Vol.1, No.4, 132-141 (2013) Advances in Enzyme Research
http://dx.doi.org/10.4236/aer.2013.14014
Escherichia coli superoxide dismutase expression
does not change in response to iron challenge during
lag phase: Is the ferric uptake regulator to blame?
Robert L. Bertrand*, Michael O. Eze#
Health Enhancement Biochemistry Laboratory, Department of Chemistry, University of Winnipeg, Winnipeg, Canada;
#Corresponding Author:
Received 19 July 2013; revised 23 August 2013; accepted 10 September 2013
Copyright © 2013 Robert L. Bertrand, Michael O. Eze. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
Intracellular iron levels and the expression of
superoxide dismutase (SOD) and hydroperoxi-
dase (HP) are regulated in Gram-negative bacte-
ria by the iron(II)-activated ferric uptake regula-
tor (Fur). We have previously observed that the
expression of SOD in exponential phase Es-
cherichia coli is dependent upon the redox state
of iron in media, consistent with the ferrous
specificity of Fur regulation (Bertrand et al., Med.
Hypotheses 78: 130 - 133, 2012). Through the
non-denaturing electrophoretic technique we
have determined the Escherichia coli expression
profiles of SOD and HP in response to iron
challenge throughout lag, logarithmic, and sta-
tionary phases of replication. Lag phase SOD
presented an unusual expression profile such
that SOD expression was unresponsive to iron
challenge, analogous to observations of mutant
strains lacking Fur and of E. coli incubated in
iron-deplete media. Challenging Escherichia coli
with iron during logarithmic phase revealed that
length of exposure to oxidants is unlikely to be
the cause of SOD unresponsiveness in lag
phase. HP activity was up-regulated two- or
three-fold throughout all growth phases in re-
sponse to iron challenge, but did not present
redox- or growth phase-specific outcomes in a
manner analogous to SOD. We hypothesize that
low Fur levels during lag phase are responsible
for unresponsive SOD.
Keywords: Antioxidant Enzymes; Oxidative Stress;
Iron Metabolism; Enzyme Expression
1. INTRODUCTION
Iron is an essential metal, required as a prosthetic
group for many metallo-proteins. However, iron potenti-
ates oxidative stress through the catalytic formation of
hydroxyl radicals from hydrogen peroxide through the
Fenton reaction [1] (Eq.1):
23
22
FeH OFeOHOH


  (1)
Reactive oxygen species such as hydroxyl radical
(OH·), superoxide


.
2
O

, and hydrogen peroxide (H2O2)
have been implicated in numerous pathologies as well as
the process of aging [2-8]. Cells must therefore carefully
regulate intracellular iron levels to meet metabolic needs
yet mitigate iron cytotoxicity [9-12]. In Gram-negative
bacteria such as Escherichia coli, this balance is medi-
ated by the ferric uptake regulator (Fur), an iron (II)-
dependent suppressor of iron uptake proteins [13-15]. As
Fur also regulates superoxide dismutase (SOD) and hy-
droperoxidase (HP) [16-23], two essential antioxidant
enzymes for respiring bacteria [24,25], Fur is de facto a
homeostatic mediator of intracellular oxidative conditions.
SOD isozymes are distinguished and named by their
metal prosthetic groups. Escherichia coli, the model spe-
cies of this study, copiously produces manganese- and
iron-bearing forms (MnSOD, FeSOD), as well as a trace
isozyme known as CuZnSOD [26,27]. These are encoded
by the genes sodA, sodB, and sodC, respectively. Unlike
FeSOD, MnSOD expression levels vary greatly in re-
sponse to a variety of oxidative stimuli [28,29]. MnSOD
expression is regulated by the protein products of six
global regulators: soxRS and soxQ, both of which acti-
vate sodA; and fur, arcA, fnr, and ihf, all of which sup-
*Present Address: Department of Chemistry, University of Mani-
toba, Winnipeg, Canada.

R. L. Bertrand, M. O. Eze / Advances in Enzyme Research 1 (2013) 132-141 133
press sodA [17,29]. E. coli produces two isozymes of
hydroperoxidase: HPI, encoded by ka tG, and HPII, en-
coded by katE [30,31]. HPI is the principle responder to
oxidative threats, and its expression is activated by the
H2O2-inducible OxyR regulon [32]. HPII is predomi-
nantly active during stationary phase and serves as part
of the starvation response controlled by the rpoS regulon
[32-36]. The rpoS regulon has also been observed to
control HPI expression to some extent [35,37]. Com-
bined, these antioxidant enzymes eliminate superoxide
and hydrogen peroxide in the following pair of reactions
(Eqs.2,3):
SOD .
2
O2H HO

 2
2
(2)
HP
222 2
2H O2H OO (3)
Sodium nitroprusside (SNP) is an iron(II)-bearing
penta-cyanide and a potential therapeutic agent for acute
cardiopulmonary emergencies [38-42]. SNP rapidly re-
leases nitric oxide (NO) in vivo by forming S-nitro-
sothiols with sulfhydryl-containing compounds such as
glutathione, transporting NO throughout the vasculature
[43]. The utility of SNP in the present study is threefold
as it allows examination of the biochemistry of both iron
and nitric oxide, and may provide insights relevant to
therapeutics. Nitric oxide, itself an oxidant, is an en-
dogenous regulator of multiple metabolic pathways and
an inhibitor of iron-bound Fur [44-46]. We have previ-
ously observed that logarithmic-phase E. coli treated
with SNP lowered expression of MnSOD, but the ex-
pression levels of FeSOD were greatly enhanced [47]. As
NO activates MnSOD transcription [48,49], and as
FeSOD expression is generally unresponsive to oxidative
stimuli [28,29], it was predicted beforehand that treat-
ment of E. coli with SNP should increase MnSOD ex-
pression and decrease FeSOD expression. That we had
observed the opposite was a most unexpected finding
[47]. Among the six regulators of MnSOD, Fur is
uniquely known, upon activation by iron, to both inhibit
MnSOD transcription and by post-transcriptional means
de-suppress FeSOD [13,16,17,20,50,51]. We therefore
suggested SNP be activating Fur, the implications of this
hypothesis on human health and disease having already
been discussed [47]. Supporting this hypothesis were the
comparative effects of potassium ferricyanide (PFi) and
potassium ferrocyanide (PFo) on E. coli, both of which
are structural analogs of SNP that cannot release NO.
Though incubating E. coli in the presence of the ferric
PFi had unremarkable effects on SOD expression, treat-
ment with ferrous PFo mimicked SNP-associated ex-
pression patterns consistent with the ferrous specificity
of Fur activation [47]. These results were obtained through
activity staining assays of E. coli extracts run through
non-denaturing electrophoresis.
We adopted this successful methodology to elucidate
the expression profiles of both superoxide dismutase
(SOD) and hydroperoxidase (HP) throughout all phases
of E. coli growth under aerobic conditions using SNP as
well as the ferric and ferrous analog standards as oxida-
tive challengers. The objective of this study is to charac-
terize antioxidant enzyme response to iron challenge
within the context of oxidative stress, and to identify
plausible regulatory elements underlying antioxidant
enzyme response patterns. Herein we have determined
that all three iron treatments failed to change the expres-
sion of either isozyme of SOD during lag phase of E. co li
replication. MnSOD expression was markedly higher
during lag phase. We hypothesize that low Fur activity
during lag phase is the cause of the recalcitrant SOD as
well as heightened MnSOD expression. Indirect evidence
supporting this hypothesis is provided in other enzyme
studies, which will be discussed. HP was responsive to
iron treatments during all phases, but no redox- or phase-
specific behaviour analogous to that of SOD was ob-
served.
2. METHODS
2.1. Culture Conditions
Escherichia coli (ATCC 8677) cultures, originating
from 1% inocula of overnight cultures, were incubated
aerobically at 37˚C on shaker bath (75 RPM) in
pre-warmed 0.2% glucose-based M9 liquid media (500
mL in 1000 mL Erlenmeyer flasks) with sterile cotton
stoppers (Overnight cultures were similarly incubated in
500 mL of M9 media). Media contained 1.00 mM SNP,
PFo, or PFi, introduced aseptically either at the start of
incubation or after five hours of incubation. Growth rates
were monitored by periodic optical density readings at
550 nm. The E. coli strain employed was chosen simply
because it was available for research.
2.2. Protein Preparation and Gel
Electrophoresis
After 1, 6, or 24 h of incubation, E. coli cells were
harvested by centrifugation, and the supernatant replaced
with a 100 mM HEPES buffer solution (pH 7.3) con-
taining 1 mM MgCl2 and protease inhibitor cocktail
(Sigma). Cells were lysed by abrasion vortexing, and the
cellular debris removed by centrifugation. Total protein
concentration was determined by the method of Bradford
[52], and the crude extracts were diluted to 0.6 mg/ml
total protein. These dilutions permitted quantitation of
the relative differences in the expression of SOD and HP
between treated and untreated cultures following elec-
trophoresis and activity assays. 20 µL of each extract
were loaded into 7.5% non-denaturing polyacrylamide

R. L. Bertrand, M. O. Eze / Advances in Enzyme Research 1 (2013) 132-141
134
gels (29:1), and electrophoresed at 200 V and 0.03 A for
approximately two hours using a TRIS-glycine running
buffer. At least three experiments were conducted per
treatment and growth phase.
2.3. Enzyme Activity Assays
SOD activity was determined by the method of
Beauchamp and Fridovich [53], with modifications as
described by Britigan and colleagues [54]: A 50 mM
phosphate buffer solution (pH 7.8) containing 0.25 mM
nitroblue tetrazolium (NBT), 1 mM ethylenediamine-
tetraacetate (EDTA), 28 mM tetramethylethylenediamine
(TEMED), and 0.03 mM riboflavin was applied to gels
in darkness for 45 minutes, stirring occasionally, fol-
lowed by destaining in darkness with phosphate buffer
for 45 minutes. Achromatic bands appear on a blue-pur-
ple background upon exposure to light. HP activity was
assayed by the method of Woodbury and colleagues [55]:
Gels were immersed in a 0.003% (w/v) H2O2 solution for
ten minutes, followed by rinsing and addition of a
freshly-prepared solution of 2% (w/v) potassium ferri-
cyanide and 2% (w/v) ferric chloride for five minutes.
Achromatic bands appear on a dark green background.
Band intensities were quantified with a band analyzer
(Vakili Gel Analysis Unit, Dept. of Chem., U. of Winni-
peg).
2.4. Enzyme Activity Ratios
To determine how much SOD isozyme concentrations
changed in E. coli in response to treatment, enzyme ac-
tivity ratios were generated by dividing the determined
isozyme concentration in treated samples by the concen-
tration in the corresponding untreated standard. For ex-
ample, if MnSOD from SNP-treated E. coli during ex-
ponential phase was half as concentrated as MnSOD
from untreated E. coli during the same phase, an enzyme
activity ratio of 0.50 would be reported. Isozyme con-
centrations are the average determination of a minimum
of three independent experiments. Data from unpub-
lished work was included in the data sets (when appro-
priate) to provide as many as 10 independent experi-
ments in some instances. The average was five experi-
ments. Data points exceeding the lower and upper quar-
tiles by 1.5 times were removed as outliers. The enzyme
concentrations were approximated by comparing band
intensity values between sample bands and that of a
known quantity of a standard enzyme loaded into gels.
These standard enzymes were 10 ng of Corynebacterium
glutamicum catalase (Fluka) and 50 ng of E. coli man-
ganese SOD (Sigma). Ratio significance was determined
through Student’s T-test and pooled standard deviation at
90 and 95 percent confidence.
3. RESULTS
3.1. E. coli Growth Curves
Untreated E. coli remained in lag phase for approxi-
mately two hours, following which cultures progressed
into an exponential phase that ended after eight addi-
tional hours of incubation (Figure 1). 1.00 mM SNP was
observed to uniquely hinder E. coli replication, evident
by mid-exponential phase, resulting in a lower optical
density at stationary plateau as compared to all other
treatments (Figure 1). These results suggest that SNP
imparts a severe oxidative and/or metabolic threat to E.
coli.
3.2. E. coli Growth Curves
E. coli cells were incubated in the presence of 1.00
mM SNP, PFo, or PFi, and harvested after 1, 6, or 24
hours of incubation in accordance with the middle of its
determined lag, exponential, and stationary periods
(Figure 1). SOD assays of electrophoresed extracts and
SOD activity ratios are provided (Figure 2; Table 1).
Unlike exponential and stationary-phase harvests (Fig-
ures 2(b) and (c)), no treatment was capable of changing
SOD isoyzme activities during lag phase, as the activity
ratio was approximately 1 and did not deviate signifi-
cantly from the untreated standard (Table 1). There are at
least two possible reasons why this occurred.
It is possible that: 1) induction of SOD is time-de-
pendent, and one hour is not enough time to observe an-
tioxidant adaptation to oxidative challenge; or that 2)
there are underlying regulatory reasons why iron chal-
lenge is unable to change SOD activities during lag
Figure 1. Growth curves of E. coli treated
with 1.00 mM sodium nitroprusside (SNP),
1.00 mM potassium ferrocyanide (PFo), or
1.00 mM potassium ferricyanide (PFi), in-
cubated as described (see “Methods”).
Growth curve is representative of at least
three independent trials.

R. L. Bertrand, M. O. Eze / Advances in Enzyme Research 1 (2013) 132-141 135
Figure 2. E. coli SOD expression profiles, presented as activity
bands on non-denaturing polyacrylamide gels. E. coli originat-
ing from overnight cultures were incubated as described (see
“Methods”) in the presence of 1.00 mM sodium nitroprusside
(SNP), 1.00 mM potassium ferrocyanide (PFo), or 1.00 mM
potassium ferricyanide (PFi), then harvested after 1, 6, or 24 h
of incubation (a-c). “6 h (mod)” are E. coli harvested after 6 h
of incubation, but only received 1 h of treatment (addition at 5
h) (d). 20 µL of 0.6 mg/ml crude protein extract were loaded
into each well, and electrophoresed and stained for SOD activ-
ity as described (see “Methods”). “Std E.” is 50 ng of E. coli
MnSOD, used as a positive standard. “No Tx” is untreated E.
coli, equivalently harvested. Under present conditions, E. coli
MnSOD is electrophoretically slowest (top band) and FeSOD
fastest (bottom band) [71,72]. As the staining technique is
light-activated, and gel backgrounds darken with more light
exposure, variations in gel background colour from trial to trial
will inevitably arise. Gels are representative of at least three
independent trials.
phase. To investigate, liquid broths of E. coli were
equivalently incubated without treatment for five hours,
following which 1.00 mM (final concentration) of SNP,
PFo, or PFi was added to each broth. These cultures were
incubated for 1 h more, and then harvested. If SOD is
unresponsive to iron challenge because 1 h of incubation
is not enough time for E. coli to change SOD expression
levels, then SOD should remain similarly unchanged
when E. coli cells are exposed to the same iron challenge
for 1 h during exponential phase. If, however, SOD is
unresponsive to iron challenge because of underlying
regulatory reasons determining SOD activity during lag
phase, then 1 h exposure to iron challenge during expo-
nential phase should result in a different expression pro-
file than that observed in lag phase. Such “modified” ex-
ponential phase trials would likely be similar to other
exponential phase trials with 6 h of exposure to treat-
ments.
This predicted outcome was indeed observed, albeit
with some statistical caution (Figure 2(d)). SNP and PFo
Table 1. Activity ratios of E. coli SOD isozymes throughout all
growth phases under treatment of 1.00 mM SNP, PFo, or PFi.
MnSOD FeSOD
Growth Phase Treatment
Ratio SD RatioSD
Lag phase (1 h)
SNP
PFo
PFi
1.07 ± 0.98
0.80 ± 0.52
0.81 ± 0.43
0.92 ± 0.55
1.20 ± 0.49
1.04 ± 0.50
Exponential
phase (6 h)
SNP
PFo
PFi
0.76 ± 0.64
0.50 ± 0.43*
0.80 ± 0.60
3.16 ± 3.15**
1.51 ± 1.75
0.60 ± 0.72
Stationary
phase (24 h)
SNP
PFo
PFi
1.59 ± 1.94
0.55 ± 0.52**
0.48 ± 0.48*
2.63 ± 1.05*
1.93 ± 1.01
1.39 ± 0.65
“Modified”
Exponential
phase (6 h)
SNP
PFo
PFi
0.66 ± 0.58
0.56 ± 0.51
0.96 ± 0.87
2.58 ± 2.02**
1.61 ± 1.21
1.01 ± 0.73
“Ratio” is defined as the average SOD isozyme concentration of the treated
E. coli divided by the average SOD isozyme concentration of untreated E.
coli under identical conditions. Significance at 90 (*) and 95 (**) percent
confidence was determined through Student’s T-test and pooled standard
deviation.
both reduced MnSOD activity by about one-third as
compared to untreated E. coli, but were only significant
at P ≤ 0.36 and P ≤ 0.25, respectively, suggesting there is
an approximate one third to one quarter chance that de-
viation was caused by random error alone. PFi failed to
change MnSOD, with an approximate 94 percent chance
of variance being due to random error. FeSOD expres-
sion was heightened by SNP and PFo treatment 2.58 and
1.61 fold, respectively, but only SNP treatment reached
statistical reliability at P ≤ 0.05 (Table 1).
We therefore conclude that the hypothesis that there
are underlying regulatory reasons distinct to lag phase
causing SOD unresponsiveness is more likely correct. It
is remarkable that PFi was also capable of decreasing
MnSOD activity alongside PFo during stationary phase
(Table 1). It is plausible that this may be caused by redox
shifts in media, changes in intracellular iron import and
export rates, changes in iron solubility influencing Fur
activation, or other causes. Strangely, SNP induced a 50
percent increase in MnSOD activity at this time; however,
deviation is likely due to random error as statistical
analysis shows an approximate 90 percent chance of be-
ing caused by random error.
Extracts of untreated E. coli harvested from all three
growth phases were electrophoresed and stained for SOD
activity. MnSOD was found to display remarkably higher
activity during lag phase (Figure 3). In summary, SOD
assays show markedly higher MnSOD expression during
lag phase. This heightened expression is immutable to
iron treatment, and SOD recalcitrance does not seem to

R. L. Bertrand, M. O. Eze / Advances in Enzyme Research 1 (2013) 132-141
136
be caused merely by inadequate time exposure to iron
challengers.
3.3. Hydroperoxidase
The expression profiles of HP isozymes from E. coli
treated with SNP, PFi, and PFo were determined in the
same manner as that of SOD. Activity ratios for HP
isozymes are provided (Ta b l e 2 ). E. coli exposed to any
treatment responded with heightened HP activity during
all phases of replication, albeit with varying levels of
statistical certainty per treatment and growth phase
(Figure 4; Ta b l e 2). Treatments during lag phase raised
HP expression by 50 to 150 percent, but failed to reach
Figure 3. Electrophoresis and
SOD staining of untreated E.
coli extracts, harvested after
(L to R) 1, 6, and 24 h of in-
cubation, as described (see
“Methods”). “Std” is 50 ng of
E. coli MnSOD. Gel is repre-
sentative of at least three trials.
Table 2. Activity ratios of E. coli HP isozymes throughout all
growth phases under treatment of 1.00 mM SNP, PFo, or PFi.
HPII HPI
Growth Phase Treatment
Ratio SD RatioSD
Lag phase (1 h)
SNP
PFo
PFi
1.64 ± 1.52
1.50 ± 1.76
1.64 ± 1.42
2.32 ± 2.35
1.54 ± 1.71
1.65 ± 1.65
Exponential
phase (6 h)
SNP
PFo
PFi
1.33 ± 1.16
2.00 ± 1.84**
2.17 ± 1.54**
1.47 ± 0.78*
2.07 ± 1.48**
2.10 ± 0.95**
Stationary
phase (24 h)
SNP
PFo
PFi
2.62 ± 1.06**
2.02 ± 0.72*
1.33 ± 0.27*
2.10 ± 1.60
2.45 ± 1.86*
1.93 ± 1.31
“Modified”
Exponential
phase (6 h)
SNP
PFo
PFi
0.92 ± 0.73
1.00 ± 0.76
0.92 ± 0.68
1.63 ± 0.75**
1.51 ± 0.66**
1.16 ± 0.70
“Ratio” is defined as the average HP isozyme concentration of the treated E.
coli divided by the average HP isozyme concentration of untreated E. coli
under identical conditions. Significance at 90 (*) and 95 (**) percent confi-
dence was determined through Student’s T-test and pooled standard devia-
tion.
Figure 4. E. coli HP expression profiles, presented as activity
bands on non-denaturing polyacrylamide gels. E. coli originat-
ing from overnight cultures were incubated as described (see
“Methods”) in the presence of 1.00 mM sodium nitroprusside
(SNP), 1.00 mM potassium ferrocyanide (PFo), or 1.00 mM
potassium ferricyanide (PFi), then harvested after 1, 6, or 24 h
of incubation (a-c). “6 h (mod)” are E. coli that were harvested
after 6 h of incubation, but only received 1 h of exposure to a
treatment (addition at 5 h point) (D). 20 µL of 0.6 mg/ml crude
protein extract were loaded into each well, and electrophoresed
and stained for HP activity as described (see “Methods”). “Std
E.” is 10 ng of Corynebacterium glutamicum catalase, used as a
positive standard. Under present conditions, E. coli HPII mi-
grates slowest (top bands), and HPI fastest (bottom bands) [73].
Gels are representative of at least three independent trials.
significance even at P ≤ 0.10. Heightened HP activity
associated with iron treatments was often significant
during logarithmic and stationary phases of growth.
Unlike SOD, no redox-specific behaviour was observed
as PFo and PFi had approximately similar effects on both
isozymes of HP. This is evident by the comparatively
similar HP activity ratios produced by PFo and PFi
throughout all growth phases (Table 2).
Remarkably, “modified” exponential phase trials an-
alogous to those of SOD also revealed phase-dependent
enzyme expression, but not in a manner anticipated. Both
lag and exponential phase trials displayed conspicuous
up-regulation of HP in response to iron challenge. How-
ever, “modified” exponential phase trials (exposed to
oxidants for only 1 h) displayed HPII activity ratios at or
near 1, and SNP and PFo induced HPI expression at a
level that barely achieved statistical significance These
results suggest that the duration of exposure to these
oxidants is a significant determining factor in HP expres-
sion during exponential phase.
4. DISCUSSION
4.1. SNP Has a Unique Oxidative Impact on
E. coli
Though iron is present in PFi, PFo, and SNP, only the

R. L. Bertrand, M. O. Eze / Advances in Enzyme Research 1 (2013) 132-141 137
NO-releasing SNP slowed exponential phase E. coli and
prematurely arrested E. coli at a lower optical density at
stationary phase (Figure 1). These results suggest that
NO, or NO combined with iron, imparts unique oxidative
consequences on E. coli. Nitric oxide is a free radical
that can directly inflict oxidative damage or combine
with other compounds to form powerful oxidative agents
[56-58]. Peroxynitrite, the product of the reaction be-
tween NO and superoxide, is one such agent that would
be rapidly formed in a NO-flooded environment [59].
Peroxynitrite can self-generate by inhibiting electron
transport chain complexes, arresting electron flow; and
consequently, allow nearby oxygen molecules to steal
electrons. Superoxide radicals adventitiously formed
then bind with NO to form more peroxynitrite [57]. Con-
sidering that exponential phase is characterized by
maximal metabolic activity and that the exhaustion of
metabolic substrates defines stationary phase, it is plau-
sible that the slowed growth and premature plateau asso-
ciated with SNP treatment is the consequence of per-
oxynitrite-associated metabolic waste. This is by no
means the only possible explanation.
4.2. SOD Expression is Immutable to Iron
Challenge during Lag Phase
Exponential phase E. coli exposed to SNP and PFo for
6 h was determined to have reduced MnSOD expression
and enhanced FeSOD expression as compared to un-
treated cultures, albeit with some statistical uncertainty
(Figure 2; Ta b l e 1 ). We have previously suggested that
such Fe(II)-associated changes should be mediated by
the activation of Fur [47]. The present observations are
consistent with studies demonstrating changes in FeSOD
and MnSOD activities in iron-replete and iron-deplete
conditions in a manner implicating Fur regulation [54,
60]. The dichotomous expression of SOD isozymes was
not observed during lag phase (Figure 2; Tab l e 1), and
this was accompanied by the observation of dramatically
higher MnSOD expression during lag phase as compared
to later growth phases (Figure 3). SOD recalcitrance was
a puzzling observation considering that iron influx oc-
curs during lag phase [61]; and therefore, dramatic
changes in isozyme activities should have been produced
through Fur induction. Several studies have suggested
that the intracellular concentration of the Fur protein is
significantly lower during lag phase of microbial replica-
tion. During lag phase there is an impressive influx of
iron (and other metals) that would be best mediated by
low Fur levels [61]. Fur has been observed to activate the
transcription of HPI and HPII, evident by the near ab-
sence of both isozymes in mutant E. coli lacking Fur
(Δfur); however, expression differences between mutant
and wildtype strains were only plainly evident after lag
phase had ended [23]. This latter observation is consis-
tent with our own observations such that iron challenge
may only induce changes in SOD expression once lag
phase has ended. Furthermore, E. coli Δfur mutants and
E. coli incubated in iron-deplete conditions show hei-
ghtened MnSOD activity akin to what is presently ob-
served in our lag phase cultures [18,23,54]. Heightened
MnSOD expression is consistent with our hypothesis that
Fur activity is low during lag phase because MnSOD
would be logically de-repressed at this time. Therefore,
based on these present observations and those of other
researchers, we hypothesize that heightened SOD ex-
pression, and recalcitrant SOD expression, during lag
phase are the result of low intracellular levels of the Fur
protein. To our best knowledge, no study of the in vivo
concentration of the Fur regulator throughout the micro-
bial growth phases has ever been conducted. Such a
study would be fortuitous for evaluating this hypothesis,
but such an experiment would be beyond the scope of the
present research. It should be noted that MnSOD would
still be inducible during this phase through other stimu-
lants such as superoxide [62].
4.3. HP Expression Patterns Suggest
Underlying Metabolic Factors
Determining HP Activity
As iron (II)-activated Fur up-regulates HP expression
[21,23], we expected to see redox-specific changes in HP
activity akin to what was observed in SOD expression
profiles. It was instead observed that both PFo and PFi
increased HP activity with approximately similar activity
ratios throughout all growth phases (Figure 4; Ta b le 2 ).
The unique regulatory role of glutathione in prokaryotes
provides a plausible explanation. Though glutathione is
commonly known as an endogenous reducing agent,
forming disulfide bridges that are then recycled by glu-
tathione reductase and NADPH, in prokaryotic cells glu-
tathione controls the expression of HP [63]. E. coli cells
treated with ferricyanide have been observed to increase
HPI activity, and glutathione has been implicated in this
up-regulation [63,64]. As PFo may induce higher HPI
expression through Fur, and not through other HP regu-
lators such as the protein products of oxyR or rpoS [21],
both iron (II) and iron (III) are capable of up-regulating
HP activity by independent means. Heightened HP activ-
ity in response to both PFo and PFi may therefore be the
consequence of the involvement of both Fur and glu-
tathione, and of the oxidative impact inflicted by these
treatments necessitating such a robust anti-oxidative re-
sponse.
Both exponential and stationary phase cultures pre-
sented substantial increases in HP activity in response to
iron treatment (Table 2; Figures 4(b) and (c)). Exponen-

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138
tial phase is the most metabolically active phase, pro-
ducing ten times more hydrogen peroxide than any other
phase [65-67]. To maintain homeostasis, cells respond to
these generated oxidative and/or free radical stresses by
dramatically up-regulating HP activity [65-67]. Through
the catalysis of radicals by the Fenton reaction, the oxi-
dative impact of iron would be greatest at this time. It
should not be surprising to see such a robust response to
these iron treatments during exponential phase (Figure
4(b); Ta ble 2). Heightened HP activity during stationary
phase is also most probably the consequence of aerobic
metabolism; and specifically, the acidification of the me-
dia. We have observed that the pH of these glucose-based
cultures decreased over time (data not shown), consistent
with other pH studies [26]. The accumulation of weak
acids in stationary phase is complacent in the up-regula-
tion of HP [34,68]. Furthermore, Fenton’s own studies
[69] and subsequent research by Walling [70] revealed
that in mildly acidic environments, hydrogen peroxide
may regenerate iron (II) from iron (III), generating per-
oxyl radicals and hydrogen cations (Eq.4):
+
2
3+ 2+
2
Fe FH OHOOe  H (4)
The low pH in stationary phase media would therefore
exacerbate the oxidative impact of iron and would fur-
ther explain why the effects of PFi and PFo on HP ex-
pression are similar.
4.4. Unexpected Findings and Study
Limitations
As studies using E. coli lacking Fur (Δfur) have shown
[23], Fur is necessary for robust HP activity during lag
phase. In accordance with our hypothesis of low Fur ac-
tivity at this time, we had expected HP activity to be
quite low compared to other phases at this time and that,
like SOD, HP expression should be unresponsive to iron
challenge. HP expression was indeed found to be much
lower during lag phase than, say, exponential phase (data
not shown); however, iron treatment did produce activity
ratios deviating considerably from the expected value of
1, with much statistical uncertainty (Ta bl e 2 ). We have
had some difficulty obtaining consistent data for HP
during lag phase, and the error margins were quite large.
We have previously observed in unpublished work that
HP expression during lag phase is highly malleable to
specific incubatory conditions. For example, the age of
the over-night starter culture used to inoculate fresh me-
dia for incubation is one such consideration: If it is old
(e.g., one week) it decreases MnSOD activity by two
thirds and doubles HPII activity, as compared to fresh
starter culture. Harvest time within the lag period is an-
other consideration: HPI activity is 10-fold higher 40
minutes post-inoculation than at 60 or 80 minutes post-
inoculation. This heightened activity may possibly be an
artifact from the starter culture as it exits stationary phase
to renew replication in a nutrient-replete environment. If
so, this would implicate this 40 to 60 minute interval as a
transitional period for HP activity, and any experimental
work during this time is bound to produce much variance
in data. Future studies harvesting at 80 minutes under
identical conditions may remove this source of error.
Temperature is yet another consideration: To illustrate
the effects of extreme environmental change, we have
observed that when a starter culture is cooled to 4˚C then
added to fresh media set at 37˚C, both HP and SOD ex-
pression are affected as compared to starter cultures kept
at 37˚C throughout. These observations may be of inter-
est in elucidating the antioxidant profile during this most
poorly understood phase of microbial replication. We did
not encounter data consistency issues when assaying
SOD activity during lag phase, which instead showed
remarkably consistent data that proved useful for formu-
lating the present hypothesis.
Each enzyme activity ratio is the mean value of a
treated condition divided by the mean value of the cor-
responding untreated standard. Hence, the standard de-
viation for each activity ratio is the product of the error
of the treated data set and the error of the untreated data
set. This approach will inevitably produce considerably
large error margins in the final activity ratios given
smaller error margins in either data set. We used the pre-
sent methodology because it has previously shown to be
a simple and successful means of investigating antioxi-
dant expression profiles in microbial culture [47]. Al-
though we are confident that SOD expression is both
heightened and unresponsive to iron challenge during lag
phase, the considerable error margins produced by the
methodological approach in many of the treatments and
growth phases stresses caution in interpretation. Some
changes in SOD or HP activity induced by iron treat-
ments proved statistically significant, but others did not.
Alternative experimental approaches could be useful for
supporting the present observations and further elucidat-
ing the role of Fur in regulating antioxidant enzymes in
response to iron threats.
5. ACKNOWLEDGEMENTS
This work was supported by the following grants which are hereby
gratefully acknowledged: Research Corporation Cottrell College Sci-
ence Award #5926 [Research Corporation, 4703 E. Camp Lowell Drive,
Suite 201, Tucson, AZ 85712, USA] to MOE; two University of Win-
nipeg (UofW) Major Research Grants #7437 and #7671 to MOE; and a
Special Grant from the UofW Research and Graduate Studies Office to
Dr. Desiree Vanderwel (Chemistry Dept. UofW) and MOE. We are also
grateful to Mr. Ramin Vakili (Chemistry Dept., UofW) and Dr. Jamie
Galka (Chemistry Dept., UofW) for valuable technical assistance. An
NSERC PGS-M (425305-2012) awarded to RLB is also gratefully ac-
knowledged. We also thank two anonymous reviewers for their insightful

R. L. Bertrand, M. O. Eze / Advances in Enzyme Research 1 (2013) 132-141 139
critiques on previous drafts of the manuscript.
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