Induction of the soxRS Regulon of Escherichia
coli by Superoxide*
Stefan I.
Liochev
,
Ludmil
Benov
,
Daniele
Touati§, and
Irwin
Fridovich
¶
From the
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 |
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 O
2 production, led to the view that
O
2 is one signal to which it responds. However, redox cycling
agents deplete cellular reductants while producing O
2, and one
may question whether the regulon responds to the depletion of some
cytoplasmic reductant or to O
2, or both. We demonstrate that
raising [O
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 O
2, directly or indirectly, can cause
induction of this defensive regulon.
 |
INTRODUCTION |
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
O
21 production, led
to the view that this regulon is capable of responding to O
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 O
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 O
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. O
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
O
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
O
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
O
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 O
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 O
2 can induce the soxRS regulon or alternately
that the induction of FumC by O
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 O
2 is ablated by mutational elimination
of the soxRS response. It follows that O
2 can
induce the soxRS regulon and that the soxRS
regulon is the sole mediator of the induction of FumC by O
2.
 |
MATERIALS AND METHODS |
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
(soxR-Zjc2204)
Zjc2205::Tn10 Km (provided by B. Demple)
(2); QC1799 = GC4468
sodA3,
sodB-kan
(16); and QC1817 = GC4468
sodA3,
sodB-kan,
sox8::cat (obtained by transduction
of the
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
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 |
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 O
2 (19) and
regenerating the paraquat dication. The rate of production of
O
2 is thus increased within aerobic E. coli by
paraquat. The net effect of paraquat on the steady state concentration
of O
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 O
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 O
2 in that
it could not elevate FumC in response to aerobic paraquat. This
establishes that O
2 induced FumC and did so via the
soxRS regulon. Hence the soxRS regulon is
responsive to O
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 O 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 |
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 O
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. O
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 O
2.
Although we are in agreement with Gort and Imlay (15) concerning the
importance of SOD as a defense against O
2, some exception must
be taken to their conclusion that induction of FumC by O
2 is
not adequate to compensate for the inactivation of FumA by O
2.
They used an sodA sodB
Ptac-sodA strain which could not induce Mn-SOD in
response to increased [O
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 O
2-generating compounds such as paraquat
cannot be unequivocally attributed to O
2, because redox
cycling agents deplete cellular reductants while producing O
2
and that depletion will interfere with the reduction of oxidized SoxR. An indication that depletion of cellular reductants can induce soxRS, independent of O
2, was the anaerobic
induction seen with paraquat plus the electron sink nitrate (20, 21).
No such ambiguity is encountered when O
2 is raised by deletion
of SOD. In that case, if cellular reductants are also diminished,
O
2 is the cause of that diminution. Thus O
2 can
induce the soxRS regulon whose members provide manifold
defenses against the oxidative damage imposed by O
2 and its progeny.
An estimation of the O
2-dependent and
O
2-independent routes of induction of soxRS can be
attempted. Thus the level of [O
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
[O
2] as discussed below, because SOD is the major sink for
O
2 in the parental strain. In the sodA
sodB strain paraquat can cause much more than a 20-fold
increase in [O
2] as compared with [O
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
[O
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 O
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 O
2
and the induction of the soxRS regulon by O
2.
The degree of protection provided by the wild type level of SOD to all
O
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 O
2 (Vf) must be equal
to the sum of its rates of consumption by SOD
(VSOD) and by all other targets
(VT) i.e.
|
(Eq. 1)
|
and
|
(Eq. 2)
|
and
|
(Eq. 3)
|
therefore
|
(Eq. 4)
|
Application of Eq. 4 would require several difficult measurements
and/or estimations so another approach is useful, from Eq. 1, as
follows.
|
(Eq. 5)
|
and
|
(Eq. 6)
|
When VSOD = VT, one-half
of all the O
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 O
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 [O
2]
is directly proportional to VT (Eq. 3), so is
[O
2]. Thus we see that [O
2] is less than
inversely proportional to [SOD] because of the effect of the multiple
targets for O
2 in the E. coli. It also follows that
O
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 [O
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 O
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 O 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.
|
|
[O
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 O
2 and the threshold for
induction of FumC via the soxRS regulon by O
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:
O
2, superoxide radical;
SOD, superoxide dismutase;
FumC, fumarase C;
FumA, fumarase A.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.