From the Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229
Received for publication, December 6, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Nitric oxide (NO) induces NO-detoxifying enzymes
in Escherichia coli suggesting sensitive mechanisms for
coordinate control of NO defense genes in response to NO stress.
Exposure of E. coli to sub-micromolar NO levels under
anaerobic conditions rapidly induced transcription of the NO reductase
(NOR) structural genes, norV and norW, as
monitored by lac gene fusions. Disruption of rpoN ( Nitric oxide (NO)1 is a
free radical with multiple and diverse biological functions (reviewed
in Ref. 1). NO serves as an intermediate in microbial denitrification
(2), a signal molecule controlling the activation of guanylate cyclases
(3), and as a broad-spectrum antibiotic, anti-viral and anti-tumor
agent secreted by host immune cells (4, 5). Sub-micromolar NO can
inactivate or inhibit critical enzymes, including [4Fe-4S]
(de)hydratases and heme-dependent terminal respiratory
oxidases, accounting at least in part for the cytotoxic actions of NO
(6-11).
Not surprisingly, organisms have evolved mechanisms for NO
detoxification. NO reductases (NORs) reduce NO to N2O and
are widely distributed in denitrifying bacteria, nitrogen-dissimilating
fungi, and pathogenic bacteria (2). Microbes also express NO
dioxygenases (NODs) that utilize O2 to convert NO to
nitrate (12-18). Escherichia coli employs both of these
enzymes. An inducible NOD (flavohemoglobin), encoded by the gene
hmp (19), detoxifies NO under aerobic growth conditions (12,
15, 20). An inducible O2-sensitive NOR activity encoded by
the norRVW operon detoxifies NO under anaerobic and microaerobic conditions (8, 20). NorV is a di-iron center-containing flavorubredoxin-type NOR with orthologues in the Archeae, strict anaerobes, and facultative anaerobes (21-24). It is distinct from the
bacterial heme/nonheme iron-containing cytochrome bc-type NORs and the fungal P450-type NOR (2). NorW functions as an NADH:flavorubredoxin oxidoreductase (21) and is required for maximal
flavorubredoxin-catalyzed NO reduction in cells (8) and in
vitro (25). Together, the O2-dependent NOD
and the O2-sensitive NOR (NorVW) detoxify NO throughout the
physiological [O2] range (7, 8, 20).
NORs and NODs are induced by NO or NO-generating agents suggesting
fine-tuned mechanisms for the coordination of microbial NO defenses to
NO stress levels. In denitrifying Pseudomonas and Rhodobacter, cytochrome bc-type NORs are
up-regulated by the Fnr-like DnrD/NnrR transcription regulators in
response to nanomolar NO (26-28). However, unlike Fnr (29), DnrD/NnrR
do not bear NO-reactive [4Fe-4S] centers, and the NO sensing
mechanism is currently unknown (26-28, 30). In the denitrifier
Ralstonia eutropha, the tripartite transcription factor NorR
regulates denitrification, norA1B1 transcription, and NOR
activity expression in a Recently, Hutchings et al. (32) reported
NorR-dependent activation of norV transcription
by the NO+ donor and NO-generating compound nitroprusside
in support of the proposed regulatory function. Interestingly,
nitroprusside-elicited norV transcription was increased
>5-fold by normoxic O2 suggesting mechanisms for NorR
activation involving O2-derived reactive nitrogen intermediates rather than NO per se. The large oxygen
enhancement of norV transcription observed with or without
nitroprusside exposure has also supported proposals for aerobic
functions for the norRVW operon, including O2
reduction and the detoxification of O2-derived reactive
nitrogen intermediates (25, 32).
We now report the rapid and robust induction of norV
and norW transcription and NorVW activity by sub-micromolar
NO via a NorR- and Chemicals and Reagents--
Bovine liver catalase (260,000 units/ml) was purchased from Roche Molecular Biochemicals. Glucose
oxidase (4,000 units/ml) and Strain and Plasmid Construction--
Strains and plasmids are
described in Table I. Chromosomal
lacZ gene fusions of norR, norV, and
norW were created as previously described (8). A
Tn10 mutation in the rpoN locus was transduced with P1 phage and mutants were selected for tetracycline resistance. The pUC19NorR construct is a 1.9-kb SalI-PstI
fragment cloned in pUC19 containing the intragenic region between
norR and norV in addition to the norR
coding region from the lambda phage 9G10 (36). To construct
pUC19NorR Media, Growth Conditions, and Gas Exposures--
Anerobic
starter cultures were grown static overnight at 37 °C in 15-ml tubes
containing 10 ml of phosphate-buffered LB medium supplemented with 20 mM glucose (7). Aerobic and microaerobic starter cultures
were grown overnight in 5 ml of phosphate-buffered LB medium in 15-ml
tubes shaking at 200 rpm at 37 °C. Chloramphenicol and ampicillin
were added as indicated at 30 and 50 µg/ml, respectively. Culture
growth was monitored by following the turbidity at 550 nm
(A550) and by plating and counting. An
A550 value of 1.0 in a 1-cm cuvette was
equivalent to 3 × 108 bacteria per milliliter for
cultures grown in phosphate-buffered LB media. Gases were mixed and
delivered to sealed 50-ml growth flasks as previously described
(20).
NO Consumption Assays--
Whole cell NO consumption rates were
measured at 37 °C with a 2-mm ISO-NOP NO electrode (World
Precision Instruments, Sarasota, FL) in the presence or absence of
O2 as previously described (12, 20).
Statistical Analysis--
Statistical significance
(p < 0.05) was determined using the Tukey Kramer
honestly significantly different method in the JMP program (SAS Institute).
NO Induces Transcription of norV and norW but Not
norR--
norV and norW transcriptional units
are arranged in a head-to-tail fashion with the start methionine of
NorW located within the coding region of norV suggesting
coordinate transcription and translation in response to NO stress (8).
In contrast, norR is divergently transcribed from
norVW (8) and is autogenously regulated (32).
Strain AG300 carrying a norV-lacZ fusion within the
norV genomic locus and lacking inducible anaerobic NOR
activity (8) was used to measure the responsiveness of norV
transcription to authentic NO. Exposure of anaerobic AG300 to 960 ppm
gaseous NO (
NO similarly induced norW-lacZ in strain AG400 under
anaerobic conditions (Table II). However,
the norW-lac fusion was induced to a 10-fold lower extent
than that observed for the norV-lac fusion following a
similar NO exposure.
The dampened response of norW-lac to NO may be explained by
the production of significant NOR activity from norV
expression within strain AG400 (8), thus resulting in a lower
steady-state NO level. It is also possible that higher steady-state NO
levels are required to activate maximal norW transcription.
Nevertheless, the results clearly demonstrate a rapid, robust, and
coordinate up-regulation of norV and norW
transcription in response to low levels of NO. The results in Table II
also demonstrate a low non-inducible level of transcription from
norR consistent with previous results obtained using
nitroprusside as the potential inducer (32).
O2 Decreases norV and norW Transcription in Response to
NO--
NO-mediated induction of norV-lac and
norW-lac fusions was significantly inhibited by
O2. Under fully aerated conditions (~200 µM
O2), induction ratios for norV-lacZ and
norW-lacZ fusions were reduced 200- and 20-fold,
respectively, with no change in the basal expression levels (Table II).
At a lower O2 concentration (~5 µM),
norV-lac and norW-lac induction ratios were
reduced 3.1- and 1.8-fold, respectively. Lower norV and
norW induction in the presence of O2 can be
explained by the decrease in cellular NO levels achieved by the
inducible NOD. Indeed, NOD expression decreased norV-lac
expression by ~96% in cells exposed to an atmosphere containing 960 ppm NO in 21% O2 for 45 min. NOD-deficient strain AG301
and control strain AG300 produced 3453 ± 493 and 149 ± 23 milliunits/mg Induction of norVW Transcription Is Dependent on
We used rpoN mutants to test the role of
NorR Activates NOR Expression in trans--
Expression of NorR
from a multicopy plasmid rescued the NO inducibility of NOR activity in
the norR deletion strain AG200 (Fig.
3A) thus confirming the
trans-acting regulatory role of NorR in the activation of
norVW transcription and NOR activity expression (8, 32). In
the absence of NO, there was no measurable NOR activity expressed (Fig.
3A, open bars) indicating that overexpression of
wild-type NorR does not by itself increase NorVW expression. However,
internal deletion of NorR, eliminating amino acids 30-164 containing
the putative signaling domain (31, 39), but retaining the entire
central
These results demonstrate the signaling function of the N-terminal
domain of the E. coli norVW transcription
regulator NorR similar to that described for other tripartite
regulators (31, 42). In addition, the results demonstrate that neither
NorR nor Our data demonstrate that the exposure of E. coli to NO
induces transcription of the norV and norW genes
via a NorR and The use of pure NO gas for the quantitative evaluation of gene
expression responses also presents challenges because of the existence
of multiple pathways for rapid and inducible NO metabolism and because
of the incipient toxicity of NO. Nevertheless, the demonstration that
the NO levels required for norV induction correspond with
levels shown to exert cellular damage strongly supports the proposed
role of the norRVW operon in NO reduction and
detoxification. Thus, 240 ppm gaseous NO ( A search of GenBankTM (NCBI) with the N-terminal 182 amino
acids of NorR identifies several NorR orthologues (Fig.
4). As in E. coli, NorR
orthologues in Salmonella typhimurium, Klebsiella pnuemoniae, Shigella flexnerei (AAN44223), and
Vibrio vulnificus CMCP6 (NP_763239) are positioned upstream
of norVW orthologues. Interestingly, NorR orthologues in the
Pseudomonas aeruginosa, Vibrio cholera,
Azetobacter vinelandii (ZP_00091183),
Burkholderia sp. strain TH2 (BAC16772), and
Burkholderia fungorum (ZP00028693) genomes are found
divergently transcribed from flavohemoglobin (hmp) genes
suggesting a potential role for NorR in regulating NODs in response to
NO. In this regard it is noteworthy that NorR was not required for the
induction of NOD activity in response to NO in E. coli (Fig.
3B), thus demonstrating the existence of one or more
separate NO-responsive regulator(s) of hmp in E. coli.
54) impaired the NO-mediated induction
of norV and norW transcription and NOR
expression, whereas disruption of the upstream regulatory gene,
norR, completely ablated NOR induction. NOR inducibility was restored to NorR null mutants by expressing NorR in
trans. Furthermore, an internal deletion of the N-terminal
domain of NorR activated NOR expression independent of NO exposure.
Neither NorR nor
54 was essential for NO-mediated
induction of the NO dioxygenase (flavohemoglobin) encoded by
hmp. However, elevated NOR activity inhibited NO
dioxygenase induction, and, in the presence of dioxygen, NO dioxygenase
inhibited norV induction by NO. The results demonstrate the
role of NorR as a
54-dependent regulator of
norVW expression. A role for the NorR N-terminal domain as
a transducer or sensor for NO is suggested.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
54-dependent
mechanism in response to exposures to sodium nitroprusside, the NO
donor compound NOC18, or during growth with nitrite or nitrate (31).
E. coli and related microbes contain norR
orthologues suggesting a global regulatory role for NorR in controlling
defenses (i.e. norVW, norBC, and
hmp) against the incipient toxicity of NO and secondarily
derived reactive nitrogen species (8, 31, 32).
54-dependent mechanism in
E. coli. We also show that a deletion within the conserved
NorR N-terminal domain activates NorVW expression independent of NO
exposure, thus demonstrating the role of the N terminus in NO sensing
and signaling. Contrary to the results obtained with nitroprusside
(32), O2 greatly diminished norV and
norW induction by NO. The results are discussed in light of the proposed NO reduction and detoxification function of the
norRVW operon within the NO defense network.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase (1190 units/ml) were
obtained from Sigma. Saturated NO stocks (2 mM) were
prepared in water as previously described (7). Compressed gas cylinders
containing 1200 ppm (±5%) of NO in ultrapure N2, 99.999%
N2, 99.993% O2, and 1.05% O2 in
ultrapure N2 were obtained from Praxair (Bethlehem, PA).
30-164, a 1.1-kb fragment encoding the C terminus was
PCR-amplified using pUC19NorR as the template and using the
oligonucleotide primers
5'-GTTGCGGATCCAACAACTGGAAAGCCAGAATATGC-3' and
5'-CATGCCTGCAGGATTTCTATCAGGCCG-3' containing
BamHI and PstI sites
(underlined), respectively. pUC19NorR was digested with Bcl1 and PstI, and the 1.1-kb PCR fragment was
subcloned. This procedure generated an in-frame fusion of NorR that
deleted amino acids 30-164 and added a glutamate residue at the
junction. A second in-frame deletion of amino acids 30-214 was created
by digesting pUC19NorR with Bcl1 and religating.
E. coli strains and plasmids used in this study
-Galactosidase Assays--
Cells were harvested by
centrifugation and washed in 100 mM sodium phosphate
buffer, pH 7.0.
-Galactosidase activity was measured according to
the method of Miller (37) with the following modifications. Frozen cell
pellets were suspended at ~1 × 1010 cells per
milliliter in 100 mM sodium phosphate buffer, pH 7.0, and
sonicated on ice. Cell-free extracts were prepared by centrifuging lysates at 12,000 × g for 5 min. Assays were incubated
for 15 min at room temperature in a 0.1-ml volume with 1-15 µg of
extract protein in a 96-well plate. Extract activities were determined using a standard curve generated with 0-3.5 milliunits of
-galactosidase. Activity is reported in milliunits per milligram of
extract protein where one milliunit cleaves 1 nmol of
o-nitrophenyl-
-D-galactopyranoside per minute
at room temperature. Background
-galactosidase activity in parental
AB1157 cells was 3.3 ± 0.6 milliunits/mg extract protein in
anaerobic cells, 3.5 ± 0.7 in low aerobic cells, and 8.3 ± 1.0 in aerobic cultures.
-Galactosidase activity remained constant in AB1157 cells irrespective of NO exposure and was subtracted from the
activities measured in lacZ fusion strains under similar growth conditions. Soluble protein was measured using Bio-Rad dye
reagent with bovine serum albumin as the standard (38).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 µM in solution) induced
-galactosidase
activity by ~50-fold within 5 min.
-Galactosidase expression
peaked after 30-45 min of exposure resulting in
1000-fold induction
(Fig. 1A). A 30-fold increase
in norV transcription was observed with 120 ppm NO (
0.25 µM in solution) (Fig. 1B). Maximal induction
of
-galactosidase activity was observed with 480 ppm NO (
1
µM in solution). Expression was blunted with 960 ppm NO
exposure suggesting toxicity of NO under these conditions.
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Fig. 1.
Expression of a norV-lacZ
fusion in anaerobic cultures exposed to NO. Strain AG300 was
exposed to 960 ppm gaseous NO for various times (A) or to
various NO concentrations for 45 min (B), and
-galactosidase activity was measured. Cultures were initiated from
static overnight cultures at an A550 = ~0.1
and were grown under an N2 atmosphere in phosphate-buffered
LB medium. At an A550 of ~0.3, cultures were
exposed to mixtures of NO in N2. Cells were harvested, and
extracts were prepared and assayed for
-galactosidase activity in
triplicate as described under "Materials and Methods." Error
bars represent the S.D. of measurements from three independent
exposures.
Expression of norV-, norW-, and norR-lacZ fusions
-galactosidase activity as described under "Materials and
Methods." Anaerobic and (micro)aerobic cultures were initiated at an
A550 = 0.1 from static and aerated overnight
cultures, respectively. Activities were determined in triplicate for
three to five independent exposures.
-galactosidase (n = 4, ±S.E.),
respectively. Thus, norV and norW are maximally
induced under conditions in which the O2-sensitive NOR
functions most effectively (8, 20), and NOD indirectly regulates NOR expression.
54--
The central domain of the tripartite NorR
protein is highly homologous with
54-dependent response regulators (31, 39)
thus suggesting an important role for
54 in the NO
response. Furthermore, the region upstream of the norVW genes contains the respective
12 and
24 elements TTGCA and TGGCA characteristic of
54-dependent promoters
(40, 41).
54 in NO-induced norV transcription and NOR
activity expression.
-Galactosidase activity was measured in AG300
and
54-deficient strain AG305 following a 45-min
exposure to 600 ppm gaseous NO under microaerobic conditions. In the
absence of
54, norV-lacZ expression was
substantially impaired (Fig.
2A). The
54-deficient strain AG500 and parental AB1157 were
similarly exposed to NO under low O2 and tested for
anaerobic NOR and aerobic NOD activity. NOR activity was significantly
reduced in strain AG500 (Fig. 2B). There was no significant
effect of
54 on NOD (hmp) expression under
these conditions (Fig. 2C). The results clearly demonstrate
a role for
54 in norV transcription. The
residual induction of norV transcription and NOR activity in
the absence of
54 suggests ancillary roles for other
factors in norV transcription or mechanisms for
post-transcriptional regulation.
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Fig. 2.
Effect of
54 on constitutive and induced
norV transcription and NO metabolism.
A,
-galactosidase activity was measured in strain AG300
and
54-deficient strain AG305 grown under a
N2 atmosphere containing 0.5% O2 balanced with
N2 in the absence (white bars) or presence of
600 ppm gaseous NO (black bars). Anaerobic (B)
and aerobic (C) NO consumption activities were measured for
the parental strain AB1157 and the
54-deficient strain
AG500 grown under an N2 atmosphere containing 0.5%
O2 (white bars) or 0.5% O2 and 600 ppm NO (black bars). Cultures were initiated at
A550 = ~0.1 from aerated overnight cultures
grown in phosphate-buffered LB medium. Cultures were grown to
A550 = ~0.3 and were either exposed to NO or
maintained under an atmosphere containing 0.5% O2 in
N2. After a 45-min exposure, cultures were shifted to a
N2 atmosphere and immediately harvested for the assay of
-galactosidase activity, or anaerobic NOR activity and aerobic NOD
activity as described under "Materials and Methods." Error
bars represent the S.D. of three independent experiments.
Asterisks indicate p < 0.05 relative to the
corresponding value for AG300 (A) or AB1157
(B).
54-interacting ATPase domain and the C-terminal
DNA binding domain, induced NOR activity in the absence of NO (Fig.
3A). Further deletion of NorR to amino acid 214, eliminating
part of the
54-interacting ATPase domain, did not induce
-galactosidase activity (data not shown) thus further delineating
the requirement for
54 interaction with NorR for
transcriptional activation. Interestingly, the NO-mediated induction of
NOD activity was significantly (p < 0.05) reduced in
strains expressing NorR and elevated NOR activity (Fig. 3B)
thus suggesting an indirect role for NorR and NOR in regulating NOD
expression by reducing NO levels.
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Fig. 3.
Effects of NorR and a NorR N-terminal domain
deletion mutant on the expression of NOR and NOD activities.
Anaerobic NOR (A) and aerobic NOD (B) activities
were measured in the norR::lac deletion
strain AG200 containing either pUC19 (control), pUC19NorR
(NorR), or pUC19NorR 30-164 (
30-164).
Cultures were either maintained under an atmosphere containing 0.5%
O2 balanced with N2 (white bars) or
exposed for 60 min to 600 ppm gaseous NO in 0.5% O2
balanced with N2 (black bars). Cultures were
initiated, grown, and harvested as described in the legend to Fig. 2,
except that ampicillin was added at 50 µg/ml. Error bars
represent the ±S.D. for three to five independent trials.
Asterisks indicate p < 0.05 relative to the
value of the corresponding control.
54 is directly involved in the NO-mediated
up-regulation of the E. coli NOD (hmp).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
54-dependent mechanism. The
data extend the results of Hutchings et al. (32)
demonstrating activation of norV transcription by the
NO+ donor nitroprusside, nitrite, or nitrate in a
NorR-dependent fashion. Given the relatively low
concentration of NO required for anaerobic norV induction
(Fig. 1B), NO is the most probable physiological signal
modulating NorR and norVW transcription. Our results differ
from those of Hutchings et al. (32) who reported that
constitutive and induced norV transcription was greater in the presence of O2. One likely explanation for the
discrepancy is that we used NO gas and Hutchings et al. (32)
used nitroprusside as a NO+ donor and potential
NO-generating agent. Nitroprusside may have deleterious effects on
transcription or, alternatively, may generate NO at higher levels in
aerobic cells. Pure NO gas is readily available and is clearly
preferred for investigations of the effects of NO on NO defense gene regulation.
0.5 µM in
solution) inactivated E. coli aconitase and
6-phosphogluconate dehydratase and inhibited growth in the absence of
the induced NorVW activity (6, 8). Furthermore, the level of NO
inducing half-maximal norV transcription (
0.7
µM) approximates the apparent Km (NO)
value of ~0.4 µM determined for NorVW-catalyzed NO
reduction (8). These results diminish the likelihood of a significant
function of the operon in O2 detoxification or in the
detoxification of unspecified reactive nitrogen intermediates generated
from nitroprusside or NO exposure as previously suggested (25, 32).
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Fig. 4.
Conservation within the N-terminal domain of
NorR orthologues. An alignment of NorR orthologues was performed
using the ClustalW program (MacVector 7.0). Dark shading,
identical amino acids; light shading, similar amino acids;
double dots, close similarities with a threshold comparison
value of 0.50. GenBankTM accession numbers are as
follows: E. coli, NP_417189 using the second start
methionine for a 504-amino acid protein; S. typhimurium,
NP_461760; K. pneumoniae, contig 661 with three base pairs
added to maintain reading frame (available at genome.wustl.edu);
R. eutropha NorR, CAC00710; R. eutropha NorR2,
CAC00712; P. aeruginosa, NP_251355; V. cholera,
NP_232582. Asterisks mark conserved aspartate residues in
position for potential phospho-acceptance. The number symbol
identifies the putative kinase-stimulating acidic site. Solid
dots indicate potential heme iron ligands.
NorR belongs to the family of two-component response regulators (42). Similar to other tripartite regulators in this family, deletion of the N-terminal signaling or inhibitory domain of NorR activated NorVW expression independent of NO (Fig. 3A). Furthermore, conserved aspartate residues in the NorR N-terminal signaling domain suggest the potential for phosphorylation by a sensor histidine-kinase similar to that described for the NtrB/NtrC pair (43). In particular, aspartates 57 and 62 are in position to accept phosphate, and the conserved acidic residue at position 14 may serve to optimize phosphorylation (Fig. 4) (44). Alternatively, the NorR N-terminal domain could activate transcription by interacting with a signal transducing protein as described for NifL/NifA (45) or by binding NO directly as the formate-sensing transcription regulator FhlA binds formate (46). The NorR N-terminal domain contains potential metal-liganding histidine and cysteine residues that could form the NO sensor module. For example, NorR contains an His111-X-Cys113 site reminiscent of the Cys75-X-His77 heme iron ligand-switch motif in the carbon monoxide-sensing CooA of Rhodospirillum rubrum (47).
Fig. 5 summarizes our current view of the
NO defense network in E. coli. NO exposure elicits the
synthesis of two major NO-metabolizing enzymes, NOD and NOR (NorVW) by
activating transcription of their corresponding genes, hmp
and norVW. The respective contribution of each enzyme to NO
detoxification depends primarily on the availability of O2.
NOD is effective under aerobic and microaerobic conditions (Km (O2) = 60-100
µM) (13, 14). The NOR activity of NorVW is unique in that
its exquisite sensitivity to O2 restricts its NO scavenging
function to anaerobic or microaerobic conditions (8, 20). The results
also support a model in which norVW and hmp
transcription are indirectly influenced by O2 availability, because O2 levels affect NorVW and NOD activities, which
ultimately determine NO steady-state levels and the activity of
transcription regulators such as NorR and Fnr (29, 48).
|
Interestingly, neither SoxRS nor OxyR, which have been persistently
proposed to be critical NO stress response sensor-regulators (49, 50)
appear to be involved in the regulation of either hmp or
norVW in E. coli (Fig. 5)
(48).2 Future investigations
will aim to further clarify the diverse roles and mechanisms of NO
defense genes, enzymes, and regulators in microbial adaptations to NO
in vitro and in various models of infection.
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ACKNOWLEDGEMENTS |
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We thank Drs. Alex Ninfa and Kenn Rudd for supplying strains and phage used in these investigations.
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FOOTNOTES |
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* This work was supported by a grant from the Children's Hospital Research Foundation Trustees and Public Health Services Grant GM-65090 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: ML 7006, Children's
Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-8712; Fax: 513-636-4892; E-mail:
Anne.Gardner@cchmc.org.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212462200
2 A. M. Gardner and P. R. Gardner, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; NOR, NO reductase; NOD, NO dioxygenase; LB, Luria-Bertani; Cmr, chloramphenicol resistance, Apr, ampicillin resistance; Tcr, tetracycline resistance; Knr, kanamycin resistance.
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