| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biology (Area 10), University of York, Heslington, York YO10 5YW, UK
2 Department of Microbiology, Faculty of Medicine, Chiangmai University, Chiangmai 50200, Thailand
3 Department of Molecular Biosciences, Centre for Molecular Biology and Neuroscience, University of Oslo, 0316 Oslo, Norway
Correspondence
James W. B. Moir
jm46{at}york.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The nasopharyngeal mucosa is habitat to a wide variety of other bacteria, including both aerobes and anaerobes (Brook, 2003). In previous work, we have determined that under aerobic conditions N. meningitidis is able to support respiration by reducing oxygen to water via the enzyme cytochrome cbb3 oxidase (which is the only oxygen reductase in N. meningitidis), and that when oxygen becomes limiting the bacterium expresses genes encoding a nitrite reductase (aniA) and a nitric oxide reductase (norB) that collectively catalyse the respiratory reduction of nitrite to nitrous oxide (Anjum et al., 2002). N. meningitidis is able to employ this partial denitrification pathway to support growth under microaerobic conditions in the presence of nitrite (Rock et al., 2005).
We have proposed an organization for the respiratory chain of N. meningitidis, based on genome sequence analysis and experimental analysis with specific inhibitors (Deeudom et al., 2006). The nitric oxide reductase of N. meningitidis appears more similar to the quinol-oxidizing nitric oxide reductase (qNOR) than the cytochrome c-oxidizing NOR (cNOR) (de Vries & Schröder, 2002), and hence it was proposed that the nitric oxide reductase receives its electrons directly from the ubiquinone pool. This was confirmed experimentally by showing that nitric oxide reduction is insensitive to the cytochrome bc1 complex inhibitor myxothiazol. Contrastingly, the reduction of the other two electron acceptors (oxygen and nitrite) is very sensitive to myxothiazol. The enzymes responsible for these reductase reactions typically receive their electrons from c-type cytochromes in other micro-organisms, and hence it was proposed that these two enzymes terminate the electron-transport chain downstream of the cytochrome bc1 complex and c-type cytochromes (Deeudom et al., 2006). The genome of N. meningitidis MC58 reveals the presence of genes encoding three putative c-type cytochromes that might mediate the transfer of electrons between the cytochrome bc1 complex and the reductases for oxygen and nitrite. These three c-type cytochromes are conserved within the genomes of other N. meningitidis strains and in Neisseria gonorrhoeae.
Gene NMB0717 (from N. meningitidis MC58) is predicted to encode a periplasmic mono-haem cytochrome with a molecular mass of 12.5 kDa. We call the putative product of this gene cytochrome cx. A homologue of NMB0717, cytochrome c552 from Thermus thermophilus, has been proposed to transfer electrons between the cytochrome bc1 complex and ba3 oxidase in that organism (Muresanu et al., 2006). BLAST searching reveals that the predicted protein sequences with closest similarity to NMB0717, outside of the Neisseria homologues, are predicted copper-type nitrite reductases in Bdellovibrio bacteriovorus, Pseudoalteromonas haloplanktis and several Burkholderia species. In these organisms, the gene for the copper nitrite reductase (homologous to the nitrite reductase gene, aniA, of N. meningitidis) is fused to a cytochrome domain homologous to NMB0717. This suggests a possible role for cytochrome cx in transfer of electrons to nitrite reductase in N. meningitidis.
Gene NMB1805 is predicted to encode a periplasmic di-haem cytochrome with a molecular mass of 21.5 kDa. We call the putative product of this gene cytochrome c4, consistent with the nomenclature for the homologous gene (also known as cycA) from N. gonorrhoeae (Turner et al., 2005). Cytochrome c4 homologues are found in many other proteobacteria and a crystal structure has been found for the protein from Pseudomonas stutzeri (Kadziola & Larsen, 1997). Both haem groups are hexacoordinated, consistent with a role in electron transfer, rather than enzyme catalysis.
Gene NMB1677 is predicted to encode a membrane-associated di-haem cytochrome with a molecular mass of 30 kDa. We call the putative product of this gene cytochrome c5, consistent with the nomenclature for the homologous gene (also known as cycB) from N. gonorrhoeae (Turner et al., 2005). The protein contains a predicted N-terminal membrane span, followed by two soluble domains containing two covalently bound haems located within the periplasm. The second haem-containing domain of cytochrome c5 bears a striking similarity (74 % identity) to a region of the cytochrome cbb3 oxidase subunit III (encoded by ccoP) from N. gonorrhoeae. CcoP from N. gonorrhoeae is predicted to contain three haem groups, whereas CcoPs from other organisms that have been characterized contain just two haem domains (Pitcher & Watmough, 2004). Indeed, the predicted CcoP from N. meningitidis contains two haems. The similarity of cytochrome c5 to a domain of the oxidase immediately suggests a role for cytochrome c5 in the electron transfer from cytochrome bc1 to the cytochrome cbb3 oxidase.
The possession of multiple c-type cytochromes as part of multiply branched respiratory chains is characteristic of many bacterial species. The unambiguous assignment of function to these cytochromes is often difficult due to their large number and apparent redundancy of function. The aim of the work presented in this paper was to investigate the roles of the three putative electron-carrier proteins of N. meningitidis in respiration in this organism, which is an important pathogen but which may also be viewed as a good model for analysis of branched respiratory metabolism due to its relatively small number of respiratory electron-acceptor reductases.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) (Table 1). N. meningitidis strains were routinely cultured at 37 °C, in air supplemented with 5 % CO2 on Columbia agar plus 5 % (v/v) horse blood plates, or in liquid culture in Mueller–Hinton Broth (MHB) supplemented with 10 mM NaHCO3. Aerobic culture was carried out in 5 ml of broth in 25 ml Sterilin McCartney bottles shaken at 200 r.p.m. Microaerobic culture was carried out using 20 ml of broth in a 25 ml Sterilin McCartney bottle, shaken at 90 r.p.m. and, where appropriate, supplemented with 6 mM NaNO2. Growth was monitored by measuring OD600 in a Jenway 6305 spectrophotometer. For whole-cell spectroscopy experiments cultures were supplemented with 5 mM glucose in order to provide a defined electron donor that could be included as physiological reductant in cuvettes. Antibiotics were used at the following concentrations: tetracycline 20 µg ml–1, erythromycin 50 µg ml–1, kanamycin 50 µg ml–1 and spectinomycin 50 µg ml–1.
|
) using Pfu polymerase (Promega). The product was cloned into pCR-Blunt II TOPO (Invitrogen). The product was cleaved with BsgI, which recognizes a site located centrally within the NMB0717 gene. Subsequently, the ends of the linearized plasmid were rendered blunt with Klenow fragment and the product was ligated with the spectinomycin resistance (SpcR) gene derived from pHP4 
(Prentki & Krisch, 1984) to yield pTOPO_cx : : SpcR. The gene encoding cytochrome c4 (NMB1805) and 500 bp flanking on each side was amplified with primers c554F and c554R (Table 1) and disrupted by essentially the same method as described for cytochrome cx except that the gene was disrupted by digestion with Bsu36I (which recognizes a site located centrally within the NMB1805 gene). Insertion of an erythromycin resistance (EryR) gene [derived by PCR from a strain of N. meningitidis in which the gene fnr was disrupted by EryR (Rock et al., 2005)] yielded pTOPO_c4 : : EryR. The gene encoding cytochrome c5 (NMB1677) and 500 bp flanking on each side was amplified with primers c555F and c555R (Table 1) and disrupted by essentially the same method as described for cytochrome cx except that the gene was disrupted by digestion with XmnI (which recognizes a site centrally within the NMB1677 gene) and insertion of a tetracycline resistance (TetR) gene derived from Tn916, yielding pTOPO_c5 : : TetR.
The recombinant plasmids containing disrupted copies of the genes encoding cytochromes were transformed into N. meningitidis MC58 using the method of Bogdan et al. (2002), selecting for recombinant strains using the appropriate antibiotic selection on plates, and verifying the correct chromosomal rearrangement by PCR.
Gels and blotting.
Whole-cell extracts of N. meningitidis were prepared by harvesting 1 ml samples of cultures in late exponential phase by centrifugation, resuspending the pellets in 500 µl 30 mM Tris/HCl (pH 8)+1 % (w/v) n-dodecyl β-D-maltoside+1 mg lysozyme ml–1+1 mg DNase I ml–1, and subjecting the suspension to 8–10 cycles of freezing and thawing. Samples were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were stained with Ponceau S to check for efficient protein transfer and destained with water. The expression of c-type cytochromes was assessed using a chemiluminescence method (Vargas et al., 1993) to measure the peroxidase activity of haem groups covalently attached to protein. Five hundred microlitres of chemiluminescence detection reagents (SuperSignal West Dura substrate, Pierce) were mixed and pipetted onto the membrane, which was incubated, sandwiched between two acetate sheets, for 5 min. The membrane was subsequently exposed to X-ray film (for 5 s to 5 min) to achieve an optimum chemiluminescence signal. Bands due to c-type cytochromes were clearly visualized upon developing the film. Western blotting was used to detect expression of nitrite reductase AniA, as previously described (Rock et al., 2007).
Activity assays.
Oxygen and nitric oxide were measured using electrode-based methods and nitrite was assayed colorimetrically as described previously (Rock et al., 2005). Nitrite reductase activity was assayed in whole-cell extracts using methyl viologen as electron donor as described previously (Moir et al., 1993).
Spectroscopy.
The spectra of cytochromes in intact cells were measured using a Jasco V550 UV–visible spectrophotometer fitted with Integrating Reflective Sphere (ISV-469) in order to compensate for the light scattering by very high-density bacterial suspensions. Bacterial cultures grown in MHB+5 mM glucose were harvested and resuspended in 25 mM HEPES+5 mM glucose (pH 7) to an OD600 of 1.0–1.2 (in this spectrophotometer, equivalent to approx. 5–10 mg protein ml–1). Spectral measurements were carried out in 1 ml cell suspensions in 1.4 ml quartz cuvettes with sealed caps (117.100F-QS, Hellma). Each spectral measurement was taken five times to allow averaging of multiple spectra to reduce noise. Reduction of respiratory components was achieved using the physiological electron donor glucose and leaving the cell suspension to exhaust the available oxygen over the course of 10 min. Oxygen-oxidized spectra were taken following vigorous shaking of the cell suspension. Oxidation of the suspension by nitrite was achieved by addition of 5 mM nitrite (in a total volume of 5 µl to minimize dilution effect).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The lane containing extract from strain N. meningitidis 
reveals the absence of a cytochrome band with molecular mass of approximately 33 kDa, consistent with the absence of cytochrome c5 (which has a predicted molecular mass of 30 kDa). The lanes containing extracts from strains N. meningitidis 
and 
. lacked an intense band with a molecular mass of approximately 23 kDa, consistent with the absence of cytochrome c4 (predicted molecular mass 21.5 kDa) from these strains. There is no difference in the appearance of lanes between and . or between MC58 and , indicating that a band due to cytochrome cx cannot be observed by this method.
|
band located near 550 nm) and b-type ( band shoulder near 560 nm) cytochromes in this organism (Fig. 2). Comparison of wild-type versus strains deficient in cx, c4 and c5 indicates the absence of specific cytochromes in each of the constructs (Fig. 2). The overall intensities of the redox difference spectra were decreased for the cytochrome-deficient strains compared to the wild-type. Furthermore, the positions of the bands in these difference spectra are shifted compared to the wild-type. For N. meningitidis MC58, the band is positioned at 553 nm, whilst the and mutants have their peak at 552 nm and the mutant has an band at 555 nm. The band of the .mutant is centred at 553 nm. It is notable that the mutation in cx causes a shift in the band as well as a decrease in spectral intensity (for MC58 versus and for versus .), providing evidence that cytochrome cx is expressed in wild-type N. meningitidis and absent from the cx mutant strains (despite giving no band on haem-stain blots).
|
, , and . were cultured under aerobic conditions and under microaerobic conditions in the presence and absence of 5 mM nitrite (Fig. 3). The growth rate of the mutant was virtually the same as that of the wild-type during the exponential phase in aerobic culture (doubling time of 43 min), whereas the , and . mutants grew relatively poorly, with doubling times of 63 min, 130 min and 174 min, respectively (Fig. 3a). N. meningitidis MC58, , and grew at similar rates to one another under microaerobic conditions in the absence but not the presence of nitrite (Fig. 3b, c). In the presence of nitrite, N. meningitidis MC58, , and . had an exponential phase of growth, as expected for cultures that have nutrient sufficiency when nitrite is present, whereas the mutant had a linear growth curve, as would be expected for a strain that is limited by availability of a nutrient that is supplied at a constant rate, such as oxygen (Fig. 3c). This has been observed previously for strains of N. meningitidis that are unable to denitrify (Rock et al., 2005), and suggests that under these growth conditions the cytochrome c5-deficient strain grows using the limiting nutrient oxygen and is unable to denitrify. Measurements of nitrite remaining in culture medium corroborated this proposition, since nitrite disappeared during the course of growth of all strains except N. meningitidis (Fig. 3d). It is notable that N. meningitidis , and . grew to lower final cell densities than did N. meningitidis MC58, and yet utilized nitrite as quickly as or quicker than the wild-type. This is consistent with the relatively poor ability of these strains to grow aerobically (Fig. 3a), and indicates that under denitrifying conditions these strains use nitrite as the major electron sink for respiration whereas the wild-type combines nitrite and oxygen utilization simultaneously.
|
and strains relative to wild-type following aerobic growth. The rates of oxygen uptake were 83±5 % and 59±5 % of the wild-type rate in strains and respectively. The growth of N. meningitidis . was so poor aerobically that we cultured the wild-type and . strains under denitrifying conditions to analyse the relative oxygen respiration rate in this strain. The rate of oxygen uptake was 45±5 % of the wild-type rate in the . strain. Clearly both cytochromes cx and c4 are involved in enabling N. meningitidis to utilize oxygen and to grow aerobically.
N. meningitidis failed to utilize nitrite during incubation under microaerobic conditions with nitrite. To determine whether this was due to an inability to respire nitrite or nitric oxide we grew strains MC58 and microaerobically in the absence of nitrite and then followed the accumulation of nitric oxide using an NO electrode after the addition of nitrite (Fig. 4). No nitric oxide accumulated from the strain, whereas, as expected, it accumulated immediately on adding nitrite to the wild-type. This indicates that the lesion in the c5 mutant relates to nitrite reduction, not nitric oxide reduction. To assess whether the defect in nitrite reduction in N. meningitidis might be due to low nitrite reductase expression and/or activity rather than to a break in the electron-transport chain to the nitrite reductase we measured nitrite reductase expression and activity. Expression of nitrite reductase was assessed by Western blotting (Fig. 1b), which showed that AniA is expressed at similar levels in all strains. Using a methyl viologen-linked nitrite reductase assay in total cell extracts we found that N. meningitidis MC58 and had similar overall rates of nitrite reductase activity (data not shown). In the absence of cytochrome c5 the electron-transport chain to nitrite reductase is disabled, presumably because cytochrome c5 is an electron carrier between the cytochrome bc1 complex and AniA nitrite reductase.
|
). The redox difference spectrum following oxidation by nitrite is less intense than that following oxidation by oxygen, consistent with nitrite being a less good oxidant than oxygen. Furthermore, the positions of the spectral maxima are different (the band is located at 560 nm), indicating that oxidation by nitrite brings about predominantly the oxidation of b-type cytochromes. The redox difference spectra following oxidation by nitric oxide (provided by treating cells with 1 mM DEA-NONOate) gives similar spectra to those seen on oxidation by nitrite. Redox difference spectra of N. meningitidis following treatment with nitrite showed no features, consistent with this strain possessing no significant electron-transport chain to nitrite reductase (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
The simplest explanation of the data we have obtained is that cytochrome c5 is a mediator that carries electrons from the cytochrome bc1 complex to the AniA nitrite reductase. Cytochromes cx and c4 are not required for the reduction or oxidation of c5. Is it feasible that cytochrome c5 might be a direct electron donor to AniA nitrite reductase? AniA consists of a trimer of water-soluble subunits (Boulanger & Murphy, 2002) associated with the outer membrane via covalent attachment of N-terminal cysteine residues to fatty acid moieties (Clark et al., 1987; Hoehn & Clark, 1992). It is presumed to be associated with the inner leaflet of the outer membrane, and hence located within the periplasmic compartment. To reduce AniA and drive nitrite reduction, electrons must be transported across the periplasm from the cytochrome bc1 complex (in the inner membrane) to AniA. Can cytochrome c5 span this gap? Analysis of the sequence of cytochrome c5 indicates that it arose by a duplication event from an ancestral gene encoding a monohaem protein. There is no structure available for a dihaem cytochrome c5, but structural information is available for a homologous monohaem cytochrome c5 from Shewanella putrefaciens (Bartalesi et al., 2002). This globular protein has a diameter of approximately 30 Å (3 nm), indicating that the dimeric cytochrome c5 from N. meningitidis may span approximately 60 Å. N. meningitidis cytochrome c5 is predicted to be attached to the membrane via a transmembrane helix, and thus it is anticipated that it can form a structure which protrudes up to approximately 60 Å into the periplasm from the inner membrane. The ingress of electrons to AniA occurs via a blue copper centre which is accessed from a site on the surface of AniA which is approximately 45 Å from the N-terminal face of the soluble protein structure (Boulanger & Murphy, 2002). Thus a complex of the globular domains of cytochrome c5 and AniA could span some 105 Å across the periplasm. The width of the Gram-negative periplasm is approximately 170 Å, based on electron microscopy measurements (Matias et al., 2003) and calculations based on the interaction of inner-membrane protein AcrB with outer-membrane protein TolC (Tamura et al., 2005). Towards the N terminus of the mature AniA polypeptide there is a 35–40 residue region rich in alanine, proline and glutamate that may form an elongated unstructured linker that will allow AniA to get access to electrons from cytochrome c5. It will be of interest to shorten this ‘linker region’ to determine whether it is required for allowing AniA to obtain electrons.
Haem staining was used successfully to identify cytochromes c4 and c5 in total extracts of N. meningitidis strains, but cytochrome cx could not be seen by this method. Haem staining relies on the peroxidase activity of the haem group, an activity that is affected by the naturation state of the protein folding around the haem group (Diederix et al., 2002). This activity varies between cytochromes and presumably is very low for cytochrome cx. Spectroscopic measurements, however, showed a significant loss of c-type cytochrome from the cx mutant (Fig. 2), confirming that this is a major cytochrome in N. meningitidis. The additive effects of mutations in cx and c4 on oxygen respiration indicate that these two proteins operate as independent parallel pathways of electrons from the cytochrome bc1 complex to the cytochrome cbb3 oxidase (Fig. 6). Additionally, there is residual oxidase activity in a . mutant, indicating that there are alternative pathways to cytochrome cbb3. Cytochrome c5 may act as an alternative electron carrier to the oxidase, and it is noticeable that growth of a mutant aerobically is slower than that of the wild-type at high optical densities in late exponential phase (Fig. 4a). We were unable to construct a .. triple mutant, lending support to this proposition. An alternative route might involve the outer-membrane-associated cupredoxin Laz (lipid-modified azurin) (Gotschlich & Seiff, 1987; Kawula et al., 1987). Mutants deficient in the gene encoding Laz grew well aerobically (Wu et al., 2005; our unpublished data) but we were unable to generate a ..laz triple mutant (data not shown). The proposed electron-transport chain of N. meningitidis is summarized in Fig. 6.
|
)]. Whilst oxygen strongly oxidizes the cytochrome c pool in steady-state cell suspensions, nitrite does not. At steady state in the presence of nitrite, the cytochrome c pool remains largely reduced. Presumably this is related to the fact that nitrite is a poor oxidant relative to oxygen (
=0.375 V; 
=0.8 V). It may also be related to the fact that nitrite reductase draws electrons from only cytochrome c5 whereas oxygen oxidizes cytochromes cbb3, cx, c4 and possibly c5. Furthermore, this spectroscopic observation is consistent with the finding that oxygen almost completely inhibits nitrite reduction in intact cells, whereas the reverse is not the case (Rock et al., 2005); i.e. oxygen is able to out-compete nitrite for electrons at the level of cytochrome c. In summary, we have identified the roles of three c-type cytochromes in the respiratory chain of N. meningitidis. Respiration is crucial for survival of N. meningitidis, and these cytochromes are highly conserved and extracytoplasmic, making them potential targets for the development of novel therapeutics against pathogenic neisseriae.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: R. J. M. van Spanning
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bartalesi, I., Bertini, I., Hajieva, P., Rosato, A. & Vasos, P. R. (2002). Solution structure of a monoheme ferrocytochrome c from Shewanella putrefaciens and structural analysis of sequence-similar proteins: functional implications. Biochemistry 41, 5112–5119.[CrossRef][Medline]
Bogdan, J. A., Minetti, C. A. & Blake, M. S. (2002). A one step method for genetic transformation of non-piliated Neisseria meningitidis. J Microbiol Methods 49, 97–101.[CrossRef][Medline]
Boulanger, M. J. & Murphy, M. E. (2002). Crystal structure of the soluble domain of the major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria: a new class of copper-containing nitrite reductases. J Mol Biol 315, 1111–1127.[CrossRef][Medline]
Brook, I. (2003). Microbial dynamics of purulent nasopharyngitis in children. Int J Pediatr Otorhinolaryngol 67, 1047–1053.[CrossRef][Medline]
Clark, V. L., Campbell, L. A., Palermo, D. A., Evans, T. M. & Klimpel, K. W. (1987). Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae. Infect Immun 55, 1359–1364.
Deeudom, M., Rock, J. & Moir, J. W. B. (2006). Organization of the respiratory chain of Neisseria meningitidis. Biochem Soc Trans 34, 139–142.[CrossRef][Medline]
de Vries, S. & Schröder, I. (2002). Comparison between the nitric oxide reductase family and its aerobic relatives, the cytochrome oxidases. Biochem Soc Trans 30, 662–667.[CrossRef][Medline]
Diederix, R. E. M., Ubbink, M. & Canters, G. W. (2002). Peroxidase activity as a tool for studying the folding of c-type cytochromes. Biochemistry 41, 13067–13077.[CrossRef][Medline]
Gotschlich, E. C. & Seiff, M. E. (1987). Identification and gene structure of an azurin-like protein with a lipoprotein signal peptide in Neisseria gonorrhoeae. FEMS Microbiol Lett 43, 253–255.[CrossRef]
Hoehn, G. T. & Clark, V. L. (1992). The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan1, is a lipoprotein. Infect Immun 60, 4704–4708.
Kadziola, A. & Larsen, S. (1997). Crystal structure of the di-haem cytochrome c4 from Pseudomonas stutzeri determined at 2.2 Å resolution. Structure 5, 203–216.[Medline]
Kawula, T. H., Spinol, S. M., Klapper, D. G. & Cannon, J. G. (1987). Localization of a conserved epitope and an azurin-like domain in the H.8 protein of pathogenic Neisseria. Mol Microbiol 1, 179–185.[CrossRef][Medline]
Matias, V. R., Al-Amoudi, A., Dubochet, J. & Beveridge, T. J. (2003). Cryo-transmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa. J Bacteriol 185, 6112–6118.
McGuinness, B. T., Clarke, I. N., Lambden, P. R., Barlow, A. K., Poolman, J. T., Jones, D. M. & Heckels, J. E. (1991). Point mutation in meningococcal porA gene associated with increased endemic disease. Lancet 337, 514–517.[CrossRef][Medline]
Moir, J. W. B., Baratta, D., Richardson, D. J. & Ferguson, S. J. (1993). The purification of a cd1-type nitrite reductase and the absence of a copper-type nitrite reductase from the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor. Eur J Biochem 212, 377–385.[Medline]
Muresanu, L., Pristovsek, P., Löhr, F., Maneg, O., Mukrasch, M. D., Rüterjans, H., Ludwig, B. & Lücke, C. (2006). The electron transfer complex between cytochrome c552 and the CuA domain of the Thermus thermophilus ba3 oxidase. A combined NMR and computational approach. J Biol Chem 281, 14503–14513.
Pitcher, R. S. & Watmough, N. J. (2004). The bacterial cytochrome cbb3 oxidases. Biochim Biophys Acta 1655, 388–399.[Medline]
Prentki, P. & Krisch, H. M. (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–313.[CrossRef][Medline]
Rock, J. D., Mahnane, M. R., Anjum, M. F., Shaw, J. G., Read, R. C. & Moir, J. W. B. (2005). The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels. Mol Microbiol 58, 800–809.[CrossRef][Medline]
Rock, J. D., Thomson, M. J., Read, R. C. & Moir, J. W. B. (2007). Regulation of denitrification genes in Neisseria meningitidis by nitric oxide and the repressor NsrR. J Bacteriol 189, 1138–1144.
Tamura, N., Murakami, S., Oyama, Y., Ishiguro, M. & Yamaguchi, A. (2005). Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide cross-linking. Biochemistry 44, 11115–11121.[CrossRef][Medline]
Turner, S. M., Moir, J. W. B., Griffiths, L., Smith, H. & Cole, J. A. (2005). Genomic analysis of the c-type cytochromes of Neisseria gonorrhoeae: mutational and biochemical analysis of cytochrome c', a nitric oxide-binding lipoprotein important for adaptation to microaerobic growth and its implications for pathogenicity. Biochem J 388, 545–553.[CrossRef][Medline]
van Deuren, M., Brandtzaeg, P. & van der Meer, J. W. M. (2000). Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev 13, 144–166.
Vargas, C., McEwan, A. G. & Downie, J. A. (1993). Detection of c-type cytochromes using enhanced chemiluminescence. Anal Biochem 209, 323–326.[CrossRef][Medline]
Wu, H. J., Seib, K. L., Edwards, J. L., Apicella, M. A., McEwan, A. G. & Jennings, M. P. (2005). Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells. Infect Immun 73, 8444–8448.
Received 7 May 2008;
revised 6 June 2008;
accepted 16 June 2008.
This article has been cited by other articles:
|
|
 |
|
  A. Vik, F. E. Aas, J. H. Anonsen, S. Bilsborough, A. Schneider, W. Egge-Jacobsen, and M. Koomey Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae PNAS, March 17, 2009; 106(11): 4447 - 4452. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
(red), 
(green), 
(blue), and 
are marked. For each cell suspension, the OD600 for the endogenously reduced sample was 1.0.
—
---
---
—
---
and NO is shown by solid arrows. A possible alternative electron transport pathway from cytochrome c5 to the oxidase is shown by the dashed arrow. UQ, ubiquinone; NIR, nitrite reductase.