From The Krebs Institute for Biomolecular Research,
Department of Molecular Biology and Biotechnology, The University
of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United
Kingdom, the
Life Sciences Division, King's College London,
Campden Hill Road, London W8 7AH, United Kingdom, and the ** Chemistry
Department, King's College London, Strand, London WC2R
2LS, United Kingdom
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ABSTRACT |
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Escherichia coli possesses a
flavohemoglobin (Hmp), product of hmp, the first microbial
globin gene to be sequenced and characterized at the molecular level.
Although related proteins occur in numerous prokaryotes and eukaryotic
microorganisms, the function(s) of these proteins have been elusive.
Here we report construction of a defined hmp mutation and
its use to probe Hmp function. As anticipated from up-regulation of
hmp expression by nitric oxide (NO),
S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP), the hmp mutant is hypersensitive to these agents. The
hmp promoter is more sensitive to SNP and
S-nitroso-N-penicillamine (SNAP) than is the
soxS promoter, consistent with the role of Hmp in protection from reactive nitrogen species. Additional functions for Hmp
are indicated by (a) parallel sensitivity of the
hmp mutant to the redox-cycling agent, paraquat,
(b) inability of the mutant to up-regulate fully the
soxS and sodA promoters in response to oxidative stress caused by paraquat, GSNO and SNP, and (c)
failure of the mutant to accumulate reduced paraquat radical after
anoxic growth. We conclude that Hmp plays a role in protection from
nitrosating agents and NO-related species and oxidative stress. This
protective role probably involves direct detoxification of those
species and sensing of NO-related and oxidative stress.
The best known members of the ancient globin superfamily are the
hemoglobins of vertebrate blood and intramuscular myoglobin (1), which
are primarily responsible for oxygen delivery and storage in animals,
although the circulating hemoglobin has also been implicated in
transport of NO1 (2). It is
now clear that homologous hemoglobins also occur in many bacteria and
yeast as well as in invertebrates and higher plants (3). Microbial
hemoglobins are divisible into two groups: dimeric hemoproteins
comprising two polypeptides each having one heme, as in
Vitreoscilla VGB (4), and monomeric, chimeric
flavohemoproteins composed of a single polypeptide having both a single
heme and FAD. The sequence of the hmp gene (5), encoding the
prototype of the latter class, Escherichia coli Hmp, has
revealed an N-terminal domain homologous to vertebrate, plant, and
Vitreoscilla globins, whereas a C-terminal domain has FAD-
and NAD(P)H-binding sites as in proteins in the ferredoxin-NADP
reductase family (6). Closely related flavohemoglobins occur in the
yeasts Saccharomyces cerevisiae (7) and Candida
norvegensis (8) and in the bacteria Alcaligenes
eutrophus (9), Erwinia chrysanthemi (10), and Bacillus subtilis (11). On the basis of polymerase chain
reaction experiments (12) and genome sequencing projects,
e.g. that on Mycobacterium tuberculosis (13),
related hemoglobins are predicted to be also present in many other bacteria.
The functions of microbial globins have been elusive. Based on
up-regulation of the Vitreoscilla globin at low oxygen
tensions (14) and the ability of this protein to restore aerobic
respiration when expressed in oxidase-deficient E. coli
mutants (15), VGB has been implicated in oxygen storage, delivery, or
reduction (16). The E. coli Hmp protein also consumes oxygen
(17) and reduces various acceptors, including cytochrome c
(18), Fe(III) (6, 19, 20), and the Azotobacter regulatory
flavoprotein NifL (21). This dual ability might allow Hmp to act as an
oxygen sensor (17, 22).
The first evidence that Hmp might function in responses to NO came from
the discovery that its expression is markedly up-regulated by NO, both
aerobically and anaerobically (23). Bacillus subtilis hmpB is also induced by nitrite (11). Furthermore, in
A. eutrophus, mutation of the hmp homologue,
fhp, results in failure to detect nitrous oxide as an
intermediate during denitrification (9). Recently the Salmonella
typhimurium flavohemoglobin has been shown to confer resistance to
acidified nitrite (and thus presumably NO) and
S-nitrosothiols (24), and E. coli Hmp has been
shown to have NO dioxygenase activity (25). These findings implicate bacterial flavohemoglobins in detoxification or utilization of NO.
However, other evidence suggests that microbial flavohemoglobins are
involved in responses to oxidative stress. Paraquat
(1,1'-dimethyl-4-4'-bipyridinium dichloride; methyl viologen) is a
strong inducer of the hmp gene, independently of the SoxRS
regulatory system (26), and Hmp itself generates superoxide anion,
detectable using a superoxide-sensitive To resolve whether E. coli Hmp is important in responses to
oxidative stress or NO, or both, we have constructed the first defined
null allele of hmp and used this mutant to test responses to
paraquat, sodium nitroprusside (SNP, a nitrosating agent) and S-nitrosoglutathione (GSNO) and
S-nitroso-N-penicillamine (SNAP), the last two
being widely used as NO-releasing agents. In addition, since the SoxRS
system has been shown to respond to both oxidative stress and NO (29,
30), we have compared the response of the hmp and
soxS promoters to challenge with "NO-releasing" agents. These data suggest that Hmp is pre-eminently involved in responses to
NO and related reactive nitrogen species.
Strains, Media, and Growth Conditions--
Strains and plasmid
used are listed in Table I. Cells were
grown in rich medium (LB) or MOPS-glucose defined medium, initial pH
7.0 (23). Kanamycin, chloramphenicol, tetracycline, spectinomycin, and
ampicillin were used at final concentrations of 100, 40, 15, 100, and
200 µg/ml, respectively. Culture optical density was measured with a
Pye-Unicam SP6-550 spectrophotometer at 600 nm, after dilution to
bring A600 to below 0.7 when measured in cells of 1-cm path length. All cultures were grown at 37 °C with shaking (200 rpm) in conical flasks containing Determination of Resistance to Paraquat, SNP, and GSNO--
GSNO
was prepared by the method of Hart (35); SNP was from Sigma. Cultures
(10 ml) of strains VJS676 (wild-type) and RKP4545 (hmp) were
grown aerobically in LB medium to an OD600 of 0.3: the
cultures were then divided, one-half being treated with paraquat, SNP,
or GSNO at the final concentrations shown under "Results." After 45 min further incubation, the cultures were serially diluted in medium
and plated on LB medium containing antibiotic, where appropriate.
Results are expressed as a percentage of the viable counts in cultures
not exposed to the agents.
Genetic Methods and DNA Manipulations--
Genetic crosses were
performed using bacteriophage P1vir-mediated transduction
(36). Transformation was carried out after CaCl2 treatment
(37). For Southern hybridizations, DNA purified using a genomic DNA kit
(Promega) was digested with endonucleases and transferred by capillary
blotting from a 1.5% agarose gel to Hybond N+ nylon membrane. The blot
was fixed (UV1800 Stratalinker, Stratagene) and probed with a 1.3-kb
DNA fragment containing the hmp gene excised from an
appropriate plasmid (see "Results") and labeled nonradioactively
with horseradish peroxidase using the Enhanced Chemiluminescent
Detection System (ECL; Amersham). Washes were performed in saline
sodium citrate (SSC) (37), pH 7.0, which contained (at 1 × strength) NaCl (8.77 g/liter) and sodium citrate (4.41 g/liter). The
blot was washed at high stringency (0.1 × SSC, 20 min, 55 °C)
and then in secondary wash buffer (2 × SSC). After 5-min
agitation at room temperature and repeat of this step, the blot was
drained and subjected to signal detection as described in the ECL kit
protocol. For Northern hybridizations, total RNA was purified using the
RNeasy mini kit (Qiagen) from cells growing exponentially. Total RNA
was run in a 1.5% (w/v) agarose gel containing 20 mM
guanidinium thiocyanate and probed with the same 1.3-kb hmp fragment.
Visible Electronic Spectroscopy of Cells Grown with
Paraquat--
These spectra were recorded using an SDB4
dual-wavelength scanning spectrophotometer (University of Pennsylvania
School of Medicine Biomedical Instrumentation Group and Current Designs Inc., Philadelphia PA 19104-2420) (39). For direct observation of the
paraquat radical in anaerobically cultivated cells, the unopened
culture bottle was mounted directly adjacent to the photomultiplier. Spectral data were analyzed and plotted using SoftSDB
(Current Designs Inc.) and CA-Cricket Graph III.
Construction of an hmp Null Mutant--
To determine the
consequences of Hmp deletion, we constructed a strain carrying a null
mutation in its structural gene, hmp. Plasmid pGS16 (34, 40)
comprises the pACYC184 vector and a 13-kb EcoRI insert
containing the hmp, glyA, and glnB
genes, with a Tn5 insertion located in that portion of
hmp encoding the heme domain (243 base pairs downstream of
the translational start site). pGS16 was digested with
EcoRI, and the gel-purified linear fragment containing the
disrupted hmp::Tn5 was used to
transform strain JC7623; this strain has mutations in the RecBCD enzyme
(product of recBC) and exonuclease I (sbcB gene
product), allowing linear DNA to have a longer half-life in
vivo and recombine (32). Six colonies were selected on plates
containing kanamycin, one of which (strain RKP4322) was selected for
further study. The KmR phenotype of strain RKP4322 was
transduced to strain VJS676 ( Growth of the hmp Mutant and Its Isogenic Parent Strain--
To
study the physiological effects of an hmp mutation, strains
VJS676 and RKP4545 were compared with respect to their growth under
common laboratory conditions. Growth of the two strains was similar
either in LB medium or MOPS minimal medium, with glucose or glycerol as
carbon and energy sources, aerobically or anaerobically, and with or
without nitrate as electron acceptor (not shown). Thus, Hmp is not
essential for aerobic or anaerobic growth under these conditions.
However, in all cases, turbidity of the hmp mutant cultures
declined slightly (data not presented), but significantly and
reproducibly, during the stationary phase (10-30 h after inoculation).
An hmp Null Mutant Shows Increased Sensitivity to Paraquat, SNP,
and GSNO--
The demonstration that hmp is induced by
paraquat and nitric oxide (23, 26) suggested that Hmp participates in
bacterial defenses against oxidative stress, NO, or the deleterious
effects of related, reactive nitrogen species. We therefore
investigated the killing action of paraquat, SNP (a nitrosating agent),
and GSNO (a compound widely used as an NO-releasing agent) on the hmp mutant strain RKP4545 and its isogenic parental strain
VJS676. We also included for comparison an fpr mutant; the
gene product, ferredoxin (flavodoxin) NADP+ oxidoreductase
(41), belongs to the ferredoxin-NADP+ reductase family as
does the flavin domain of Hmp. Mutation of fpr results in
increased sensitivity to paraquat (31). The wild-type strain VJS676
gave a biphasic response to paraquat; as the paraquat concentration was
increased to 800 µM, viability in LB medium decreased to
about 80% of the control value after 45-min incubation (Fig.
2A). At higher concentrations,
the decline in viability was precipitous, reaching 25% of the control
value at 1 mM paraquat. No viable cells could be cultured
after 45-min treatment with 2 mM paraquat. The
hmp mutant was markedly more sensitive, particularly at
lower paraquat concentrations. Most sensitive was the fpr
mutant, the viability of which was decreased to about 5%, even at 800 µM (Fig. 2A). The hmp and
fpr mutants were also more sensitive to GSNO (Fig.
2B) and SNP (Fig. 2C). Exposure for 45 min to 2 mM GSNO (Fig. 2B) and 2 mM SNP (Fig.
2C) gave similar results. A 25% loss of viability was
measured for the wild-type strain, whereas viabilities of the
hmp and fpr mutants were reduced by about 70 and
80%, respectively.
Since expression of the yeast flavohemoglobin is enhanced by
H2O2 (27), even though a YHB1 mutant
is more resistant to this agent (28), we compared the growth of the
hmp mutant and its isogenic parent strain in a wide range of
H2O2 concentrations. No significant differences
were evident (data not shown).
The hmp Mutation Prevents Full Response by the Sox System to
Oxidative Stress--
The sensitivity of the hmp mutant to
paraquat, SNP, and GSNO may result from Hmp protein being directly
involved in combating these agents (e.g. by reaction with
superoxide or detoxifying NO and related species) and/or because Hmp is
needed to elicit the SoxRS response to oxidative stress (17).
Therefore, we investigated the effects of an hmp mutation on
the Sox system, exploiting the KmR-marked mutant
hmp allele and transducing it to a strain carrying
Similar results were found when SNP or GSNO was used (not shown). SNP
(200 µM final concentration) increased
Transcription of hmp in the Presence of SNP and NO--
Since
hmp is important in the response to paraquat, NO, and
NO-related species and is up-regulated by these agents (Fig. 2; Refs.
23 and 26), we sought direct evidence at the level of the
hmp transcript for hmp up-regulation by growing
strains VJS676 or RKP4545 (hmp) and challenging them with
SNP in the exponential phase of growth. After 20-min incubation with
SNP, total RNA was isolated and subjected to Northern blot analysis
using the hmp gene as probe (see Fig. 1A). RNA
(10 µg) loaded to each well gave uniform loading (Fig.
4A). Northern hybridization
(Fig. 4B) revealed that both strains exhibited low levels of
hmp expression during exponential phase in the absence of
SNP, consistent with maximal expression of hmp in the
stationary phase of growth (42). SNP resulted in strong induction of
the hmp gene in strain VJS676 (mRNA of about 1.2 kb;
indicated by the arrow in Fig. 4B). When strain
RKP4545 was similarly challenged, no mRNA at 1.2 kb was detected,
confirming that strain RKP4545 was carrying an hmp null mutation.
Up-regulation of hmp Transcription Is Triggered by Lower Levels of
SNP and NO than Is soxS Transcription--
The above results show that
Hmp is synthesized in response to SNP and that Hmp is needed for full
expression of sodA in response to paraquat and for full
expression of soxS, whose gene product is required for
up-regulation of sodA and superoxide dismutase synthesis. A
possible explanation is that Hmp is part of a regulatory cascade and
that hmp transcription, triggered by low levels of oxidative
stress, is required so that the Hmp may participate in the induction
processes. We therefore compared, under identical conditions, the
responsiveness of Spectral and Respiratory Consequences of an hmp Mutation--
Hmp
is a soluble flavohemoglobin (5) with features in the visible spectral
region attributable to heme B and FAD (17, 43). In yeast, deletion of
the flavohemoglobin Yhb results in the loss of the characteristic
spectral forms of the oxyhemoglobin at about 575 nm (28), and so we
sought spectral changes in soluble extracts of ultrasonically disrupted
cells. Reduced minus oxidized difference spectra of extracts
from wild-type cells showed a Soret peak at 421 nm, an
To determine the contribution of the oxidase activity (17, 44) of Hmp
to total cellular respiration, oxygen uptake rates in the wild-type
strain VJS676 and the hmp mutant RKP4545 were compared.
Respiration rates were unaffected whether measured using endogenous
substrates present in cells harvested from overnight aerobic cultures
in rich medium or after stimulation with exogenous succinate (10 mM). The oxygen uptake rates of "soluble" supernatant fractions from ultrasonically disrupted cells were low and were stimulated by NADH by 4-fold (to about 4 nmol of O2/min/mg
of protein) in soluble fractions from both wild-type and hmp
mutant cells (not shown).
Paraquat Reduction Is Affected in the hmp Mutant--
Hmp is an
oxidoreductase with a broad specificity for electron acceptors. Since
NADPH-dependent paraquat diaphorases, i.e. enzymes capable of reducing the paraquat cation (PQ2+),
have been reported in E. coli (45, 46), we tested the
possibility that Hmp might be such a diaphorase. To detect directly the
reduction of paraquat by the hmp mutant and its parent
strain, cells were grown aerobically in rich medium for 6 h and
then subcultured (4% inoculum) into fresh medium containing 1 mM paraquat. The tubes were sealed and growth allowed to
continue in the anoxic atmosphere of an anaerobic jar. It was
anticipated that paraquat would increase synthesis of paraquat
diaphorase(s) and that, on exhaustion of oxygen, reduced paraquat would
accumulate as the relatively stable blue radical (PQ+).
After overnight growth at 37 °C, the hmp mutant culture
was straw-colored, whereas the culture of the parent strain was deep blue. The absorbance spectrum from the sealed tubes (Fig.
6) revealed a prominent peak at 605 nm in
the wild-type strain that was absent in the hmp mutant. This
absorbance corresponds to that of the reduced form of paraquat (47). To
demonstrate that these color differences were not due to differences in
growth yield in the presence of paraquat, the vials were opened (upon
which the blue color disappeared in a few minutes) and the OD measured.
The hmp mutant and parent gave similar growth yields (data
not shown), despite the marked color differences in the anoxic
cultures. This test illustrates an experimentally useful phenotype of
hmp mutants.
Several possible functions have been proposed for bacterial
hemoglobins, including oxygen storage and delivery (4), as terminal
oxidase (15), in denitrification (9) and as oxygen sensor (17). While
this work was being reviewed, Gardner et al. (25) reported
that Hmp has NO dioxygenase activity and that a mutant carrying an
undefined deletion that extends into hmp is more sensitive
to growth inhibition by NO and lacks the NO-consuming activity of the
parent strain. In this paper we directly addressed the function of
E. coli Hmp by constructing a genetically marked null allele
of hmp and characterizing the hmp mutant strain.
No microbial globin described to date appears to be essential for
either aerobic or anaerobic growth under normal laboratory conditions.
For example, destruction of the S. cerevisiae globin by
ethyl hydrogen peroxide (48) or mutation (27, 49) did not alter
respiration rates, cell viability, or growth under a variety of oxygen
conditions and with various carbon sources. Mutation of hmpX
in the plant pathogenic bacterium Erwinia chrysanthemi does
not affect growth in either aerobic or microaerobic conditions, but
hmpX mutants are compromised in their pathogenic effects
(10). Likewise, we show here that loss of Hmp is not detrimental to respiration or growth under common laboratory conditions. The only
growth defect found thus far for the hmp mutant is in the stationary phase of growth where it attained a slightly lower population density; it may be significant that hmp is
normally induced in stationary phase (42). The possible roles for Hmp in stationary phase survival and the possibility of more subtle effects
of loss of Hmp on growth under certain conditions remain to be studied.
An important outcome of this work is that the hmp mutant is
more sensitive than its isogenic parent to SNP and GSNO. Although the
latter is widely used as an NO-releasing agent, both compounds may act
similarly as nitrosating agents (50). Responses to SNP and GSNO are of
special interest, since we have recently elucidated a novel mechanism
for hmp up-regulation via nitrosation of homocysteine thus
modulating binding of MetR to the glyA-hmp intergenic region (50). Other mechanisms of toxicity of SNP and GSNO are poorly understood, but such nitrosating agents are expected to be reactive with thiols and may interact with the Fe-S cluster of SoxR preventing full induction of soxS, sodA, and other stress-responsive genes.
The finding that Hmp is involved in surviving the oxidative stress
caused by paraquat or "NO releasers" is consistent with the results
of previous studies with the hemoglobins of S. cerevisiae and E. chrysanthemi (10, 27). Mutations in the S. cerevisiae YHB gene conferred increased sensitivity to oxidative
stress from the thiol oxidants diamide and diethylmaleate, but paraquat
had only a minor effect on the YHB mutant (27). In E. chrysanthemi, mutations in the gene hmpX conferred loss
of plant pathogenicity (10); it was speculated that this was due to
increased sensitivity to oxygen radicals, but the recent discovery (51)
that plants utilize NO to resist pathogenic bacteria suggests that
hmpX might also be involved in NO responses in
Erwinia.
Following reaction of paraquat with oxygen to generate superoxide, the
oxidized divalent paraquat cation formed can be re-reduced leading to
redox cycling and sustained superoxide generation. Paraquat reductases
(diaphorases) identified in E. coli are
ferredoxin:NADP+ oxidoreductase, thioredoxin reductase, and
NADPH:sulfite reductase (31, 45, 52). Although hmp is
up-regulated by paraquat, purified Hmp is not itself an effective
reductant of paraquat with NADH or NADPH as
substrate,2 consistent with
the much higher midpoint potential of the flavin (around A paradoxical aspect of the induction of hmp by paraquat is
that Hmp itself generates free superoxide (20). This, and the present
finding that an hmp mutant fails to elicit full responses to
the presence of paraquat (Fig. 3), may be reconciled by considering Hmp
as an amplifier of oxidative stress. In this model, paraquat (possibly
via superoxide anion) induces synthesis of Hmp, which generates further
superoxide, resulting in the activation of SoxR and the cascade of
regulatory processes that result in oxidative stress responses. Such a
mechanism may explain the increased sensitivity of the hmp
mutant to paraquat (Fig. 2). The extreme paraquat sensitivity of the
fpr mutant confirms the findings of Bianchi et
al. (31), and we report for the first time the additional
sensitivity of the fpr mutant to GSNO and SNP; the mechanism
for neither effect is known.
Interestingly, hmp up-regulation appears to respond to lower
concentrations of SNP and SNAP than does transcription of
soxS. This lends support to the view that Hmp may be an
early component of the regulatory cascade. Several possible mechanisms
can be envisaged. First, superoxide generation by Hmp might directly facilitate conversion of SoxR to the active form, perhaps by reaction with the FeS cluster (55). NAD(P)H:flavin oxidoreductase activity (Fre)
has also been demonstrated to induce soxRS-regulated genes by superoxide generation (56). Second, Hmp is an NADH (43) and NADPH
oxidase (57), and its activity will contribute to reducing the anabolic
reduction charge ([NADPH]/[NADPH] + [NADP+]) (58)
that has been regarded as one of the possible signals for SoxR. Such
regulatory influences of Hmp on genes involved in stress response would
be reinforced by the ability of Hmp to directly convert NO to the
relatively innocuous NO3
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(sodA-lacZ)
fusion or with the purified protein (20). The yeast flavohemoglobin
encoded by the YHB1 gene is also induced by agents that
promote oxidative stress and antimycin A (27), but a subsequent re-examination has produced conflicting views (28).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
to
of
their own volume of medium.
Bacterial strains and plasmid used in this study
-Galactosidase Assays--
-Galactosidase activity
measurements (36, 38) were carried out at 21 °C on CHCl3
and sodium dodecyl sulfate-permeabilized cells by monitoring the
hydrolysis of
o-nitrophenyl-
-D-galactopyranoside. Activities are expressed per A600 of cell
suspensions (36).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
lac) to give strain RKP4545.
Confirmation that the genomic copy of hmp was disrupted was
obtained by comparing genomic Southern blots of strains VJS676 and
RKP4545. Digestion of genomic DNA from VJS676 (hmp+) with PvuII and
EcoRI, which cut outside the hmp gene (Fig.
1A), yielded a single band,
equivalent to a fragment of about 2.5 kb, when probed with the
hmp gene (Fig. 1B). Digestion of genomic DNA from
RKP4545 with PvuII and EcoRI yielded two bands
(3.3 and 2.1 kb; Fig. 1B), the expected result for
interruption of hmp. The larger is the
PvuII-EcoRI fragment comprising 1.42 kb from Tn5, 1.1 kb from the interrupted hmp gene, and
0.82 kb from the region flanking the 3' end of the hmp gene.
The 2.1-kb band represents the PvuII-PvuII
fragment comprising 1.42 kb from the Tn5, 0.2 kb from the
interrupted hmp gene, and 0.47 kb from the region flanking
hmp at the 5' end. Fragments generated from the
PvuII sites internal to the Tn5 are not revealed
with this probe.
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Fig. 1.
Disruption of hmp.
A, schematic diagram of the chromosomal locus of the parent
strain VJS676 and the location of the Tn5 gene insertion in
the hmp::Tn5 gene disruption strain
RKP4545. Restriction sites for PvuII (P) and
EcoRI (E) are shown. B, Southern blot
analysis of genomic DNA from RKP4545 (left) and VJS676
(right). Genomic DNA was digested with PvuII and
EcoRI and probed with the hmp probe shown in
A.
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Fig. 2.
Effects of paraquat, GSNO, and SNP on
viability. Exponentially growing cultures of VJS676 (wild-type,
open squares), RKP4545 (hmp, filled
squares), and C-6007 (fpr, open
circles) were challenged with the indicated concentrations
of paraquat (A), GSNO (B), and SNP (C)
for 45 min and then plated on LB medium. Colony-forming units plotted
are expressed as a percentage of the values for untreated cultures. The
experiment was repeated at least three times with similar
results.
(soxS-lacZ) and monitoring
-galactosidase activity.
Strain TN530 (hmp+) gave an activity of about
200 Miller units, which was increased 9-fold by 200 µM
paraquat (Fig. 3A). In
contrast, strain RKP4324, having
(soxS-lacZ) but also the
hmp mutant allele, had a similar basal level of 140 Miller
units, which was up-regulated only 5-fold. A more marked difference was
observed when the hmp mutation was transduced into a strain
(QC772) carrying a
(sodA-lacZ) fusion, which is
up-regulated by the SoxS protein. This strain had a basal
-galactosidase activity of 160 Miller units, which was increased 10-fold on challenge with paraquat (Fig. 3B). In the
hmp mutant (strain RKP4323), the same fusion had a similar
basal level of 230 units, which was increased only 2.6-fold on adding
paraquat (Fig. 3B). Therefore, the Hmp protein is essential
for full aerobic activation of the SoxRS system and resistance to
paraquat.
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Fig. 3.
Activities of (soxS-lacZ) and
(sodA-lacZ) fusions in response to paraquat. Strain
TN530
(soxS-lacZ) and its hmp derivative (RKP
4324) (A) and strain QC772
(sodA-lacZ) and its
hmp derivative (RKP4323) (B) were grown in rich
medium. In the exponential phase, cells were treated with or without
200 µM paraquat, and the incubation was continued for 45 min. Cells were pelleted, and
-galactosidase activity was determined
and expressed as Miller units. The experiment was repeated at least
three times with similar results.
(sodA-lacZ) and
(soxS-lacZ) activities by
3.3- and 2.1-fold, respectively, but the presence of the hmp
mutation gave increases of only 1.7- and 1.3-fold, respectively. GSNO
(500 µM final concentration) increased
(sodA-lacZ) and
(soxS-lacZ) activities by
3.5- and 1.5-fold, respectively, but the presence of the hmp
mutation gave increases of only 1.5- and 1.2-fold, respectively.
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Fig. 4.
Levels of hmp mRNA in the
absence or presence of SNP in strains VJS676 (wild-type) and RKP4545
(hmp). A, ethidium bromide-stained gel as a
control for RNA loading. Lane 1, RNA markers; lanes
2-4, RNA from strain VJS676; lanes 5-7, RNA from
strain RKP4545. Numbers below lane labels (0, 50, 100)
indicate the concentration of SNP (micromolar) used to treat cultures.
B, autoradiography of the blot hybridized with the probe
shown in Fig. 4A. Lane descriptions are the same as
described in A. The arrow at the left
indicates hmp mRNA.
(hmp-lacZ) and
(soxS-lacZ) to SNP and SNAP, an NO-releasing agent.
Expression of
(hmp-lacZ) in strain RKP2178 was enhanced
above the basal level by 18-fold at 50 µM SNP and by
37-fold at 200 µM SNP (Fig.
5). In contrast,
(soxS-lacZ) fusion activity in strain TN530 was enhanced
only 2-fold at 200 µM SNP (Fig. 5).
(soxS-lacZ) fusion activity was significantly increased
only at 1000 µM SNP (not shown). The NO releaser SNAP
enhanced
(hmp-lacZ) activity in strain RKP2178 12-fold at
concentrations as low as 50 µM (Fig. 5). Again,
millimolar concentrations of SNAP were required for induction of
(soxS-lacZ) fusion activity (not shown).
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Fig. 5.
Activities of (hmp-lacZ) and
(soxS-lacZ) fusions in response to SNP and SNAP.
Strains RKP2178
(hmp-lacZ) and TN530
(soxS-lacZ) were grown in rich medium. In the exponential
phase, cells were treated with the indicated concentrations of SNP
(left panels) or SNAP (right panels). The
incubation was continued for 45 min. Cells were pelleted, and the
-galactosidase activity was determined. The experiment was repeated
at least three times with similar results.
-peak at
about 553 nm, and a weak
-band (not shown). These signals probably
arose from low levels of cytochrome c and are not
characteristic of pure Hmp (Soret,
-, and
-bands at 435.5, 560, and about 590 nm, respectively, in reduced minus oxidized
difference spectra; Ref. 43). These signals were quantitatively and
qualitatively similar in the mutant (not shown), indicating that Hmp
does not make a significant spectral contribution in such extracts. The
region at wavelengths > 553 nm was featureless, indicating the
absence of cytochrome bd, a marker for contamination by
membrane fragments (22). Reduced minus oxidized spectra of intact cells also revealed no significant differences between wild-type
and mutant cells.
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Fig. 6.
Absorption spectra of cultures grown
anaerobically in the presence of paraquat. Strains RKP4545
(hmp) and VJS676 (wild-type) were grown in LB medium
anaerobically in the presence of paraquat for 16 h at 37 °C.
The base line (unlabeled) is the spectrum of LB medium.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
150 mV) for
the 2-electron reduction from FAD to FADH2 (53) than of
paraquat (E0'
446 mV) (54). It is
therefore unlikely that the accumulation of reduced paraquat in anoxic
cell suspensions (Fig. 6) is due to direct reduction by Hmp. More
likely, the presence of Hmp is required for full induction of
components of the oxidative stress response (Fig. 3). One candidate for
the paraquat-reducing enzyme is NADPH:ferredoxin oxidoreductase (45), a
member of the soxRS regulon.
ion by
reaction of NO with oxy-Hmp, recently demonstrated in Ref. 25, but such
a mechanism cannot explain the anaerobic roles of Hmp in protecting
cells from NO reported recently (24, 25). Such mechanisms are currently
under investigation in this laboratory.
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ACKNOWLEDGEMENTS |
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We thank Drs. Vera Bianchi, Bruce Demple, George Stauffer, and Daniéle Touati for providing the strains and plasmids used in this work.
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FOOTNOTES |
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* This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grant P05184 and by a BBSRC Studentship (to T. M. S.).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.
§ Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115.
¶ Recipient of Biochemical Society Krebs Memorial Scholarship and an Overseas Research Student Award.
To whom correspondence should be addressed: The Krebs Institute
for Biomolecular Research, Dept. of Molecular Biology & Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10
2TN, UK. Tel.: 44-114-222-4447; Fax: 44-114-272-8697; E-mail: r.poole{at}sheffield.ac.uk.
The abbreviations used are: NO, nitric oxide; SNP, sodium nitroprusside; GSNO, S-nitrosoglutathione; SNAP, S-nitroso-N-penicillamine; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase pair(s).
2 S. O. Kim and R. K. Poole, unpublished data.
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REFERENCES |
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