Bacterial cells respond to the deleterious effects of reactive oxygen species by inducing the expression of antioxidant defence genes. Here we show that treatment with hydrogen peroxide leads to a transient decrease in DNA negative supercoiling. We also report that hydrogen peroxide activates topA P1 promoter expression. The peroxide-dependent topA P1 activation is independent of oxyR, but is mediated by Fis. This nucleoid-associated protein binds to the promoter region of topA. We also show that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide. Our results suggest that topA activation by Fis is an important component of the Escherichia coli response to oxidative stress.
Reactive oxygen intermediates, such as superoxide anion, hydrogen peroxide and hydroxyl radical, lead to damage of proteins, nucleic acids and cell membranes, and have been implicated in cancer, ageing and numerous diseases (Aruoma and Halliwell, 1998). To counter oxidative stress, both prokaryotic and eukaryotic cells maintain inducible defence systems to detoxify the oxidants and repair the damage. In Escherichia coli, the key regulator of the adaptive response to protect against hydrogen peroxide is the OxyR transcription factor. Upon exposure to hydrogen peroxide, the OxyR protein induces the expression of a set of antioxidants encoding genes including katG (hydroperoxide I), ahpCF (alkylhydroperoxide reductase), gorA (glutathione reductase), dps (a non-specific DNA binding protein), grxA (glutaredoxin I), fur (Fur repressor) and oxyS (a regulatory RNA) (Storz and Imlay, 1999). The phenotypes of mutations in dps and oxyS indicate that the DNA-binding protein Dps and the regulatory small RNA OxyS protect against mutagenesis (Altuvia et al., 1997; Martinez and Kolter, 1997).
Bacterial adaptation to environmental stresses requires rapid changes in protein expression. DNA supercoiling is considered to be one link between the environmental changes and gene expression. A number of environmental factors including osmolarity, temperature anaerobiosis and nutrients can alter the level of DNA supercoiling (Yamamoto and Droffner, 1985; Balke and Gralla, 1987; Higgins et al., 1988; Bhriain et al., 1989; Mizushima et al., 1993; 1997; Ogata et al., 1994). The changes in supercoiling in turn affect processes such as transcription, replication and recombination. The key components in determining the degree of DNA supercoiling are Gyrase encoded by gyrA and gyrB that introduces negative superhelical turns, and the topA-encoded Topoisomerase I that relaxes DNA (Luttinger, 1995). Changing DNA topology, in response to stress, may require other cellular factors, in addition to Gyrase and TopA. In E. coli exposed to cold shock, an increase in the negative supercoiling of DNA is observed which is mediated by the function of Gyrase and the DNA binding protein HU (Mizushima et al., 1997). Bacterial adaptation to heat shock requires the action of both Topoisomerase I and Gyrase, inducing a transient DNA relaxation, and resupercoiling (Mizushima et al., 1993; Ogata et al., 1994). DNA resupercoiling is also stimulated by the heat shock protein DnaK (Ogata et al., 1996).
Because E. coli adaptation to oxidative stress also requires rapid changes in protein expression, we investigated the effect of hydrogen peroxide on DNA supercoiling and TopA expression. TopA expression is under the control of four promoters, of which P1 is induced in response to heat shock (Qi et al., 1997). Here we show that hydrogen peroxide activates topA P1 transcription and affects negative supercoiling levels of DNA in a transient manner. topA induction by hydrogen peroxide is mediated by the DNA binding protein Fis, that binds to the promoter region of topA. We were also able to show that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide. We propose that topA activation by Fis, leading to DNA relaxation and possibly to selective gene expression, is important to the response to oxidative stress.
Hydrogen peroxide affects DNA architecture
Exposure to hydrogen peroxide leads to a variety of lesions however, DNA is a particular target. We examined the effect of hydrogen peroxide on DNA topology in E. coli MC4100 cells carrying a small reporter plasmid. Cultures at mid log phase were treated with hydrogen peroxide, and at different time intervals, plasmid samples were isolated and analysed on chloroquine gels. The results show that the negative supercoiling of DNA decreases within 2 min after exposure to 1 mM hydrogen peroxide (Fig. 1A). A further decrease is observed at 15 min of treatment. However, at 30 min after exposure, the DNA resupercoils back to its original state.
The OxyR transcription factor is central to the adaptive response to hydrogen peroxide and oxyR deficient mutants are highly sensitive to this oxidant. Using an oxyR mutant, we asked whether the inability to remove hydrogen peroxide affects on the kinetics of DNA relaxation or rewinding. We found that DNA relaxation is similar to that in wild-type cells and is independent of OxyR. However, resupercoiling was delayed. The DNA in the oxyR deficient mutant remained unwound even 60 min after exposure to 1 mM hydrogen peroxide (Fig. 1B).
The oxyR regulon includes activities that detoxify the oxidants as well as activities that protect against mutagenesis. We tested the effect of two oxyR targets: katG encoding for hydroperoxide I that degrades hydrogen peroxide; and dps encoding for a non-specific DNA binding protein. DNA resupercoiling was delayed in the katG mutant similarly to the oxyR mutant. However, the absence of Dps protein had no effect on the pattern of DNA relaxation, or rewinding (Fig. 1B). We found that oxyR and katG mutant strains were unable to resume growth even 1.5 h after exposure to 1 mM hydrogen peroxide, while wild type and dps mutant cells recovered shortly after treatment (Fig. 1C). These results demonstrate that exposure to hydrogen peroxide leads to a transient decrease in the negative supercoiling of DNA, and suggest that resupercoiling and growth occur only after hydrogen peroxide levels decrease.
Hydrogen peroxide activates topA P1
The results presented above show that oxidative stress causes transient DNA relaxation. Given that TopA is known to induce relaxation in DNA, we examined the effect of hydrogen peroxide on topA transcription. Exponentially growing cells were exposed to 1 mM hydrogen peroxide, and at different time intervals, total RNA was isolated and subjected to primer extension, using a topA primer. The gel shows that the P1 promoter of topA is induced shortly after exposure to hydrogen peroxide, reaching the highest levels between 5 and 15 min and declining thereafter (Fig. 2A and C). To test whether accumulation of hydrogen peroxide has an affect on topA P1 activation, we assayed topA mRNA levels in oxyR mutant strains. Primer extension analysis of total RNA isolated from oxyR strains demonstrated that topA P1 is greatly induced by hydrogen peroxide (Fig. 2B and C). However, in contrast to wild-type cells, P1 transcript levels remained constant for a longer period of time (≥ 60 min). Given that the pattern of topA P1 induction by hydrogen peroxide correlates with the kinetics of DNA topology shifts, DNA relaxation probably results from the activation of topA P1. Our results also suggest that a decrease in the levels of hydrogen peroxide leads to P1 inactivation, resulting in resupercoiling and subsequent growth.
DNA architecture and topA P1 induction in mutants of DNA repair
To minimize the toxicity of reactive oxygen species, cells utilize DNA repair enzymes. Oxidative damage to DNA is known to induce the recA-dependent SOS pathway. Some oxidative DNA lesions are repaired by MutM and MutY DNA glycosylases. To examine the effect of DNA repair activities on the oxidative stress response, we monitored both the kinetics of DNA supercoiling, and topA P1 expression, in strains mutated in DNA repair genes. In contrast to the plasmid from wild-type cells which resupercoils within 30 min after exposure to hydrogen peroxide, the DNA in both the recA and the mutM mutY double mutant remains unwound (Fig. 3A). In agreement with the observation that the mutM mutY double mutant is more sensitive to DNA damage than either mutator alone (Michaels et al., 1992), we found that the strain carrying a single mutation in mutM exhibits only a minor delay in resupercoiling. These observations suggest that increased oxidative DNA damage due to repair deficiencies can inhibit the rewinding step as observed for the accumulation of hydrogen peroxide. Quantitative measurements of primer extension assays of RNA isolated from the double mutant, show that topA P1 expression increases with time, reaching highest levels at 30 min (Fig. 3B). The high transcript levels of topA, at 30 min after exposure, correlates well with the prolonged DNA relaxation observed with both of the mutants, recA and mutM mutY, further confirming that topA P1 induction leads to DNA relaxation. These observations suggest that repair activities can affect the kinetics of the oxidative stress response.
topA P1 activation by hydrogen peroxide is Fis dependent
The E. coli nucleoid-associated proteins including Fis (factor for inversion stimulation), H-NS, IHF (integration host factor) and Lrp (leucine-responsive regulatory protein) are known to be involved in DNA compaction as well as in transcriptional control of gene expression. To learn more about the signal for topA activation by hydrogen peroxide, we examined whether these proteins are involved in the peroxide-dependent P1 activation. Primer extension assays showed that P1 induction by hydrogen peroxide occurs in the absence of Lrp, IHF and H-NS (Fig. 4A and B). However, activation of P1 by hydrogen peroxide is abolished in the absence of Fis.
A computer search for Fis binding sites in the promoter region of topA P1 indicated several possible sites, each matching the loose Fis consensus sequence proposed by Hübner and Arber (1989). To test for Fis binding to the promoter region of topA, we carried out DNase I footprinting experiments using purified Fis (Fig. 4C). Fis was found to protect three sites (I, II and III) centered at approximately −62, −93 and −129, upstream of the transcription start site of P1 (Fig. 4C and D). The binding of Fis to the promoter also induced enhanced DNase I cleavage, possibly reflecting Fis-induced DNA distortion as has been noted for other Fis targets (Finkel and Johnson, 1992).
A fis mutant strain is sensitive to hydrogen peroxide
Because the expression of topA in response to oxidative stress is Fis dependent, we examined whether fis mutant strains showed altered sensitivity to peroxides. Interestingly, we found that a fis mutant is extremely sensitive to hydrogen peroxide (Fig. 5A). The calculated generation time of fis::kan increases by 20-fold upon exposure to 1 mM hydrogen peroxide, whereas wild-type cells are hardly affected (1.4-fold). Viability assays of cells exposed to 2.5 mM hydrogen peroxide demonstrated that ≈ 85% of the fis::kan cells were killed within 30 min after treatment (Fig. 5B). The results presented here indicate that a fis mutant is extremely sensitive to hydrogen peroxide, a sensitivity that has been observed with mutants of antioxidant encoding genes (see Fig. 1C).
A previous study demonstrated that topA P1 is induced by heat shock (Qi et al., 1997). We tested whether heat shock induction of P1 is Fis dependent by looking at heat induced RNA levels in a fis mutant. Our primer extension assays showed that topA P1 induction by heat shock is independent of Fis (not shown). Given this result, we asked whether a prior treatment of heat shock, that leads to Fis-independent topA P1 activation, could assist a fis mutant strain to challenge a hydrogen peroxide treatment. We found that fis mutant cells exposed to heat shock, prior to hydrogen peroxide treatment, were able to resume growth faster than cells treated with hydrogen peroxide only (Fig. 5C). Taken together, these results suggest that topA induction is an important component of the response to oxidative stress.
We have shown that the response of E. coli to hydrogen peroxide involves activation of topA P1 promoter, which leads to a transient decrease in the negative supercoiling of the DNA. The peroxide dependent topA P1 activation is mediated by Fis, the nucleoid-associated protein, which also binds to the promoter region of topA, in at least, three sites. We have also shown that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide, and that exposure to heat shock, prior to hydrogen peroxide treatment, assists with the oxidative stress response.
Exposure of E. coli to oxidative stress induces the expression of multiple genes to detoxify reactive oxygen species and to repair the damage. We find that oxidative stress in E. coli leads to topA P1 activation and to DNA relaxation. The kinetics of DNA topology shifts, after treatment with hydrogen peroxide (i.e. relaxation and resupercoiling), correlates with the pattern of topA P1 induction and with final recovery. topA P1 induction and DNA relaxation are simultaneous and transient in wild-type cells, which also recover shortly after hydrogen peroxide treatment (see Fig. 1C). In an oxyR mutant, and to some extent in mutM mutY mutants, both events are continuous, indicating that accumulation of hydrogen peroxide or lack of repair, can signal for prolonged activation of Topoisomerase I and DNA relaxation. The result that growth resumption is also delayed in these mutants, suggests that Topoisomerase I acts as a check point that selectively enables the function of convalescence activities, before normal growth is resumed.
The transcription factor Fis has been implicated mostly in the regulation of several stable RNA promoters, acting as a trans-activator of rRNA and tRNA synthesis (Nilsson et al., 1990; Ross et al., 1990; Zacharias et al., 1992). The number of Fis binding sites diverse from one site at the promoter region of leuV, to three sites at rrnB P1 and at least seven sites at the promoter of H-NS (Ross et al., 1990; 1999; Falconi et al., 1996). We found that Fis regulates the peroxide-dependent induction of P1 promoter of topA. Fis binds to at least three sites in the topA promoter region from −50 to −140 upstream of the start site. Our preliminary data indicate that Fis may also bind to additional sites. The relevance of these sites to topA P1 activation remains to be seen. Interestingly, the most distal site (site III) has the largest similarity (14 out of 15) to the modified Fis consensus derived by Finkel and Johnson (1992), whereas the other two sites, sites I and II, deviate by three and four mismatches respectively.
It is intriguing that the P1 promoter of topA is also induced by heat shock. We found that activation of P1 by heat shock is independent of Fis (not shown). The sequence of the P1 promoter resembles the consensus sequence recognized by the heat shock sigma factor, σ32, and the induction is σ32 dependent (Qi et al., 1997). However, the promoter also resembles the core promoter recognized by σ70, with suboptimal −35 and −10 hexamers and a spacer of 17 bases (see Fig. 4D). Therefore, it is conceivable, that in response to hydrogen peroxide, Fis activates P1 transcription with σ70-holoenzyme, while at heat shock the promoter is recognized by σ32.
The signal that triggers Fis to initiate transcription at the P1 promoter, in response to hydrogen peroxide, is unknown. The variables affecting activities of the fis-dependent promoters are the superhelical density of the DNA template and the occupation of Fis binding sites (Travers and Muskhelishvili, 1998). It has been argued, that Fis acts as a topological homeostat (Schneider et al., 1997) that lowers the overall chromosomal superhelical density, and compensates locally, by stabilizing domains of higher superhelicity, in promoter regions sensitive to supercoiling levels. Whether Fis alone is sufficient for the peroxide-dependent P1 activation, is also not clear. Interestingly, we have noticed that in mutants of DNA repair, the activation of topA P1 is somewhat delayed (see Fig. 3B). Whether DNA repair activities take a share in the signaling for the initiation of topA activation, through changes in DNA topology, is an important direction for future studies. Based on our findings we propose a putative model for E. coli adaptation to oxidative stress; exposure to hydrogen peroxide activates both the oxyR regulon pathway and the topoisomerase pathway (Fig. 6). topA P1 induction via Fis and yet unknown signals, possibly also through DNA damage and repair, results in DNA relaxation and selective gene expression, which in the presence of an active oxyR regulon leads to the disposal of reactive oxygen species (ROS) and to DNA repair. These events subsequently lead to P1 inactivation and to resupercoiling. The molecular details of various aspects of this working model are currently being studied.
The findings that a mutation in fis leads to increased sensitivity to oxidative stress, and that topA P1 induction by heat shock assists fis mutant cells to challenge hydrogen peroxide, support our conclusion that topA activation by Fis is an important component of the oxidative stress response, independent of the oxyR regulon.
Shifts in DNA supercoiling, in response to environmental factors, could coordinate expression of stress response networks, and have implications for the adaptation of bacterial cells to changing environments. It is possible, that Fis and topA as pleiotropic regulators, able to control gene expression through shifts in DNA topology, act to integrate the oxidative stress response, with other cellular stress networks, leading to bacterial adaptation to environmental changes.
Strain construction and media
The bacterial strains used in this study are listed in Table 1. Strains were routinely grown at 37°C in Luria–Bertani (LB) medium. Ampicillin (amp, 50–100 µg ml−1), tetracycline (tet, 10 µg ml−1), or kanamycin (kan, 25 µg ml−1) was added where appropriate. Mutations were moved to MC4100 by P1 transductions.
Table 1. Bacterial strains.
Source or reference
MC4100 mutM::kan mutY::mini-Tn10
MC4100 ΔrecA srlC300::Tn10
To construct pGEM-5′topA (pSA15) the topA promoter fragment (491 nucleotides) was amplified from MC4100 chromosomal DNA by PCR (5′-TTG TGA ATT CTT TAC TCC TTA AAC and 5′-GGC GGA TCC CGG ATG TGA CCG ACG CT) and subcloned into unique EcoRI and BamHI sites of pGEM-3 (Promega).
Bacterial cultures grown to mid log phase in LB were treated with 1 mM hydrogen peroxide or transferred to 42°C. At the indicated time points, total RNA was isolated by acid–phenol extraction as described (Storz and Altuvia, 1994). Primer extension assays were carried out based on total RNA measurements as described for other hydrogen peroxide regulated genes (Altuvia et al., 1994). The RNA samples (30 µg) were subjected to primer extension (at 42°C for 45 min) using AMV-RT (Promega) and end-labelled topA oligonucleotide (5′-CAT ATT CAC CTT ACC). The extension products together with sequencing reactions primed with the same end-labelled primer were separated on 6% sequencing gel.
Analysis of DNA supercoiling
MC4100 carrying pKK177-3 were grown to mid log phase in LB and treated with 1 mM hydrogen peroxide for the indicated time points. Plasmid DNA was isolated by the alkaline lysis procedure and analysed on 1.4% agarose gel (BRL) containing 10 µg ml−1 chloroquine (sigma). The gels were run at 2.5 v cm−1 in 50 mM Tris phosphate pH 7.2 containing 10 µg ml−1 chloroquine, for 19 h (Shure et al., 1977). The buffer was recirculated. For documentation the gels were soaked for 2 h in water and stained with ethidium bromide (1 µg ml−1) for 1 h.
Footprints of FIS
topA promoter fragment was polymerase chain reaction (PCR) amplified using 32P end-labelled top strand primer (5′ TTG TGA ATT CTT TAC TCC TTA AAC) or 32P end-labelled bottom strand primer (5′ GCG GAT ATC AAC CCC T). The fragments were purified using DNA purification columns (Promega). Footprint reactions were carried out at 22°C in a solution of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT and 100 µg ml−1 BSA. Fis (kindly provided by Georgi Muskhelishvili) was present at the concentrations indicated. The fragments were partially digested by DNase I (Promega) (0.2 U for 3 min). The results obtained with top and bottom strands are shown.
We thank Georgi Muskhelishvili and Gad Glaser for purified Fis. This work was supported by the Human Frontier Science Program and by grant number 95-00092 from the United States–Israel Binational Science Foundation and by The Israel Science Foundation founded by The Academy of Sciences and Humanities – Centers of Excellence Program (SA).