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J. Biol. Chem., Vol. 278, Issue 32, 30193-30198, August 8, 2003
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From the Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Spain
Received for publication, April 25, 2003 , and in revised form, May 30, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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katG
strain) and increased protein carbonyl content. This pleiotropic phenotype
disappeared when the adhE gene was reintroduced into the defective
strain. The purified enzyme was highly reactive with hydrogen peroxide (with a
Ki of 5 µM), causing inactivation
due to a metal-catalyzed oxidation reaction. It is possible to prevent this
reactivity to hydrogen peroxide by zinc, which can replace the iron atom at
the catalytic site of AdhE. This can also be achieved by addition of
ZnSO4 to cell cultures. In such conditions, addition of hydrogen
peroxide resulted in reduced cell viability compared with that obtained
without the Zn treatment. We therefore propose that AdhE acts as a
H2O2 scavenger in Escherichia coli cells grown
under aerobic conditions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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When E. coli cells are shifted from anaerobic to aerobic conditions, transcription of the adhE gene is reduced and maintained within 10% of the range found under anaerobiosis (12–15). Translation is also regulated and requires RNase III (15, 16). AdhE has been identified as one of the major targets when E. coli cells were submitted to hydrogen peroxide stress (17). In fact, under aerobic conditions, AdhE has no assigned function, accounts for about 1% of total protein, and is inactivated by metal-catalyzed oxidation (18). This apparent wastefulness prompted us to investigate why this enzyme has been maintained under aerobic conditions. Evidences about the relationship between AdhE and protection against oxidative stress are provided.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(adhE-lacZ)) was used
as the wild type parental strain
(18,
19). Isogenic strains were
used in all experiments. A previously obtained null adhE::kan strain
(ECL4002) was also used (20).
An E. coli strain katG::tet (PEL1) was constructed by P1
transduction (21) from UM202
(22). The DHB4 strain was a
kind gift from J. Beckwith and was also used as a wild type strain
(23). S. typhimurium
LT2 was from ATCC (catalog number E23564) and Serratia marcescens was
from Colección Española de Cultivos Tipo (Valencia, Spain,
catalog number 846). Cells were grown at 30 °C either in Luria broth (LB) medium (0.5% yeast extract, 1% NaCl, and 0.5% tryptone) or minimal medium (64 mM K2HPO4, 34 mM NaH2PO4, 20 mM (NH4)2SO4, 0.1 mM MgSO4, 10 µM CaCl2, and 1 µM FeSO4, pH 7.4) supplemented with one of the following carbon and energy sources: glucose (10 mM), glucuronate (10 mM), gluconate (10 mM), acetate (30 mM), succinate (15 mM), fucose (10 mM), manitol (10 mM), glycerol (20 mM), or casein acid hydrolysate (0.1% w/v). Solid media contained 1.5% Bacto-agar (Difco) in LB. Aerobic cultures of 10 ml were grown in 100-ml flasks shaken at 250 rpm. Anaerobic cultures were grown in 100-ml flasks filled to the brim. When appropriate, antibiotics (Sigma) were added at the following concentrations: 100 µg/ml kamamycine and 25 µg/ml tetracycline. To study ECL4002 auxotrophies amino acids were added to minimal medium-glucose at 100 µg/ml each and cells were aerobically cultured.
Sensitivity to Stress Conditions—Exponentially growing cells (A600: 0.3) were treated with the stressing compound, which was directly added to the growth medium at the concentrations and periods indicated for each experiment. Untreated cultures were incubated in parallel over the same periods. Sensitivity to the treatment was determined by serially diluted (1/10) bacterial suspension with phosphate-buffered saline and then plated by triplicates on LB agar.
Enzymatic Activity Assays—Cells were disrupted by sonication in an ice bath, and the centrifuged extracts were assayed for the following enzyme activities, as described: ethanol dehydrogenase (15), aconitase (24), malate dehydrogenase (25), catalase, and superoxide dismutase (26). Protein concentration was determined by the Bradford method, using bovine serum albumin as standard.
Western Blot Analysis—Oxidized proteins in cell extracts were revealed immunochemically by their carbonyl content (27) after derivatization with dinitrophenylhydrazine (17). The anti-dinitrophenyl (DNP) antibody (Dako) was used at 1:5,000 dilution. Anti-AdhE Western blot was performed using the primary antibody at 1:2,000 dilution. In both cases, the secondary antibody was a goat anti-rabbit conjugated with alkaline phosphatase (Tropix) used at 1:25,000 dilution.
Measurement of Intracellular Oxidation Level—The oxidant-sensitive probe H2DCFDA was used to measure the intracellular peroxide levels (28). Cells growing aerobically in LB (A600: 0.3) were washed with 10 mM potassium phosphate buffer, pH 7.0, and incubated for 30 min in the same buffer with 10 µM H2DCFDA (dissolved in dimethyl sulfoxide). Cells were washed, resuspended in the same buffer, and disrupted by sonication. Cell extracts (100 µl) were mixed in 1 ml phosphate buffer and fluorescence intensity was measured using a Shimadzu RF 5000 spectrofluorimeter (excitation, 490 nm; emission, 519 nm). Emission values were normalized by protein concentration.
In Vitro Inactivation Assays of AdhE—Purified AdhE (2.5 µM) in 50 mM Tris-HCl, 160 mM NaCl, pH 8.5, was incubated for 4 min at room temperature with different concentrations of either KO2 (superoxide donor) plus catalase (50 units) or H2O2 and ethanol dehydrogenase was assayed. Potassium superoxide (KO2) was maintained in dimethyl sulfoxide due to its instability in aqueous solutions. KO2 concentration was monitored spectrophotometrically (e260 = 2086 M–1 cm–1) (29).
Other Methods—Total cellular iron was determined under reducing conditions (30), with bathophenanthroline sulfonate as chelator (31). Quantification of AdhE expression was carried out by Laurell rocket immunoelectrophoresis (32), according to a calibration curve (not shown) obtained with AdhE purified from strain ECL4000 as described previously (18). For DNA staining, cells were resuspended for 10 min in 70% (v/v) ethanol, washed and incubated with 1 µg/ml diaminophenylindole in phosphate-buffered saline for 10 min, and then rinsed with the same buffer. Fluorescence was viewed using a Nikon fluorescence microscope and images were taken using a Sony camera system. To obtain scanning electron microscopy images, exponentially growing cells were washed in 0.1 M phosphate potassium buffer, pH 7.0, and fixed for 30 min in phosphate-buffered glutaraldehyde (2.5% v/v). Cell suspension was transferred to silanized glass. After washing with potassium phosphate, cells were postfixed with osmium tetraoxide (1% v/v) for 30 min, followed by 10 min washes with 30, 50, 70, and 90% acetone. Cells were then washed 3 times with 100% acetone (30 min each) and dried. After carbon evaporation, gold was added to the cells (Balzers ScD 050 Sputer Coater), and samples were observed using a scanning electronic microscope (DMS 940 A Zeiss).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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10,000
molecules of AdhE per cell, of which 70% were inactive. It thus seemed
inefficient to synthesize such amounts of a 96-kDa protein that apparently had
no physiological role. An explanation for this apparent paradox was found
during the study of the adhE strain (ECL4002).
ECL4002 cells were unable to grow aerobically in minimal medium on any of
the carbon sources tested (glucose, succinate, fucose, acetate, glucuronate,
gluconate, manitol, and glycerol). Aerobic metabolism of these sugars did not
require any AdhE activity. This mutant strain grew under aerobic conditions in
LB medium, but with a duplication time of 60 min (whereas wild type cells
duplicated in 35 min in this medium). Addition of 0.1% casein acid hydrolysate
to the minimal medium-glucose allowed growth but duplication time increased
progressively over time. Analysis of amino acids needed for growth revealed
that adhE cells presented auxotrophies for Arg, His, Leu, Pro,
and Thr, and growth without Lys was extremely limited.
As expected, the adhE strain was unable to grow
anaerobically in minimal medium plus glucose, but it did grow when glucuronate
was the carbon source (33).
The control lysogen ECL4063 (adhE::kan
att::adhE+) grew as the wild type strain in all
the media and showed aerobic and anaerobic levels of AdhE activity
indistinguishable from those of the wild type strain ECL4000
(18)
The growth defects of adhE cells were also associated with
changes in size and shape. When grown in LB medium, these mutant cells
presented a filamentous appearance in comparison with wild type cells.
Although, as known, wild type cells displayed a homogeneous size of 1–2
µm long (Fig. 1A),
adhE cells were, on average, 4–5 µm long, but cells
20–50 µm long could also be observed
(Fig. 1D). DNA stain
with the fluorochrome diaminophenylindole revealed that in these filamentous
cells, replication and segregation of DNA was apparently normal
(Fig. 1E). The
filamentous phenotype was probably due to a problem in septum formation.
Indeed, transmission electron microscopy revealed that there was no septum in
the long adhE cells (not shown). The same phenotype was also
observed in DHB4 cells used as a parallel control.
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AdhE Deletion Causes an Oxidative Stress Phenotype—During
our study of adhE cells, we observed that they showed less
viability after freeze-thawing and lysozyme treatment than ECL4000 cells,
which could indicate that their cell membrane was defective. This phenomenon
and the previously described growth defects led us to hypothesize an internal
oxidative stress problem in cells deficient in AdhE. To analyze this
possibility, wild type and adhE cells were tested for
viability after treatment with 2 mM H2O2 for
60 min. The mutant strain had a decreased viability (around two orders of
magnitude) both in aerobic (Fig.
2A) and anaerobic conditions (not shown) compared with
the wild type cells. No differences in viability between the two strains were
observed when cells were treated with paraquat, a superoxide generator (not
shown).
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The fluorescent probe H2DCFDA was used to measure intracellular
peroxide levels (Fig.
2B). Basal levels of endogenously generated peroxides
were 5 times greater in adhE cells compared with wild type
cells. When treated with 10 mM H2O2 for 60
min, peroxides in the mutant strain were only slightly higher than those found
in wild type cells (not shown).
Several enzyme activities known to be affected by reactive oxygen species
were measured in cells grown aerobically in LB
(Fig. 2C). Aconitase,
an enzyme with an Fe/S cluster, has been reported to be very sensitive to
oxidative stress (31,
34,
35). In adhE
cells, aconitase activity was reduced 5-fold with respect to wild type strain.
As a control, malate dehydrogenase, which has been reported to be resistant to
oxidative stress (31,
35,
36), showed no differences
between wild type and adhE cells. Catalase (HPI) and
Mn-superoxide dismutase are enzymes regulated by the transcriptional
regulators OxyR (activated in response to H2O2) and
SoxRS (activated in response to superoxide), respectively. Levels of catalase
activity in adhE cells grown either in LB medium or in
glucose-minimal medium supplemented with casein acid hydrolysate, were,
respectively, two and four times greater with respect to those displayed in
wild type cells. However, there was no difference in superoxide dismutase
activity between both strains, either when grown in LB or in glucose-minimal
medium. This indicated that H2O2, but not
levels, were higher than normal in the adhE cells
(Fig. 2C).
The endogenous oxidative stress measured with H2DCFDA could have
been due to a defective iron metabolism, resulting in iron accumulation within
the cell, as reported in several E. coli mutant strains
(37). To investigate this
possibility, total iron was measured using batophenanthroline sulfonate (see
"Experimental Procedures"). Results demonstrated that there were
no significant differences between wild type and adhE strains
grown in LB with respect to total cell iron (3.3 nmol Fe/6 x
108 cells).
One of the markers under oxidative stress conditions is the formation of
carbonyl groups on proteins due to modification of some amino acid side chains
(27). Anti-DNP Western blot
experiments revealed that in adhE cells, grown aerobically in
LB and stressed with 10 mM H2O2 for 60 min,
showed increased levels of protein damage as compared with stressed wild type
cells (Fig. 2D). As
shown, even in non-stressed cells, protein carbonylation was slightly higher
in adhE cells than in wild type cells.
Many microorganisms, including human pathogens, also synthesize a multifunctional alcohol dehydrogenase. These enzymes are members of the "iron-dependent" dehydrogenase family and share a high sequence homology to AdhE from E. coli. To verify whether these homologues were also major targets under H2O2 stress conditions, wild type cells from other two enterobactericeae, S. typhimurium and S. marcescens, were submitted to 10 mM H2O2 for 60 min, and protein oxidation was analyzed by anti-DNP Western blot and compared with E. coli (Fig. 2E). As expected, a band corresponding to AdhE became strongly carbonylated in both S. typhimurium and S. marcescens, which means that homologues of AdhE exhibit the same reactivity to H2O2.
Comparison Between adhE and katG
Cells—From the above results one can hypothesize that strain
ECL4002 was deficient at detoxifying endogenously generated peroxides. In this
context, to compare the role of AdhE with that of catalase, a katG
defective mutant was constructed (see "Experimental Procedures").
This gene encoded for the exponential phase catalase, HPI. Several parameters
were measured to compare katG cells with adhE
cells. Cultures of katG cells were grown aerobically in LB
with a duplication time of 45 min (as compared with 60 min for
adhE and 35 min for wild type strain). The HPI-deficient cells
grew on minimal medium with glucose as a carbon source under aerobic
conditions, although only at the permissive temperature of 30 °C
(adhE strain did not grow in this medium at any temperature).
When observed under the electron microscope, a minor fraction (fewer than
0.1%) of the cells were longer than normal when grown in this minimal medium
(Fig. 1G). No apparent
defects were observed when grown in LB (not shown).
Peroxide levels measured in katG cells growing under
aerobic conditions in LB (Fig.
2B) were found slightly increased compared with wild type
cells, although smaller than those found in adhE cells. It is
known that cells submitted to a non-lethal oxidative stress by
H2O2 transiently arrest their growth. After a lag period
cells recover from the stress and growth is restored. This lag time was
measured in wild type, katG and adhE cultures,
grown aerobically in LB and stressed with various concentrations of
H2O2 (Fig.
3). Wild type cells presented only slight growth delay at the
highest dose of H2O2 used (1 mM) and almost
no effect at lower concentrations. On the contrary, both adhE
and katG cells arrested their growth after stress. As
expected, at 0.5 and 1 mM H2O2, HPI was more
important than AdhE, because katG cells arrested growth longer
than adhE cells. However, at 0.1 mM
H2O2, growth of adhE cells was almost as
equally affected as katG cells. In addition,
adhE strain had a duplication time higher than
katG strain. These results indicated that AdhE was more
important than HPI for maintaining basal levels of
H2O2.
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AdhE is Highly Reactive to H2O2—The reactivity between AdhE and H2O2 was observed in vivo measuring the rapid inactivation of ethanol dehydrogenase activity following the addition of 0.5 or 1 mM H2O2 for 5 min to ECL4000 cultures. An inactivation of 55 and 80% was, respectively, obtained. Under these conditions, aconitase was only inactivated 25 and 40%, respectively. AdhE inactivation by H2O2 was irreversible because addition of 10 µg/ml chloramfenicol to ECL4000 cells simultaneous to H2O2 treatment prevented the recovery of ethanol dehydrogenase activity. When no antibiotic was added, AdhE activity increased in parallel with the duplication of cells (not shown).
Using pure preparations of AdhE, the plot of H2O2 concentration versus enzyme inactivation gave an inactivation constant (Ki) for H2O2 of 5 µM (Fig. 4A). However, when KO2 was used as a superoxide donor (in the presence of catalase to remove spontaneously formed H2O2), AdhE reactivity was clearly lower (Ki for superoxide was 120 µM) (Fig. 4A). As expected, inactivation of AdhE by H2O2 resulted in carbonyl formation (Fig. 4B). Taken together, these results were consistent with the idea that AdhE plays an important role in combating H2O2 stress, but not superoxide stress.
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Zn Inactivation of AdhE Generates Cells Highly Sensitive to Oxidative
Stress—Assuming the aerobic role of AdhE as an
H2O2 scavenger, the addition of any inhibitor of its
scavenging activity should result in cells highly sensitive to oxidative
stress, resembling adhE cells. In previous studies
(38,
39) on ADHII, a dehydrogenase
from Zymomonas mobilis, which is also a member of the iron-activated
dehydrogenase family, our group demonstrated that the Fe-ion was easily
exchanged by Zn, which results in alcohol dehydrogenase inactivation and
inability to carry out Fenton reaction. As expected, when ZnSO4 was
added to ECL4000 cultures, AdhE was inactivated in a dose-dependent manner
(Fig. 5A). In
addition, carbonyl groups formation in AdhE under oxidative stress were
undetectable when zinc was present (Fig.
5B). Fig.
5C shows that the effect of Zn treatment prior to
H2O2 stress reduced cell viability (around 1 order of
magnitude), demonstrating the importance of Fe-AdhE as an
H2O2 scavenger.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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adhE could not be explained solely on the basis of
a higher susceptibility to inactivation of enzymes implicated in the amino
acid biosynthesis. This could also have been due to membrane permeation
produced by reactive oxygen species as described in sod cells
(46). The results clearly demonstrated that AdhE was able to react with H2O2, both in vivo and in vitro, producing an irreversible inactivation of the enzyme. This was indicated by the lack of recovery when chloramfenicol and H2O2 were simultaneously added to the culture. This scavenging power would imply an equimolar reaction between H2O2 and AdhE. Although a reducing cycle (with Fe3+ being recycled to Fe2+, perhaps by NAD(P)H+ or superoxide) would make the system more efficient by allowing to scavenge more than 1 molecule of H2O2 per molecule of AdhE, several indirect results seemed to rule out this possibility. First, AdhE has lower binding affinity to ferrous ion than to ferric ion.2 And second, propanediol oxidoreductase, an enzyme with the same iron-binding signature is inactivated by metal-catalyzed oxidation due to the modification of a highly conserved histidine residue, which is essential for iron binding (39, 47).
AdhE is important for controlling H2O2 homeostasis
under normal aerobic conditions because reactive oxygen species produced in
the electron transport chain are of the same order of magnitude as
Ki for H2O2 (5
µM). A similar role has been proposed for alquil hydroperoxide
reductase (AhpCF) (48).
Although it would be difficult to establish which is more important, in
contrast to adhE strain, ahpCF mutants grow
aerobically in LB as well as a wild type strain
(48). AhpCF is regulated by
OxyR (AdhE is not), which means that at intermediate
H2O2 concentrations
(10–5–10–4
M), the amount of AhpCF would rise considerably and, consequently,
will perform its physiological role. At high concentrations, catalases are the
predominant enzyme against H2O2 as demonstrated in the
case of our HPI-deficient cells and by other authors
(48,
49). HPI has a
Km of 5.9 mM
(50), and HPII (reported as
important only in stationary phase, when it is induced by RpoS)
(48,
51) has similar kinetic
parameters (52). One can
hypothesize that these sets of enzymes could constitute a three-step barrier
against H2O2, with AdhE being the most important at
lowest H2O2 concentrations. An additional advantage,
such as the fast adaptability to an anoxic situation, can be deduced from the
maintenance of such an expensive system as AdhE under aerobic conditions. In
this context, it must be remembered that 30% of AdhE molecules remain active
and can start immediately working as a dehydrogenase.
The observation that these mutant cells exhibited lower resistance to
freeze-thawing and to lysozyme treatment (not shown) led us to believe that
adhE cells were very fragile as a result of the continuous
attacks from H2O2 generated inside the cell (the
membrane was probably affected). The decreased viability observed by a strong
stress (2–10 mM H2O2), would be the
result of this fragility and not strictly due to the lack of AdhE scavenging
activity, because in this situation HPI is by far the most important
enzyme.
There is an increasing list of microorganisms with orthologues to AdhE sharing a 60–90% sequence homology (Fig. 6). These include human pathogens, which give this enzyme clinical importance. It seems reasonable to assume that all these enzymes should act in the same way that AdhE did in E. coli. The results obtained in S. typhimurium and S. marcescens are in agreement with this assumption. Additionally, in a systematic study of genes involved in survival to macrophage attack in S. typhimurium, it was observed that adhE mutant cells were more sensitive than wild type cells (53). The role of these orthologues as H2O2 scavengers provide clues to explain why even microorganisms that neither produce alcohols nor use them as carbon sources (Listeria, Staphylococcus, and Streptococcus) have maintained this enzyme.
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It would be especially interesting to be able to inactivate these AdhEs in bacterial pathogens making them more susceptible to the immune system. From previous studies (38), we know that Zn can replace Fe easily in these dehydrogenases in vitro. As we have demonstrated in this study, treatment of E. coli cells with ZnSO4 inactivated AdhE, and this Zn-AdhE was not able to perform the Fenton reaction. More interestingly, these Zn-treated E. coli cells became more susceptible to oxidative stress. It is possible to speculate on the possible role of Zn treatment in infections involving such pathogen microorganisms. Developing specific inhibitors to AdhE could be of great interest in the near future.
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FOOTNOTES |
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A recipient of a Ph.D. scholarship from the Ministerio de Educación
y Cultura (Spain).
To whom correspondence should be addressed: Dept. Ciències
Mèdiques Bàsiques, Facultat de Medicina, Av. Rovira Roure 44,
25198 Lleida, Spain. Tel.: 34-973-702-275; Fax: 34-973-702-426; E-mail:
joaquim.ros{at}cmb.udl.es.
1 The abbreviations used are: AdhE, alcohol dehydrogenase E; AhpCF, alquil
hydroperoxide reductase; H2DCFDA,
2',7'-dichlorofluorescein diacetate; LB, Luria broth; DNP,
dinitrophenyl.
2 P. Echave, J. Tamarit, E. Cabiscol, and J. Ros, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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), 0.5 (
), or 1 (
)
mM H2O2 added. Results are the average of
three independent experiments with a variation of less than 15%.
), and ethanol dehydrogenase
activity was immediately assayed (A). Samples of AdhE treated with
H2O2 as described above, were submitted to anti-DNP
Western blot to measure carbonyl formation and densitometric analysis of the
Western blot was performed. One arbitrary unit corresponds to the signal
intensity given by the unstressed sample (B).
