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Infection and Immunity, September 2000, p. 5241-5246, Vol. 68, No. 9
Department of Microbiology and Immunology,
School of Medicine and Dentistry, University of Rochester,
Rochester, New York 14642
Received 16 March 2000/Returned for modification 10 May
2000/Accepted 13 June 2000
The gene encoding a nitric oxide reductase has been identified in
Neisseria gonorrhoeae. The norB gene product
shares significant identity with the nitric oxide reductases in
Ralstonia eutropha and Synechocystis sp. and,
like those organisms, the gonococcus lacks a norC homolog.
The gonococcal norB gene was found to be required for
anaerobic growth, but the absence of norB did not dramatically decrease anaerobic survival. In a wild-type background, induction of norB expression was seen anaerobically in the
presence of nitrite but not anaerobically without nitrite or
aerobically. norB expression is not regulated by FNR or
NarP, but a functional aniA gene (which encodes an
anaerobically induced outer membrane nitrite reductase) is necessary
for expression. When aniA is constitutively expressed,
norB expression can be induced both anaerobically and aerobically, but only in the presence of nitrite, suggesting that nitric oxide, which is likely to be produced by AniA as a product of
nitrite reduction, is the inducing agent. This was confirmed with the
use of the nitric oxide donor, spermine-nitric oxide complex, in an
aniA null background both anaerobically and aerobically. NorB is important for gonococcal adaptation to an anaerobic
environment, a physiologically relevant state during gonococcal
infection. The presence of this enzyme, which is induced by nitric
oxide, may also have implications in immune evasion and
immunomodulation in the human host.
Denitrification is the process
whereby microorganisms form dinitrogen (N2) from nitrate
(NO3 Neisseria gonorrhoeae, the etiologic agent of the sexually
transmitted disease gonorrhea, is now known to be a facultative anaerobe (17) which possesses a copper-containing nitrite
reductase, AniA, in its outer membrane (5). aniA
is very tightly regulated by oxygen and is only expressed under
anaerobic or microaerobic conditions (13, 15). Antibodies to
AniA have been found in sera from patients with gonococcal disease,
indicating that AniA is expressed within the host and that anaerobiosis
is a physiologically relevant environment in gonococcal infections
(7). We were interested in denitrifying enzymes because
copper-containing nitrite reductases are found in some of the
dentrifying bacteria such as Pseudomonas aureofaciens and
Rhodobacter sphaeroides, and these enzymes reduce
NO2 NO is known to be toxic to bacterial cells, often through its reaction
with dioxygen, superoxide, or the metal centers in various enzymes.
Bacteria which produce NO therefore also possess an NO reductase to
eliminate the molecule. This two-subunit enzyme reduces NO to
N2O and is encoded by the norC and
norB genes (33).
Since it is likely that AniA produces NO and since it has been reported
that gonococci produce nitrous oxide as a result of nitrite reduction
(19), it is logical that the gonococcus would also possess
an NO reductase. It is known that murine macrophages produce NO, and
there is evidence that this is also true in human macrophages (reviewed
in references 11 and 20).
Therefore, an NO assault could be part of the innate immune system
response to a bacterial invader. It was of interest to us to determine if the gonococcus, a strictly human pathogen, possesses this
denitrifying enzyme, which could also have a role in the organism's
survival in the face of an immune response. This study describes the
identification and the basic regulation of the gonococcal
norB gene and discusses the implications of the presence of
the NorB enzyme in the pathogenesis of the organism.
Growth of gonococcal strains.
All gonococcal strains (Table
1) were derived from strain F62 and were
grown on GC medium base (Difco Laboratories, Detroit, Mich.) plates
with 1% Kellogg's supplement (GCK) (16). When necessary,
chloramphenicol or erythromycin was added at 1 or 2 µg
ml
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Gonococcal Nitric Oxide Reductase Is Encoded by a
Single Gene, norB, Which Is Required for Anaerobic
Growth and Is Induced by Nitric Oxide
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) via nitrite
(NO2), nitric oxide (NO), and nitrous oxide
(N2O) intermediates. Denitrifiers possess separate
reductases which catalyze each intermediate step. Denitrification is
usually seen in facultative anaerobes or microaerophilic organisms, but
aerobic denitrification has been documented. The process and regulation
of denitrification has been extensively studied in pseudomonads,
Paracoccus spp., and Rhodobacter spp.; however,
many other organisms, including fungi, denitrify. (See the recent
comprehensive review by Zumft [33] for details of this
process and the properties of its enzymes.)
to NO (33).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1, respectively. Aerobic plate cultures were grown at
37°C in a 5% CO2 incubator. Aerobic broth cultures were
grown in GCK broth plus 0.042% (wt/vol) NaHCO3 in baffled
flasks which were incubated with shaking at 240 rpm in a Gyrotory water
bath shaker (New Brunswick Scientific Co., Edison, N.J.) at 37°C.
Anaerobic cultures were incubated in a Coy anaerobic chamber (Coy
Laboratory Products, Grass Lake, Mich.) at 37°C for 20 h.
Nitrite was provided for anaerobically grown cultures by placing 40 µl of a 20% (wt/vol) NaNO2 solution on a sterile
cellulose disk in the center of the plate. Cultures provided with
nitrite grow in a characteristic halo around the nitrite disk, while
cultures without nitrite remain viable but do not grow (24).
TABLE 1.
Gonococcal strains used in this study
Gonococcal transformation. Type 1 gonococcal cells were transformed as previously described (15) and plated on GCK plates containing the appropriate antibiotic.
Construction of norB'-'lacZ fusion.
The region
upstream of the norB open reading frame (ORF) was amplified
by PCR, using primers which created BamHI sites on both
ends. The amplified fragment contained 177 bp upstream of the putative
start codon and the first three codons of the ORF. This DNA fragment
was digested with BamHI and ligated to pLES94 (25), which had also been digested with BamHI, to
create a translational lacZ fusion. This ligation mixture
was used to transform Escherichia coli MC1061. Transformants
were selected by plating on Luria-Bertani medium plates containing
ampicillin at 100 µg ml1 and
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal) at 40 µg ml1. After overnight incubation at 37°C,
blue colonies were screened for plasmids containing the insert in the
correct orientation. When an appropriate plasmid was identified (pEF1),
it was used to transform gonococcal strain F62. Transformants were
plated on GCK plates containing chloramphenicol. Chromosomal DNA was isolated from chloramphenicol-resistant colonies (15), and
PCR was used to confirm the presence of the reporter construct. The resulting PCR product was sequenced to confirm the desired nucleotide sequence. The resulting strain was designated RUG7500.
Construction of a norB null mutant. norB was amplified in two fragments by PCR with primers which created a ClaI site within the coding region. The two DNA fragments were purified and digested with ClaI. These digested fragments were ligated to an erythromycin resistance cassette (ermC) which had been amplified from pHSS23 (30) and digested with HpaII. This ligation mixture was transformed into N. gonorrhoeae RUG7001. This resulted in interruption of the norB gene with the erythromycin resistance cassette and the creation of strain RUG7058. The interruption of norB was confirmed by PCR.
The norB null mutant was also combined with an aniA null mutation. The insertionally inactivated norB was amplified by PCR from strain RUG7058. The resulting PCR product was used to transform RUG7002 which contains a 493-bp deletion in the aniA gene and the aniA'-'lacZ reporter construct. This norB aniA double mutant was designated RUG7060. Both mutations were confirmed by PCR.Construction of the aniA null and constitutive strains. To combine the norB'-'lacZ reporter with an aniA null background, the aniA gene insertionally inactivated with the ermC gene from strain RUG7011 (15) was amplified by PCR. This PCR product was used to transform RUG7500, which created strain RUG 7501. The presence of both the reporter construct and the ermC cassette was confirmed by PCR. To create the combination with the aniA constitutive mutant, pEF1 (norB'-'lacZ) was used to transform the F62 strain containing the previously described constitutive aniA (15), creating strain RUG7502. PCR was used to confirm the presence of both the reporter construct and the constitutive construct.
Construction of the narP null strain. The norB'-'lacZ reporter was combined with a narP (encodes the response regulator of a two component system [15, 19]) null background by amplifying the insertionally inactivated narP from strain RUG7036 (15) by PCR. This PCR product was used to transform RUG7500, creating RUG7503. The presence of both the reporter construct and the ermC gene within narP was confirmed by PCR.
Anaerobic survival plate counts. Nonpiliated colonies of desired strains were suspended in GCK broth at an optical density at 600 nm of approximately 0.1. Suspensions were serially diluted, and the appropriate dilutions were plated onto GCK plates in duplicate. Each strain was plated onto GCK with or without 2 mM NaNO2 and incubated both aerobically and anaerobically. Aerobic cultures were incubated for 48 h before enumeration. Anaerobic cultures were incubated for 24 h anaerobically and then 48 h aerobically to allow for visible colony formation. The number of CFU from the anaerobic cultures was compared to the CFU counts in the corresponding aerobic culture. The results are reported as the percentage of aerobic CFU and are the means of three independent assays for each strain.
Induction of norB by NO.
For each plate tested,
10 mg of the NO generator, spermine-nitric oxide complex (SPER-NO;
Sigma RBI, St. Louis, Mo.) was dissolved in 50 µl of 0.1 N NaOH and
used to saturate a cellulose disk. This disk was placed in the center
of a plate which had been inoculated with a heavy suspension of
organism, and the plate was incubated for 20 h either in the
CO2 incubator or the anaerobic chamber. Cells were
harvested with a sterile swab from around the disk and tested for
-galactosidase (-Gal) activity. Disks saturated with 50 µl of
0.1 N NaOH were used for the no SPER-NO controls.
-Gal assays.
Gonococcal cultures were assayed for -Gal
by the method of Miller (22) as previously described
(15). Activity is reported in Miller units. The results
reported are the averages of at least three independent assays for each strain.
Oligonucleotides and DNA sequencing. All oligonucleotide synthesis and DNA sequencing was performed by the University of Rochester Core Nucleic Acid Laboratory.
Molecular biology techniques. General techniques were performed according to standard protocols (2, 23). Plasmid preparations were obtained with the Wizard Plus Minipreps kit (Promega Corp., Madison, Wis.) or the QIAprep Spin Miniprep kit (Qiagen, Inc., Valencia, Calif.). DNA fragments were purified with Wizard PCR Preps kit (Promega), or with GENECLEAN Spin or MERmaid Spin kits (Bio 101, Vista, Calif.).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of the norB gene in N. gonorrhoeae. The work in our laboratory has focused on the aniA gene and its gene product in N. gonorrhoeae F62. Since AniA is a copper-containing nitrite reductase (5), it is likely that the product of nitrite reduction is NO. The next enzyme in the denitrifying pathway is the NO reductase (i.e., Nor). The sequence of what appears to be the entire coding region of a norB homolog was recently completed in the gonococcal genome (Gonococcal Genome Sequencing Project [http://dna1.chem.ou.edu/gono.html]). Surprisingly, N. gonorrhoeae does not encode the expected NorCB NO reductase which can be found in denitrifying organisms such as Pseudomonas aeruginosa (1), Pseudomonas stutzeri (4, 34), Paracoccus denitrificans (9), R. sphaeroides (3), and others. Instead, the gonococcus seems to encode a single subunit NO reductase, consisting only of NorB.
The predicted NorB protein has a calculated molecular mass of 84.3 kDa and an amino-terminal extension of approximately 260 to 270 amino acids. A BLAST search revealed significant identity to the Ralstonia eutropha (Alcaligenes eutrophus) and Synechocystis NO reductases along the entire length of the protein (61 and 42%, respectively) (Fig. 1). As in the gonococcus, the R. eutropha norB and norZ (genes encoding two isofunctional NO reductases) and Synechocystis norB predicted gene products have amino-terminal extensions, and the organisms lack norC genes (8). Like the R. eutropha NorB, the gonococcal NorB contains 14 potential transmembrane domains (data not shown) and all six conserved histidine residues (Fig. 1) and lacks a heme c binding motif (C-X-X-C-H).
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11 from the putative
translational start, and a potential 
70 10 sequence,
TACAAT, at position 61 (Fig.
2B). In contrast to other NO reductase
genes, we did not find a complete FNR binding site, only a potential
half site, TTGAA, at position 96.
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aniA is located upstream of norB. After locating the norB gene in the gonococcal genome (contig 17 on 03/03/00), we investigated the other ORFs surrounding norB. Considering our work with aniA, it was interesting to find aniA positioned about 375 bp upstream of norB, transcribed in the opposite direction (Fig. 2). This is similar to other organisms in which the nor genes are found in proximity to nitrite reductase (nir) genes. The two ORFs immediately downstream of norB are not homologous to other nir or nor genes.
norB is required for the anaerobic growth, but not the survival, of N. gonorrhoeae. It has been reported previously that aniA is required for anaerobic growth in the gonococcus (15, 21). We were interested in determining if norB was also required. The norB gene was insertionally inactivated with the ermC gene. The resulting strain, RUG7058, was not attenuated in aerobic growth either in shaken broth cultures or on plates (data not shown) but, like the aniA mutant, was unable to grow anaerobically. However, RUG7058 survived anaerobic incubation and grew into visible colonies when transferred to a CO2 incubator (data not shown).
To quantify anaerobic survival, strains RUG7001 (wild type) and RUG7058 (norB) were plated and counted as described in Materials and Methods. Plate counts from the anaerobic plates were compared to the plate counts of the corresponding aerobic cultures. As seen in Table 2, in the absence of nitrite, only approximately 20% of both RUG7001 and RUG7058 (norB) survived anaerobic incubation. In contrast, in the presence of nitrite, virtually 100% of RUG7001 survived, while about 65% of RUG7058 (norB) survived. The morphology and size of the colonies of the two anaerobically incubated strains were identical when the cultures were subsequently incubated aerobically. Interestingly, this was not the case with the cultures that were grown aerobically. In the absence of nitrite, both strains grew with normal colony morphology and to normal size. In the presence of nitrite, however, the norB mutant exhibited a small colony phenotype (0.5 versus 3 mm) only when grown aerobically, while the colonies of the wild-type strain were normal. RUG7058 (norB) plates counts did not differ in the presence or absence of nitrite (data not shown), but the colony morphology was distinct and consistent.
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norB is induced anaerobically in the presence of
nitrite.
-Gal assays were performed on RUG7500
(norB'-'lacZ) grown both aerobically and anaerobically
measuring norB expression (Table 3). In highly aerated broth cultures, the
level of norB expression was about 20 Miller units both with
and without 2 mM NO2. In contrast, when
RUG7500 was incubated anaerobically in the presence of nitrite,
expression increased to about 1800 Miller units, while levels in
cultures without nitrite remained low at 34 Miller units. In all cases
the promoterless lacZ control (RUG7021) expression was
virtually nil.
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-Gal activity
when nitrite was added to the cultures, while norB remained
at a basal level of expression during anaerobiosis with significant
induction seen only in the presence of nitrite (Table 3). This suggests
a different method of regulation.
norB is not regulated by NarP.
Since induction of
norB was seen in the presence of nitrite, we investigated
whether norB is activated by NarP. The narP gene was insertionally inactivated in the norB'-'lacZ reporter
strain. The -Gal activity obtained from this strain, RUG7503, was
not greatly decreased compared to the wild-type background (Table 3),
indicating that NarP plays no role in norB regulation.
AniA is required for the induction of norB.
To determine
the effects of an aniA null mutation on norB
expression, we created strain RUG7501, as described in Materials and
Methods. Interestingly, when aniA was inactivated, we no
longer observed the induction of norB in the presence of
nitrite. Levels of -Gal activity were 42 and 29 Miller units with
and without NO2, respectively (Table 3).
There was no effect aerobically. Since it is likely that AniA produces
NO as a product of NO2 reduction, these
results suggest that NO may be the inducer for norB.
Induction of norB is seen aerobically when AniA is
constitutively expressed.
When gonococcal cultures are grown in a
highly aerated culture, no induction of the aniA gene is
detectable due to the requirement for FNR (15). To produce
AniA, and thus NO, aerobically and to determine the effects on
norB expression, we utilized the previously described
aniA constitutive mutant in which aniA is under
the control of the tac promoter (15). RUG7502
contains the norB'-'lacZ fusion in an aniA
constitutive background. Anaerobically, RUG7502 produced over 1,000 Miller units of -Gal activity in the presence of nitrite. In highly
aerated cultures, norB induction was seen at levels of over
150 Miller units only in the presence of nitrite, while levels in
cultures without nitrite remained low. This suggested that AniA was
indeed producing NO as a result of nitrite reduction, which in turn
induced norB.
An NO generator induces norB expression in the absence
of AniA.
To confirm that NO was responsible for norB
induction, we utilized the NO generator SPER-NO. To eliminate the
potential role of AniA in norB induction, we tested the NO
generator on strain RUG7501, in which aniA is insertionally
inactivated. For each plate tested, 10 mg of SPER-NO was solubilized in
0.1 N NaOH and was used to saturate a paper disk, a technique similar
to the method used for adding nitrite to gonococcal plated cultures. While the actual levels of activity in the anaerobic cultures were
somewhat variable, we consistently obtained significant induction of
norB in the presence of SPER-NO both aerobically and
anaerobically. Aerobic -Gal levels (Miller units ± the
standard error of the mean [SEM]) were 464 ± 91 in the presence
of SPER-NO and 24 ± 3.3 in the absence of SPER-NO. Anaerobic
levels were 884 ± 199 in the presence of SPER-NO and 28 ± 3.7 in the absence of SPER-NO. This provided convincing data that the
gonococcal norB gene is induced by NO.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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As a copper-containing nitrite reductase, it is highly probable that AniA produces NO as a result of its nitrite reduction, and it has been demonstrated that gonococci produce nitrous oxide as the product of nitrite reduction (19). We therefore set out to identify the NO reductase gene and to investigate the role of this enzyme in the anaerobic growth of the organism. It is also intriguing to speculate about the potential role of this enzyme in host immune evasion by the gonococcus.
The gene for the gonococcal NO reductase was identified by BLAST analysis of the gonococcal genome project. Surprisingly, we found only a norB homolog, while most denitrifiers possess a two-subunit enzyme encoded by norC and norB. The gonococcal NorB has significant identity (61%) to R. eutropha NorB and NorZ along the entire length of the predicted protein. In contrast, when gonococcal NorB is compared to the NorB found in P. denitrificans, for example, there is only 28% identity, and the gonococcal NorB has an amino-terminal extension of 271 amino acids (data not shown). Cramm et al. speculated that the amino-terminal extension may serve the same role as the NorC subunit and proposed further studies to explore this possibility (8).
When the norB gene is inactivated in N. gonorrhoeae, the organism can no longer grow anaerobically. However, a norB mutant survives anaerobic incubation in the presence of nitrite better than an aniA mutant (Table 2). This indicates that NO is not extremely toxic to the gonococcus under anaerobic conditions. In other organisms the inability to grow anaerobically in the absence of an NO reductase was attributed to the toxic effects of NO accumulation (4, 18). This does not seem to be the case in N. gonorrhoeae under these culture conditions, as shown by the anaerobic survival data.
Our studies on AniA indicate that it is anchored in the outer membrane of the gonococcus (5, 6, 14). This external location makes it unlikely that AniA is linked to anaerobic respiration and energy production. However, the gonococcal norB gene product appears to have multiple transmembrane domains indicative of a protein integral to the cytoplasmic membrane. From this location in the cytoplasmic membrane, it is quite feasible that NorB is involved in anaerobic respiration. Thus, the lack of NorB would result in the inability to respire under anaerobic conditions, preventing the growth of the organism until it is switched to environmental conditions which are permissive for aerobic respiration.
Our initial experiments looking at norB regulation revealed significant induction of the gene in the presence of nitrite but none due to anaerobiosis alone (Table 3). This is in contrast to aniA, which is induced anaerobically by FNR. Our previous work indicated that aniA was the only gene induced by FNR that was required for anaerobic growth (15). It was not surprising, therefore, that we could not locate an FNR binding site upstream of norB and that norB was not induced due to anaerobiosis alone. The data indicating that norB expression can be induced aerobically also rule out involvement of FNR. Since aniA requires NarP for the induction due to nitrite (15), we investigated whether or not norB also required NarP. Insertionally inactivating narP (RUG7503) had only a small effect on norB expression (Table 3). This decrease from 1,800 to 1,100 U is most likely due to the decrease in aniA expression due to the lack of NarP (15), resulting in diminished NO production and norB induction.
The results obtained with the aniA null and constitutive
strains (Table 3) suggested that norB was induced by NO.
Confirmation of this was achieved by providing exogenous NO through the
use of a NO generator in the absence of AniA. When SPER-NO was added to
either aerobically or anaerobically grown plated cultures of RUG7501,
significant induction of norB was observed (see Results) compared to the low -Gal activity observed both without an inducer and in the presence of nitrite (Table 3). This observation is not
without precedence, since the NO reductases from both R. sphaeroides and P. denitrificans have been shown to be
induced by NO (18, 27, 29).
The question that remains is which activating protein is responding to NO and inducing the gonococcal norB? The R. sphaeroides and P. denitrificans NO reductases are activated by members of the FNR family of proteins (NnrR [27] and NNR [28], respectively). These proteins bind to sequences which are very similar to the FNR consensus in E. coli (summarized in reference 33). In N. gonorrhoeae, it seems unlikely that a member of the FNR family is involved in norB induction since we are unable to locate a full FNR binding site upstream of norB, although we cannot rule out a different binding consensus in the gonococcus. We have also searched the gonococcal genome with the protein sequence of several members of the FNR family of proteins and have only found the previously reported FNR (15, 19). Two other well-characterized NO-responsive regulators are SoxR and SoxS in E. coli (reviewed in reference 31). Again, BLAST analysis of the gonococcal genome found no homologs to these regulators. Further experimentation will be required, including pinpointing a binding site upstream of norB, in order to identify the norB activator.
We are intrigued by the possibility that the gonococcal NorB may function in immune evasion. Flavohemoglobin (Hmp), SoxRS, Cu,Zn-superoxide dismutase, and homocysteine have all been found to provide some pathogenic bacteria protection against NO (reviewed in reference 31). Specifically, E. coli soxRS mutants are more sensitive to macrophage killing, and the Salmonella enterica serovar Typhimurium Cu,Zn-superoxide dismutase protects against phagocyte-derived NO. In addition to SoxRS, we have also been unable to locate an Hmp homolog in the gonococcal genome, and although there is a partial coding region in GenBank for a gonococcal sod gene (accession no. U11277), the sod ORF identified in the genome appears to contain a frameshift, and no one has reported a functional superoxide dismutase in N. gonorrhoeae. In the absence of any of these known proteins involved in the defense against NO, it is possible that the gonococcal NorB is the means by which the organism protects itself against NO assault from macrophages.
It is estimated that approximately 50% of all primary gonococcal infections in women are asymptomatic, which leads to higher rates of complicated gonococcal disease, such as pelvic inflammatory disease and disseminated gonococcal infection (10). The effects of NO on the inflammatory response are dependent on the concentration. Low levels of NO appear to be anti-inflammatory, while higher levels such as those produced by iNOS (NOS-2) in response to bacterial lipopolysaccharide, can be proinflammatory (reviewed in reference 12). Gonococci, with the ability to both produce and reduce NO, may exert a modulatory effect on NO levels and thus on the host inflammatory response. This may result in the development of symptomatic or asymptomatic infections, depending on the relative activities of gonococcal nitrite reductase and NO reductase.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant RO1 AI11709 from the National Institutes of Health to V.L.C. T.C.H. was supported by National Institutes of Health training grant T32 AI07363.
We thank Lin Silver and Lori Wright for their expert technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642. Phone: (716) 275-3154. Fax: (716) 473-9573. E-mail: Ginny_Clark{at}urmc.rochester.edu.
Present address: Department of Biological Sciences, University of
Wisconsin-Milwaukee, Milwaukee, WI 53201.
Editor: D. L. Burns
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Arai, H., Y. Igarashi, and T. Kodama. 1995. The structural genes for nitric oxide reductase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 1261:279-284[Medline]. |
| 2. | Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1987. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y. |
| 3. |
Bartnikas, T. B.,
I. E. Tosques,
W. P. Laratta,
J. Shi, and J. P. Shapleigh.
1997.
Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3.
J. Bacteriol.
179:3534-3540 |
| 4. |
Braun, C., and W. G. Zumft.
1992.
The structural genes of the nitric oxide reductase complex from Pseudomonas stutzeri are part of a 30-kilobase gene cluster for dentrification.
J. Bacteriol.
174:2394-2397 |
| 5. | Cardinale, J. A. 1999. Ph.D. thesis. University of Rochester, Rochester, N.Y. |
| 6. |
Clark, V. L.,
L. A. Campbell,
D. A. Palermo,
T. M. Evans, and K. W. Klimpel.
1987.
Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae.
Infect. Immun.
55:1359-1364 |
| 7. | Clark, V. L., J. S. Knapp, S. Thompson, and K. W. Klimpel. 1988. Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb. Pathog. 5:381-390[CrossRef][Medline]. |
| 8. |
Cramm, R.,
R. A. Siddiqui, and B. Friedrich.
1997.
Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.
J. Bacteriol.
179:6769-6777 |
| 9. | de Boer, A. P. N., J. van der Oost, W. N. M. Reijnders, H. V. Westerhoff, A. H. Stouthamer, and R. J. M. van Spanning. 1996. Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans. Eur. J. Biochem. 242:592-600[Medline]. |
| 10. | Division of STD Prevention. 1999. Sexually transmitted disease surveillance, 1998. Department of Health and Human Services, Atlanta. Centers for Disease Control and Prevention, Atlanta, Ga. |
| 11. | Fang, F. C. 1997. Mechanisms of nitric oxide-related antimicrobial activity. J. Clin. Investig. 99:2818-2825[Medline]. |
| 12. |
Grisham, M. B.,
D. Jourd'heuil, and D. A. Wink.
1999.
Nitric oxide I. Physiological chemistry of nitric oxide and its metabolites: implications in inflammation.
Am. J. Physiol.
276:G315-G321 |
| 13. |
Hoehn, G. T., and V. L. Clark.
1992.
Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae.
Infect. Immun.
60:4695-4703 |
| 14. |
Hoehn, G. T., and V. L. Clark.
1992.
The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan 1, is a lipoprotein.
Infect. Immun.
60:4704-4708 |
| 15. |
Householder, T. C.,
W. A. Belli,
S. Lissenden,
J. A. Cole, and V. L. Clark.
1999.
cis- and trans-acting elements involved in regulation of aniA, the gene encoding the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae.
J. Bacteriol.
181:541-551 |
| 16. |
Kellogg, D. S., Jr.,
W. L. Peacock, Jr.,
W. E. Deacon,
L. Brown, and C. I. Pirkle.
1963.
Neisseria gonorrhoeae I. Virulence genetically linked to clonal variation.
J. Bacteriol.
85:1274-1279 |
| 17. |
Knapp, J. S., and V. L. Clark.
1984.
Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction.
Infect. Immun.
46:176-181 |
| 18. |
Kwiatkowski, A. V., and J. P. Shapleigh.
1996.
Requirement of nitric oxide for induction of genes whose products are involved in nitric oxide metabolism in Rhodobacter spheroides 2.4.3.
J. Biol. Chem.
271:24382-24388 |
| 19. | Lissenden, S., S. Mohan, T. Overton, T. Regan, H. Crooke, J. A. Cardinale, T. C. Householder, D. O'Conner, V. L. Clark, H. Smith, and J. A. Cole. Identification of transcription activators which regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. Mol. Microbiol., in press. |
| 20. | MacMicking, J., Q.-W. Xie, and C. Nathan. 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323-350[CrossRef][Medline]. |
| 21. | Mellies, J., J. Jose, and T. F. Meyer. 1997. The Neisseria gonorrhoeae gene aniA encodes an inducible nitrite reductase. Mol. Gen. Genet. 256:525-532[CrossRef][Medline]. |
| 22. |
Miller, J. H.
1972.
Assay of -galactosidase, p. 352-355.
In
Experiments in molecular genetics Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 23. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 24. |
Short, H. B.,
V. L. Clark,
D. S. Kellogg, Jr., and F. E. Young.
1982.
Anaerobic survival of clinical isolates and laboratory strains of Neisseria gonorrhoeae: use in transfer and storage.
J. Clin. Microbiol.
15:915-919 |
| 25. | Silver, L. E., and V. L. Clark. 1995. Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae. Gene 166:101-104[CrossRef][Medline]. |
| 26. | Squadrito, G. L., and W. A. Pryor. 1998. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic. Biol. Med. 25:392-403[CrossRef][Medline]. |
| 27. |
Tosques, I. E.,
J. Shi, and J. P. Shapleigh.
1996.
Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3.
J. Bacteriol.
178:4958-4964 |
| 28. | Van Spanning, R. J. M., A. P. N. De Boer, W. N. M. Reijnders, S. Spiro, H. V. Westerhoff, A. H. Stouthamer, and J. Van der Oost. 1995. Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators. FEBS Lett. 360:151-154[CrossRef][Medline]. |
| 29. |
Van Spanning, R. J. M.,
E. Houben,
W. N. M. Reijnders,
S. Spiro,
H. V. Westerhoff, and N. Saunders.
1999.
Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans.
J. Bacteriol.
181:4129-4132 |
| 30. | Wainwright, L. A., K. H. Pritchard, and H. S. Seifert. 1994. A conserved DNA sequence is required for efficient gonococcal pilin antigenic variation. Mol. Microbiol. 13:75-87[CrossRef][Medline]. |
| 31. | Watmough, N. J., G. Butland, M. R. Cheesman, J. W. B. Moir, D. J. Richardson, and S. Spiro. 1999. Nitric oxide in bacteria: synthesis and consumption. Biochim. Biophys. Acta 1411:456-474[Medline]. |
| 32. | Wink, D. A., and J. B. Mitchell. 1998. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 25:434-456[CrossRef][Medline]. |
| 33. | Zumft, W. G. 1997. Cell biology and molecular basis of dentrification. Microbiol. Mol. Biol. Rev. 61:533-616[Abstract]. |
| 34. | Zumft, W. G., C. Braun, and H. Cuypers. 1994. Nitric oxide reductase from Pseudomonas stutzeri. Eur. J. Biochem. 219:481-490[Medline]. |
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