Induction of the soxRS Regulon of Escherichia coli by Superoxide*

Stefan I. LiochevDagger , Ludmil BenovDagger , Daniele Touati§, and Irwin FridovichDagger

From the Dagger  Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the § Department of Microbiology, Institut Jacques Monod, CNRS, the Universités Paris 6 and Paris 7, France

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of ~15 genes. The induction of this regulon by redox agents, known to mediate Obardot 2 production, led to the view that Obardot 2 is one signal to which it responds. However, redox cycling agents deplete cellular reductants while producing Obardot 2, and one may question whether the regulon responds to the depletion of some cytoplasmic reductant or to Obardot 2, or both. We demonstrate that raising [Obardot 2] by mutational deletion of superoxide dismutases and/or by addition of paraquat, both under aerobic conditions, causes induction of a member of the soxRS regulon and that a mutational defect in soxRS eliminates that induction. This establishes that Obardot 2, directly or indirectly, can cause induction of this defensive regulon.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The soxRS (superoxide response) regulon positively controls ~15 genes in Escherichia coli. The inductions of this regulon by redox cycling agents, such as paraquat, plumbagin, and phenazine methosulfate, which are capable of mediating Obardot 21 production, led to the view that this regulon is capable of responding to Obardot 2 (1-3). This conclusion was strengthened by the observation that H2O2, heat shock, or ionizing irradiation did not induce the soxRS regulon (1-4). Moreover FumC was induced by paraquat more strongly in a sodA sodB strain than in its SOD-replete parent (5), and its induction in the parental strain was eliminated by mutational deletion of the soxRS response (6), thus indicating that Obardot 2 could cause induction of soxRS. However it was also noted (6) that marked overproduction of SodA did not diminish induction of members of this regulon such as fumarase C and glucose-6-phosphate dehydrogenase, an indication that Obardot 2-independent induction was also a reality. In accord with this view was the finding that NADPH could diminish in vitro transcription/translation of the sodA gene (7). Nitric oxide has also been shown to induce soxRS and to do so in the absence of dioxygen (8).

There is strong evidence that the SoxR protein, which is the sensor of the soxRS regulon (4, 9, 10), occurs in oxidized and reduced forms and that the oxidized form is the activator of soxS transcription (11-14). The balance between the oxidized and reduced forms of SoxR within E. coli can undoubtedly be influenced in multiple ways. For example either by oxidation of reduced SoxR or by reduction of oxidized soxR. Obardot 2 could accelerate the former process and, by inhibition of the oxidized SoxR reducing systems, the latter. Yet there seems to be disagreement about whether Obardot 2 is one of the factors that influences the redox status of SoxR. Thus, Nunoshiba et al. (4) used an operon fusion, of the soxS promoter to the lacZ gene, to show that redox cycling agents induced this system in an Obardot 2-dependent manner and that dioxygen itself was a stronger inducer in a SOD-deficient (sodA sodB) strain than in a SOD-replete strain. Thus supporting the view that Obardot 2 could, directly or indirectly, induce soxRS. Wu and Weiss (10) also presented evidence supporting this view. Gort and Imlay (15), using a soxS::lacZ fusion strain, reported that lack of SOD did not cause induction of soxS under aerobic conditions. Thus we have several groups reporting that elevating Obardot 2 by elimination or diminution of SOD was sufficient to cause this induction, and another group (15) reporting that this was not the case.

Fumarase C is a member of the soxRS regulon (6), and we have previously noted that it was induced under aerobic conditions by mutational deletion of SodA + SodB (5). Furthermore the induction of FumC by paraquat was greater in the sodA sodB than in the SOD-replete parental strain. These results support the view that Obardot 2 can induce the soxRS regulon or alternately that the induction of FumC by Obardot 2 was mediated by some other regulon. We explore this further by investigating the effect of deleting soxRS upon the induction of FumC. Our finding is that FumC induction by Obardot 2 is ablated by mutational elimination of the soxRS response. It follows that Obardot 2 can induce the soxRS regulon and that the soxRS regulon is the sole mediator of the induction of FumC by Obardot 2.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Paraquat was obtained from Sigma and malate from ICN. Bactotryptone, casamino acids, and yeast extract were from Difco. The strains of E. coli used were as follows: GC4468 = parent (16); DJ 901 = GC4468 Delta  (soxR-Zjc2204) Zjc2205::Tn10 Km (provided by B. Demple) (2); QC1799 = GC4468 Delta  sodA3, Delta  sodB-kan (16); and QC1817 = GC4468 Delta  sodA3, Delta  sodB-kan, Delta  sox8::cat (obtained by transduction of the Delta  sox 8::cat mutation into QC1799). (The soxRS deletion was provided by B. Weiss (3).) Strains were grown overnight at 37 °C, with shaking in air, in LB, or in M9CA media containing 50 µg/ml kanamycin and/or 30 µg/ml chloramphenicol where required. These cultures were diluted as described in the figure legends into media not containing antibiotics, and paraquat was added after 1 h, and incubation was continued for 75 min. Cells were then harvested, washed in 50 mM potassium phosphate, 0.1 mM EDTA at pH 7.8, and then resuspended in this buffer and lysed in a French press. The extracts were clarified by centrifugation, and protein (17) and fumarase C (5, 6) were assayed. One unit of fumarase was taken to be the activity that converted 1 µmol/min of L-malate to fumarate using Delta E250 nm = 1.62 mM-1 cm-1. The initial concentration of L-malate was 50 mM, and the assay buffer was 50 mM sodium phosphate, pH 7.3, at 25 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of FumC by Dioxygen and Paraquat-- Paraquat can be univalently reduced, at the expense of NADPH, by a number of diaphorases present in E. coli (18), and the paraquat monocation radical rapidly autoxidizes producing Obardot 2 (19) and regenerating the paraquat dication. The rate of production of Obardot 2 is thus increased within aerobic E. coli by paraquat. The net effect of paraquat on the steady state concentration of Obardot 2 and on the redox status of the cell will be greater in an sodA sodB mutant than in its SOD-replete parent. Therefore, we should expect that paraquat should induce a member of the soxRS regulon such as FumC more strongly in an sodA sodB strain than in the parental strain. Bars 1, 2, and 3 in Fig. 1 show that 10 and 25 µM paraquat caused a dose-dependent induction of FumC in the parental strain, whereas bars 4-6 show the greater response to paraquat exhibited by an sodA sodB strain. It is also noted that the lack of SOD, in the absence of paraquat, caused a 3-fold induction of FumC (compare bars 1 and 4). It follows that raising the steady state concentration of Obardot 2, whether by introducing paraquat or by removing SOD, was sufficient to cause induction of FumC. Bars 7 and 8 show that the sodA sodB soxRS triple mutant was unresponsive to Obardot 2 in that it could not elevate FumC in response to aerobic paraquat. This establishes that Obardot 2 induced FumC and did so via the soxRS regulon. Hence the soxRS regulon is responsive to Obardot 2. No induction by paraquat was seen in the soxRS-deficient but otherwise SOD-proficient strain DJ901 (results not shown). This confirms our previous conclusion (6) that the induction of FumC in wild type strains of E. coli is entirely soxRS dependent.


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Fig. 1.   Inductions of Fumarase C by Obardot 2: dependence upon soxRS. Overnight cultures in LB medium containing the appropriate antibiotics were diluted 20-fold into fresh LB without antibiotics and grown for 1 h before the addition of 10 or 25 µM paraquat as specified below. After an additional incubation period of 75 min, cells were collected and extracted, and extracts were assayed for FumC as specified under "Materials and Methods." The figure presents the results of a typical experiment. Repetitions under somewhat different conditions gave very similar results. Bars 1-3, parental strain (bar 1, without; bar 2,= 10 µM; and bar 3, 25 µM paraquat); bars 4-6, sodA sodB strain (bar 4, without; bar 5, 10 µM; and bar 6, 25 µM paraquat); bars 7 and 8, sodA sodB soxRS strain (bar 7, without; bar 8, 25 µM paraquat).

The induction of FumC caused by deletion of SodA and SodB was greater in cells that had been grown in M9CA rather than in the richer LB medium. This is made apparent by comparison of bars 1 and 2 in Fig. 2 with bars 1 and 4 in Fig. 1. Thus there was a ~3-fold induction, caused by the deletion of SOD activity, in the LB-grown cells and a 7-fold induction in the M9CA-grown cells. Bar 3 in Fig. 2 shows that soxRS was as essential for the induction of FumC in the M9CA-grown cells as it was in the LB-grown cells. The experiment shown in Fig. 2 was repeated under dioxygen-depleted conditions. This was done by placing 0.2% inocula, in fresh M9CA medium in a BBL gas pack jar, which was then incubated for 5.5 h before the cells were harvested and extracts prepared for FumC assay. The gas pack jars were not evacuated before incubation so hypoxic, rather than anoxic, conditions prevailed. The sodA sodB extracts were found to have 0.026 units/mg protein of FumC activity, whereas the parental extracts had 0.014 units/mg. Thus dioxygen depletion diminished the ratio of FumC in the sodA sodB extracts from ~7-fold to ~2-fold, as compared with the parental extracts.


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Fig. 2.   Inductions of fumarase C in M9CA medium. Overnight cultures were grown in M9CA and were then diluted 33-fold into fresh M9CA and incubated until A600 m reached 0.7-1.0. Cells were then collected, washed and lysed, and lysates were assayed for FumC. Bar 1, parental strain; bar 2, sodA sodB strain; bar 3, sodA sodB soxRS strain.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because the induction of FumC, whether by addition of paraquat or by deletion of SodA and SodB, was dependent on soxRS and dioxygen, it follows that Obardot 2 can induce soxRS. The induction of the soxRS regulon depends upon the oxidation of the reduced form of SoxR, because the oxidized SoxR is the transcriptional activator of soxS. There must be a pathway for the reduction of oxidized SoxR, and the steady state will depend on the balance between the rates of oxidation of reduced SoxR and of reduction of the oxidized SoxR. Obardot 2, or some product thereof, might effect this steady state by directly oxidizing reduced SoxR and/or inhibiting the reduction of oxidized SoxR. Although the mechanism remains unknown, it is clear that the soxRS regulon can be induced by Obardot 2.

Although we are in agreement with Gort and Imlay (15) concerning the importance of SOD as a defense against Obardot 2, some exception must be taken to their conclusion that induction of FumC by Obardot 2 is not adequate to compensate for the inactivation of FumA by Obardot 2. They used an sodA sodB Ptac-sodA strain which could not induce Mn-SOD in response to increased [Obardot 2]. In an SOD-competent wild type strain, in contrast, the inactivation of FumA would be lessened by the induction of Mn-SOD, which combined with the induction of FumC, should then be adequate to balance the decrease in FumA.

Inductions caused by Obardot 2-generating compounds such as paraquat cannot be unequivocally attributed to Obardot 2, because redox cycling agents deplete cellular reductants while producing Obardot 2 and that depletion will interfere with the reduction of oxidized SoxR. An indication that depletion of cellular reductants can induce soxRS, independent of Obardot 2, was the anaerobic induction seen with paraquat plus the electron sink nitrate (20, 21). No such ambiguity is encountered when Obardot 2 is raised by deletion of SOD. In that case, if cellular reductants are also diminished, Obardot 2 is the cause of that diminution. Thus Obardot 2 can induce the soxRS regulon whose members provide manifold defenses against the oxidative damage imposed by Obardot 2 and its progeny.

An estimation of the Obardot 2-dependent and Obardot 2-independent routes of induction of soxRS can be attempted. Thus the level of [Obardot 2] in the sodA sodB strain is ~20-fold higher than in the parental strain (22), and this caused an ~3-fold induction of FumC. Gort and Imlay (15), by using a strain in which the level of SOD could be modulated, reported that a 10-fold diminution of [SOD] was a threshold for induction of FumC and resulted in modest induction. A 10-fold decrease in [SOD] would correlate with a more than 5-fold increase in [Obardot 2] as discussed below, because SOD is the major sink for Obardot 2 in the parental strain. In the sodA sodB strain paraquat can cause much more than a 20-fold increase in [Obardot 2] as compared with [Obardot 2] in the wild type and this allows dramatic induction of FumC, as shown in Fig. 1. In the SOD-replete parental strain, in contrast, the increase in [Obardot 2] because of paraquat is strongly limited by the action of SOD and by the further induction of SodA elicited by paraquat. Thus the induction of the soxRS regulon by paraquat in the parental strain must largely be because of the depletion of cellular reductants by paraquat rather than to Obardot 2. Of course, this is even more emphatically the case in strains overproducing SOD and explains why overproduction of SOD does not prevent induction of the soxRS regulon by paraquat (6). The induction of SodA is finely tuned so as to minimize both the toxic effects of Obardot 2 and the induction of the soxRS regulon by Obardot 2.

The degree of protection provided by the wild type level of SOD to all Obardot 2-sensitive targets in E. coli has been estimated (22) and that leads to a number of interesting deductions. Thus the rate of formation of Obardot 2 (Vf) must be equal to the sum of its rates of consumption by SOD (VSOD) and by all other targets (VT) i.e.
V<SUB>f</SUB>=V<SUB><UP>SOD</UP></SUB>+V<SUB>T</SUB> (Eq. 1)
and
V<SUB><UP>SOD</UP></SUB>=k<SUB><UP>SOD</UP></SUB>[<UP>SOD</UP>][<UP>O</UP>&cjs1138;<SUB>2</SUB>] (Eq. 2)
and
V<SUB>T</SUB>=k<SUB>T</SUB>[<UP>T</UP>][<UP>O</UP>&cjs1138;<SUB>2</SUB>] (Eq. 3)
therefore
[<UP>O</UP>&cjs1138;<SUB>2</SUB>]=<FR><NU>V<SUB>f</SUB></NU><DE>k<SUB><UP>SOD</UP></SUB>[<UP>SOD</UP>]+k<SUB>T</SUB>[<UP>T</UP>]</DE></FR> (Eq. 4)
Application of Eq. 4 would require several difficult measurements and/or estimations so another approach is useful, from Eq. 1, as follows.
<FR><NU>V<SUB>f</SUB></NU><DE>V<SUB>T</SUB></DE></FR>=<FR><NU>V<SUB><UP>SOD</UP></SUB></NU><DE>V<SUB>T</SUB></DE></FR>+1 (Eq. 5)
and
V<SUB>T</SUB>=V<SUB>f</SUB> <FR><NU>V<SUB><UP>SOD</UP></SUB></NU><DE>V<SUB>T</SUB></DE></FR>+1 (Eq. 6)
When VSOD = VT, one-half of all the Obardot 2 flux is being scavenged by SOD and in analogy to the classical assay for SOD activity (23) in which SOD competes with cytochrome c for the flux of Obardot 2, we can define this amount of SOD activity as 1 biological unit. We have previously found that wild type E. coli contains 19 biological units of SOD on the basis of its inhibition of lucigenin luminescence (22). Hence in these cells VT = 0.05 Vf, whereas in sodA sodB cells VT = Vf.

Fig. 3 presents (100) VT/Vf as a function of the number of biological units, which is the ratio VSOD/VT. This plot ignores changes in biological units because of enzyme inductions and changes in VT because of consumption of targets. A 10-fold decrease in [SOD] leaves 1.9 biological units and then VT is increased 7-fold and, because [Obardot 2] is directly proportional to VT (Eq. 3), so is [Obardot 2]. Thus we see that [Obardot 2] is less than inversely proportional to [SOD] because of the effect of the multiple targets for Obardot 2 in the E. coli. It also follows that Obardot 2 is both more deleterious and a better inducer of the soxRS response than would be concluded on the basis of a simple inverse relationship between [Obardot 2] and [SOD]. Moreover the wild type level of [SOD] is seen as providing 95% protection rather than the 99% protection deduced by Gort and Imlay (15). This 5-fold difference in amount of Obardot 2 damage to targets is certainly explicable on the basis of the existence of targets in addition to the [4Fe-4S] containing dehydratases considered by Gort and Imlay (15).


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Fig. 3.   Percent of Obardot 2 scavenged by targets other than SOD: a theoretical curve. Calculated as (100) VT/Vf as a function of [SOD] in biological units according to Eq. 6.

[Obardot 2] in wild type E. coli has been estimated to be ~1 × 10-10 M (15). SOD-null E. coli will contain 20 times more, or ~2 × 10-9 M Obardot 2 and the threshold for induction of FumC via the soxRS regulon by Obardot 2 will be at ~7 × 10-10 M. Variation of these numbers will, of course, occur as growth conditions change. Thus the ratio of VSOD/VTappeared to be approximately 40/1 when the cells were suspended in 0.25% glucose but was much less when they were suspended in LB or in succinate (22); we therefore used 19/1 as an average approximation. Several papers (15, 24, 25) allow estimation that VSOD/VT lies in the range 10-20, in agreement with our present estimate.

    FOOTNOTES

* This work was supported by grants from the Amyotrophic Lateral Sclerosis Association, National Institutes of Health, Council for Tobacco Research-U. S. A., Inc., and North Carolina Biotechnology Center Collaborative Funding Assistant Program; support was received from Aeolus Pharmaceuticals, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 919-684-5122; Fax: 919-684-8885.

    ABBREVIATIONS

The abbreviations used are: Obardot 2, superoxide radical; SOD, superoxide dismutase; FumC, fumarase C; FumA, fumarase A.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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