- Email: [email protected]
Role of SOS and OxyR systems in the repair of Escherichia coli submitted to hydrogen peroxide under low iron conditions
Asa
e Almeida”, AC LeitW*
UGr~ersidmir Federcll do Rio de Jmeiro. 21949-900 Rio de Jurzeiro. RJ; h Dept Bioji~sictr e Biontetrk lnstituto de Biologic, Uni\~er.sidade do Estcldo do Rio de Jmwirc,, 20551-030 Rio de Jmeiro, RJ, Brazil
(Received 22 October IO%; accepted 7 May 1997) Sunlnlary - There are at least two mechanisms by which H;?@induces DNA lesions in Escherich co/i: one in the presence of physiological iron levels und the other in low iron conditions. The survival as well as tile induction of SOS response in different DNA repair mutant strain> of E coli was evaluated after Hz02 treatment under low iron conditions (pretreatment with tin iron chelator). Our results indicate that, in norm4 iron conditions RecA protein has :I relevant role in recombination repair events, while in low iron conditions RecA protein is important as ;l positive regulator of the SOS response. On the other hand, the oq+AR mutant is sensitive to the lethal effects of Hz02 only in low iron conditions and this sensitivity camiot be correlated with DNA strand breaks.
hltroduction
?‘he consecutive univalt’nt to water in the rt2sgiration
reduction
ot’ inolccular
oxygen
cycle pwduces three active ints‘rmediates: superoxide anion (O:! * ), hydrogen peroxide (HzOz), and hydroxyl radical (OH “). These so-called oxidative oxygen spccics are potent oxidants of lipids, proteins, and nucleic acids 113). Several studies have shown that the killing at’c&s cxposed to H&I:! is mainly due to damage on DNA 112, 161, Furthwmorc, the prticipution of the CM-l ( as the main clamage agcant has been suggcstcd by studies using scuvcngcrs of6H ‘$I? 4, 221. OH ’can bc generated through the Fenton reaction with the participation of transition metals, such as Fe(lI) [I, 17, 19j. III hct, a variety of metal chelators as dipyridyl, o-phenantroline and desferrioxamine, can block the Fenton reaction by occupying metal coordination sites and thus protect the cells against the lethal effects of HzOr, confirming the role of iron in the generation of active oxygen species [ 141. It is known that the recombinational repair is an important pathway in repairing DNA lesions produced by H;?Oz, the enhancement of RccA protein being the most important SOS response, since other components of the SOS system
* Correspondence
and reprints
appear to be of little or no importance for r~~os~~bi~lati~~ repair 1141. The exposure of E co/i 6 low concentrations of Hz02 induces an adaptive response which confers increased bacterial resistance to subsequent exposure to high concentrations of II~Q 171. This response is accompanied by the induction of at least 30 proteins [ I I, 2 I], nine of which arc under the control of the OxyR protein 161.OxyR is a transcriptional activator of genes such as kcrfG,which encode% cntal;Ise-~~ydropcroxide1 i Z%]. Tlnr=it~crc~sedbacterial survival is Ilrre, at least in part, to an ~~~duc~d DNA repair capacity, since h phagc dama ed exl9~~s~dto Ha02 have a higher plating efficiency on pretrcate(~cells th1 011naive cells 17, (,I. As the QxyR controltcd proteins do not a~~~~~r to be involved in DNA repair, this rcsponsc must bc due to other peroxide induced proteins. We have shown that wild type cells and different DNA repair mutants strains arc less sensitive TVMaOa(5 IIIR~) when the cells are pretreated with iron chelators. However, in this condition, DNA-single strand breaks were observed, suggesting that there are at least two mcchanisnls of H~Q~ induced DNA lesions in E cdi: one through iron oxidation and OH 0 production and the other in low iron conditions, probably without OH. production [ 11.So, different lesions may be formed in each case and thus different DNA repair tnechanisms should be involved. The role of SOS and OxyR rcgulons in the repair of lesions produced by HzOzunder low iron conditions is unknown. In this work we evaluate
GwGJp~* “F thr-1 l&$6 thi-1 argE3 his-4 proA lack’1 galk2 xyl-5 ara- I3 rspL13 tsx-33 supE44 As A61 157, but recA13
As Al31 157, but lexA1 (Ind-) As AB 1157, but recA 142 As ABI 157, but xthA mD139 (urgF-lac)ZX flBS301 non gytA2 19 re1A 1 rpsL1 SO metE70 btuB::TnlO As RK4936, but oxy A3 uB6 thi-t his-4 pyrD @E galY rpoB IacAU 169 trp::MuC+ Phoc sGA::Mud (Ap lac)Ct” FAs , but AoxyR4 [A (oxyR btuB)4] , but AkuG3. kutE 12::TnltI AS _“_-._ _ -_xI.~____ --___ __xl_~ ll..“_-_-___“^____-_ _ -. - ____ll*l~- ----_ - -___I...__ _I-II__.__-Ic_..II”III_*.._ obtuined from P Howard-Flanders (University of Yale, New Haven, USA). The RK49.36 and TA41 I2 strains were lifarnia, Berkeley, USA). The PQ65, OG 100 and OG140 strains were ohtuined from P Quilliirdct mes (Universi 1 struin was obt;tined from Bernard W&s (University of ~~i~hi~i~~M~d~~~l School. WA). The France). The j~~~~~ strain was obtained from R Devoret (lnstitut Curie, Orseur, Fruncc).
cipation of these two mechanisms in DNA repair 2 treatment under low iron conditions (pretreatun iron chelutar). We also analyzed the induction c response to H$& in low iron conditions,
trifugcd. wsuspcudcd in M9S and trwtcd with dipyridyl illId HzOz. as described in the survival experiments.
5 H $+
i 10
I 15
wncantration ( m
Fig 1.Survival of lSclrc~ic*lricrc*olicells treated with dipyridyl and PIJQz. Cultures in the mid-exponential phase of growth were trated in YS medium at 37°C with I mM dipyridyl for 20 min and then submitted to H&I2 for 20 min. Wild type (AB I 157). mcaCa(AB~463), : )Y*c’142 A (JC4728). 0; IaA I (AB2494), U
in low iron con
1h
with vigorous agitation. The measurements of P-galactosidase and alkaline ~hos~hatase activities were performed as previously detion factor of .$~::I~&? expression is the ratio of the activities of /Sgalact tive concentration of
idase and alkaline phosphatase at the respec202 divided by its value at zero concentration.
Cultures of the AB2463 (wAI_2) strain were pretreated with an iron chelator ( 1 mM of dipyridyl) and then exposed
5
10
H2 O2 concentration
5
15 (
mM
inability to induce SOS functions and/or to its inability to carry out DNA recombinational repair. These possibilities were evaluated using cultures of the K4728 (wcAI42) strain, which is defective in DNA recombination but is able to promote self-cleavage of LexA repressor 1241, and cultures of the Al32494 (IexAI) strain, defective in SOS induction by virtue of yieldin a LexA protein resistant to self-cleavage promoted by ecA [5]. The sensitivity of the IYC;~I42 mutant was close to that of the wild type strain (fig 1B). On the other hand, the IexA / mutant was as sensitive to Hz02 in low iron conditions as the ret;913 mutant (fig 1). indicating that induction of SOS functions, but not recombinational repair, is important for DNA repair in bacterial cells treated with Hz01 in low iron conditions.
1
10
H, O2 concentration
15 ( m
cells treated with dipyridyl and &02. Cultures in the Fig 2. Survival of Esc~herichirr coli wild type (PQ65), ; and A(‘XVR4K=1()()), mid-exponential phase of growth were treated in M9S medium at 37OC with I mM dipyridyi for 20 nlin and then submittcclto I-kQ:!for 20 min (A) or only treated with Hz02 for 20 min (B).
imn conditions wus distinct from that ~~b~~~v~~~ in the ologic~l iron levels itr wild type c&. In the prese norm1 iron levels, a significant induction is observed with
1 mM H$&, while in low iron conditions the induction is only observed with IOto 12.5 mM Hz02 (fig 3 We next evaluated the effects of a\:\A induction of expression of sfiA by Hz02 in normal and low iron conditions. We found that the induction of &golactosidase by H@:! in the o_@R mutant was significantly higher than in the parental strain in the normal iron conditions, while in the low iron conditions the induction was similar to the parental strain (tlg 3B).
We evaluated the nduptivc ~~‘s~9011se induced by H$J:! whether the Fenton reaction is blocked or not. A Hz01 hypersensitive mutant, the BW909I (s&A) strain 181.was pretreated with low H$&concentration in normal or in low iron conditions, followed by treatment with higher HzO:! concentrations in low and normal iron conditions. In both conditions the pretreatment protected the cells against the lesions induced by H$& in low iron conditions (fig 4) as well as in normal condition (data not shown). This protection is dependent on the aq# gene, since ~q+!k’?mutants did not express the adaptive response either in physiological or low iron conditions (dutu not shown).
1
2.5
a
7.5
IQ
12.5
-
_-_
Surwiving
fraction
(
.i
13 Halliwell B. Gutteridge JMC ( 1989~Fwc ~4~4~~4,4~~s in 14 1s
16 17
18 19
20 21
32
23 24
25
~~0~4~~~
m4i
Clilrendon Press, Oxford tmlny JA, Linn S ( lVS7) Mutugencsis and stressresponses induced in Ewkrrichia culi by hydrogen peroxide. J Bactwiol 169.29~7-297~ fmlay J. Linn S (19%) Bimodr\l pattern of killing of DNA-repair-defective or anoxictltly grow Esclwrichin coli by hydrogen peroxide. 9 Bacrerioi 166.5 19-527 tm\uy J, Linn S (\%I) DNA ddmqe and oxygen radial toxicity. S&&X? 240, 1302- 1309 Loeb L, James EA. Waltersdorph AM, Klebanoff S ( 1988) Mutagenesis by the autoxidation of iron with isolated DNA. Pm Mctl Aced Sci USA 85.39 18-3922 Marcovich H (1056) Etude radiobiologique du systeme lisogene $‘khrrichia i.00 K12, i\trn last Pwtwr 90, 303-3 I9 Mel10 Filho AEC. Meneghini R ( 1985)Protection of mammalian cells by o-phenanthroline from lethal and DNA-dnmuging effects produced by ucrive oxygen species. Miller JH (1972) i13??~~~~~~~~u~~~~ ~~~v~ff9~~~.,~. Cold Spring z!d Sprites Harbor, NY Jacobson FS, Storp, G. Ames Mtdicirw.
e Almeida”, AC LeitW*
UGr~ersidmir Federcll do Rio de Jmeiro. 21949-900 Rio de Jurzeiro. RJ; h Dept Bioji~sictr e Biontetrk lnstituto de Biologic, Uni\~er.sidade do Estcldo do Rio de Jmwirc,, 20551-030 Rio de Jmeiro, RJ, Brazil
(Received 22 October IO%; accepted 7 May 1997) Sunlnlary - There are at least two mechanisms by which H;?@induces DNA lesions in Escherich co/i: one in the presence of physiological iron levels und the other in low iron conditions. The survival as well as tile induction of SOS response in different DNA repair mutant strain> of E coli was evaluated after Hz02 treatment under low iron conditions (pretreatment with tin iron chelator). Our results indicate that, in norm4 iron conditions RecA protein has :I relevant role in recombination repair events, while in low iron conditions RecA protein is important as ;l positive regulator of the SOS response. On the other hand, the oq+AR mutant is sensitive to the lethal effects of Hz02 only in low iron conditions and this sensitivity camiot be correlated with DNA strand breaks.
hltroduction
?‘he consecutive univalt’nt to water in the rt2sgiration
reduction
ot’ inolccular
oxygen
cycle pwduces three active ints‘rmediates: superoxide anion (O:! * ), hydrogen peroxide (HzOz), and hydroxyl radical (OH “). These so-called oxidative oxygen spccics are potent oxidants of lipids, proteins, and nucleic acids 113). Several studies have shown that the killing at’c&s cxposed to H&I:! is mainly due to damage on DNA 112, 161, Furthwmorc, the prticipution of the CM-l ( as the main clamage agcant has been suggcstcd by studies using scuvcngcrs of6H ‘$I? 4, 221. OH ’can bc generated through the Fenton reaction with the participation of transition metals, such as Fe(lI) [I, 17, 19j. III hct, a variety of metal chelators as dipyridyl, o-phenantroline and desferrioxamine, can block the Fenton reaction by occupying metal coordination sites and thus protect the cells against the lethal effects of HzOr, confirming the role of iron in the generation of active oxygen species [ 141. It is known that the recombinational repair is an important pathway in repairing DNA lesions produced by H;?Oz, the enhancement of RccA protein being the most important SOS response, since other components of the SOS system
* Correspondence
and reprints
appear to be of little or no importance for r~~os~~bi~lati~~ repair 1141. The exposure of E co/i 6 low concentrations of Hz02 induces an adaptive response which confers increased bacterial resistance to subsequent exposure to high concentrations of II~Q 171. This response is accompanied by the induction of at least 30 proteins [ I I, 2 I], nine of which arc under the control of the OxyR protein 161.OxyR is a transcriptional activator of genes such as kcrfG,which encode% cntal;Ise-~~ydropcroxide1 i Z%]. Tlnr=it~crc~sedbacterial survival is Ilrre, at least in part, to an ~~~duc~d DNA repair capacity, since h phagc dama ed exl9~~s~dto Ha02 have a higher plating efficiency on pretrcate(~cells th1 011naive cells 17, (,I. As the QxyR controltcd proteins do not a~~~~~r to be involved in DNA repair, this rcsponsc must bc due to other peroxide induced proteins. We have shown that wild type cells and different DNA repair mutants strains arc less sensitive TVMaOa(5 IIIR~) when the cells are pretreated with iron chelators. However, in this condition, DNA-single strand breaks were observed, suggesting that there are at least two mcchanisnls of H~Q~ induced DNA lesions in E cdi: one through iron oxidation and OH 0 production and the other in low iron conditions, probably without OH. production [ 11.So, different lesions may be formed in each case and thus different DNA repair tnechanisms should be involved. The role of SOS and OxyR rcgulons in the repair of lesions produced by HzOzunder low iron conditions is unknown. In this work we evaluate
GwGJp~* “F thr-1 l&$6 thi-1 argE3 his-4 proA lack’1 galk2 xyl-5 ara- I3 rspL13 tsx-33 supE44 As A61 157, but recA13
As Al31 157, but lexA1 (Ind-) As AB 1157, but recA 142 As ABI 157, but xthA mD139 (urgF-lac)ZX flBS301 non gytA2 19 re1A 1 rpsL1 SO metE70 btuB::TnlO As RK4936, but oxy A3 uB6 thi-t his-4 pyrD @E galY rpoB IacAU 169 trp::MuC+ Phoc sGA::Mud (Ap lac)Ct” FAs , but AoxyR4 [A (oxyR btuB)4] , but AkuG3. kutE 12::TnltI AS _“_-._ _ -_xI.~____ --___ __xl_~ ll..“_-_-___“^____-_ _ -. - ____ll*l~- ----_ - -___I...__ _I-II__.__-Ic_..II”III_*.._ obtuined from P Howard-Flanders (University of Yale, New Haven, USA). The RK49.36 and TA41 I2 strains were lifarnia, Berkeley, USA). The PQ65, OG 100 and OG140 strains were ohtuined from P Quilliirdct mes (Universi 1 struin was obt;tined from Bernard W&s (University of ~~i~hi~i~~M~d~~~l School. WA). The France). The j~~~~~ strain was obtained from R Devoret (lnstitut Curie, Orseur, Fruncc).
cipation of these two mechanisms in DNA repair 2 treatment under low iron conditions (pretreatun iron chelutar). We also analyzed the induction c response to H$& in low iron conditions,
trifugcd. wsuspcudcd in M9S and trwtcd with dipyridyl illId HzOz. as described in the survival experiments.
5 H $+
i 10
I 15
wncantration ( m
Fig 1.Survival of lSclrc~ic*lricrc*olicells treated with dipyridyl and PIJQz. Cultures in the mid-exponential phase of growth were trated in YS medium at 37°C with I mM dipyridyl for 20 min and then submitted to H&I2 for 20 min. Wild type (AB I 157). mcaCa(AB~463), : )Y*c’142 A (JC4728). 0; IaA I (AB2494), U
in low iron con
1h
with vigorous agitation. The measurements of P-galactosidase and alkaline ~hos~hatase activities were performed as previously detion factor of .$~::I~&? expression is the ratio of the activities of /Sgalact tive concentration of
idase and alkaline phosphatase at the respec202 divided by its value at zero concentration.
Cultures of the AB2463 (wAI_2) strain were pretreated with an iron chelator ( 1 mM of dipyridyl) and then exposed
5
10
H2 O2 concentration
5
15 (
mM
inability to induce SOS functions and/or to its inability to carry out DNA recombinational repair. These possibilities were evaluated using cultures of the K4728 (wcAI42) strain, which is defective in DNA recombination but is able to promote self-cleavage of LexA repressor 1241, and cultures of the Al32494 (IexAI) strain, defective in SOS induction by virtue of yieldin a LexA protein resistant to self-cleavage promoted by ecA [5]. The sensitivity of the IYC;~I42 mutant was close to that of the wild type strain (fig 1B). On the other hand, the IexA / mutant was as sensitive to Hz02 in low iron conditions as the ret;913 mutant (fig 1). indicating that induction of SOS functions, but not recombinational repair, is important for DNA repair in bacterial cells treated with Hz01 in low iron conditions.
1
10
H, O2 concentration
15 ( m
cells treated with dipyridyl and &02. Cultures in the Fig 2. Survival of Esc~herichirr coli wild type (PQ65), ; and A(‘XVR4K=1()()), mid-exponential phase of growth were treated in M9S medium at 37OC with I mM dipyridyi for 20 nlin and then submittcclto I-kQ:!for 20 min (A) or only treated with Hz02 for 20 min (B).
imn conditions wus distinct from that ~~b~~~v~~~ in the ologic~l iron levels itr wild type c&. In the prese norm1 iron levels, a significant induction is observed with
1 mM H$&, while in low iron conditions the induction is only observed with IOto 12.5 mM Hz02 (fig 3 We next evaluated the effects of a\:\A induction of expression of sfiA by Hz02 in normal and low iron conditions. We found that the induction of &golactosidase by H@:! in the o_@R mutant was significantly higher than in the parental strain in the normal iron conditions, while in the low iron conditions the induction was similar to the parental strain (tlg 3B).
We evaluated the nduptivc ~~‘s~9011se induced by H$J:! whether the Fenton reaction is blocked or not. A Hz01 hypersensitive mutant, the BW909I (s&A) strain 181.was pretreated with low H$&concentration in normal or in low iron conditions, followed by treatment with higher HzO:! concentrations in low and normal iron conditions. In both conditions the pretreatment protected the cells against the lesions induced by H$& in low iron conditions (fig 4) as well as in normal condition (data not shown). This protection is dependent on the aq# gene, since ~q+!k’?mutants did not express the adaptive response either in physiological or low iron conditions (dutu not shown).
1
2.5
a
7.5
IQ
12.5
-
_-_
Surwiving
fraction
(
.i
13 Halliwell B. Gutteridge JMC ( 1989~Fwc ~4~4~~4,4~~s in 14 1s
16 17
18 19
20 21
32
23 24
25
~~0~4~~~
m4i
Clilrendon Press, Oxford tmlny JA, Linn S ( lVS7) Mutugencsis and stressresponses induced in Ewkrrichia culi by hydrogen peroxide. J Bactwiol 169.29~7-297~ fmlay J. Linn S (19%) Bimodr\l pattern of killing of DNA-repair-defective or anoxictltly grow Esclwrichin coli by hydrogen peroxide. 9 Bacrerioi 166.5 19-527 tm\uy J, Linn S (\%I) DNA ddmqe and oxygen radial toxicity. S&&X? 240, 1302- 1309 Loeb L, James EA. Waltersdorph AM, Klebanoff S ( 1988) Mutagenesis by the autoxidation of iron with isolated DNA. Pm Mctl Aced Sci USA 85.39 18-3922 Marcovich H (1056) Etude radiobiologique du systeme lisogene $‘khrrichia i.00 K12, i\trn last Pwtwr 90, 303-3 I9 Mel10 Filho AEC. Meneghini R ( 1985)Protection of mammalian cells by o-phenanthroline from lethal and DNA-dnmuging effects produced by ucrive oxygen species. Miller JH (1972) i13??~~~~~~~~u~~~~ ~~~v~ff9~~~.,~. Cold Spring z!d Sprites Harbor, NY Jacobson FS, Storp, G. Ames Mtdicirw.