such as recA and sfiA. The adaptive pretreatment did not affect the level of umuDC gene induction when cells were treated with UV radiation. Perhaps a low level of activated RecA protein is enough to trigger umuDC gene expression. In this respect, it has been reported that recA and sulA operators bind to the LexA protein more strongly than does the umuDC operator (6, 7, 25). The slower kinetics of the UV-mediated induction of umuDC, in comparison with recA and sfiA (Fig. 1), could perhaps also explain the differences between the responses of these genes to the inhibitory effect of the adaptive response. This subject deserves further investigations. ACKNOWLEDGMENTS We thank M. Blanco, R. D'Ari, S. J. Elledge, B. Salles, B. Sedgwick, and G. C. Walker for their generous gifts of several strains, and J. M. Cuartero for drawing figures. This work was supported by grant BT85-0028 of the Comisi6n Asesora de Investigaci6n Cientifica y Tecnica (to J.B.). We acknowledge the help of the Comissi6 Interdepartamental per a la Recerca i Innovaci6 Tecnologica de la Generalitat de Catalunya for different grants for the purchase of equipment.
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1.
2. 3. 4.
5. 6.
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