The ability of Neisseria meningitidis to utilize both oxygen and nitrogen oxides as respiratory substrates allows it to thrive in the diverse environment of the human host. Genome analysis highlighted genes encoding a cbb3 cytochrome oxidase, the aniA nitrite reductase gene and the norB nitric oxide reductase gene. In the present study, we used myxothiazol as an inhibitor of the bc1 complex in intact cells and demonstrated that electron flow to nitrite reductase and the cytochrome oxidase, but not NO reductase, passes via the cytochrome bc1 complex. UV–visible spectrophotometry of intact cells demonstrated that oxygen oxidizes c-type and b-type cytochromes. Oxidation of cytochromes by nitrite was only seen in microaerobically precultured whole cells, and the predominant oxidizable cytochromes were b-type. These are likely to be associated with the oxidation of a b-haem-containing nitric oxide reductase. Nitrite inhibits the oxidation of cytochromes by oxygen in a nitrite reductase-independent manner, indicating that nitrite may inhibit oxidase activity directly, as well as via the intermediate of denitrification, nitric oxide.
We have shown that the human pathogen Neisseria meningitidis is able to use nitrite as a terminal electron acceptor during respiration, as an alternative to oxygen respiration . Analysis of the N. meningitidis genome has lead to the identification of three possible terminal electron acceptors: (i) an oxidase of the cytochrome cbb3-type, (ii) a nitrite reductase (AniA) and (iii) a single subunit nitric oxide reductase (NorB). The genome also reveals a number of possible electron transport chain components, including genes encoding a cytochrome bc1 complex (complex III), a number of putative c-type cytochromes (none of which appear similar to the mitochondrial cytochrome c, homologues of which are found in many proteobacteria) and an outer-membrane-associated cupredoxin [Laz (lipid-modified azurin)]. In this paper, we present evidence regarding the organization of the respiratory chain in this important pathogen.
Materials and methods
N. meningitidis MC58 was grown aerobically on Columbia agar+5% horse blood plates at 37°C in an air atmosphere supplemented with 5% CO2. For liquid cultures, N. meningitidis was grown in Müller–Hinton broth+10 mM NaHCO3 which was sometimes supplemented with 1% glucose. Cultures were incubated at 37°C. For aerobic growth, 5 ml of the medium was incubated in 25 ml McCartney flasks shaken at 200 rev./min. For growth by denitrification, 20 ml cultures supplemented with 5 mM NaNO2 in 25 ml McCartney flasks were shaken at 90 rev./min. Oxygen respiration was monitored using a Clark-type electrode (Rank, Bottisham, U.K.). Nitrite was assayed colorimetrically . Nitric oxide was monitored with an iso-NO electrode (World Precision Instruments, Stevenage, U.K.). The role of the cytochrome bc1 complex was investigated using the specific inhibitor myxothiazol (Sigma–Aldrich), which was kept as a solution dissolved in methanol and used at various concentrations. Other chemicals were obtained from Sigma–Aldrich. UV–visible spectra were obtained using a Jasco V-550 spectrophotometer. For spectra of intact cells, the spectrophotometer was fitted with a reflective sphere for collection of light scattered by the highly turbid cell suspensions.
Results and discussion
Nitrite and oxygen respiration depend on electron flow via the bc1 complex
In order to investigate the role of the cytochrome bc1 complex in the flow of electrons through the respiratory chain in N. meningitidis, we used the inhibitor myxothiazol, which is specific for the cytochrome bc1 complex . We monitored oxygen respiration by suspensions of N. meningitidis using a Clark-type electrode and found that oxygen respiration was essentially completely blocked by 1 μM myxothiazol (Figure 1A). This effect occurred within 1 min of adding the myxothiazol to the cell suspension. A control in which cells were treated with the solvent methanol had no effect on oxygen respiration. Nitrite respiration was followed in N. meningitidis grown under denitrifying conditions. The effect of myxothiazol on depletion of nitrite was monitored non-continuously by taking aliquots from an anaerobic cell suspension and measuring nitrite colorimetrically. Figure 1(B) shows that myxothiazol inhibits nitrite respiration to a large extent. Immediately the rate of nitrite reduction is decreased to approx. 10% of the uninhibited rate, and after 2–3 h of incubation, the rate of nitrite disappearance decreases to zero. This long-term effect is likely to be due to a decrease in cellular viability over this long incubation period, but it is clear from the immediate effect that most of the flux of electrons to nitrite reductase is dependent upon the bc1 complex (although there appears to be a low-flux alternative pathway). The reduction of NO by cultures of N. meningitidis grown under denitrifying conditions was monitored using an NO electrode, and it was found that myxothiazol has no impact on NO respiration (Figure 1C). This indicates that NO reduction is independent of the bc1 complex and also serves as a control to assure us that the myxothiazol is acting specifically rather than behaving as a general inhibitor of respiration. The evidence from these studies supports a respiratory chain organized as shown in the scheme in Figure 1(D), which is similar to that proposed in our earlier model .
Role of the cytochrome bc1 complex in N. meningitidis
Spectroscopic analysis of the respiratory chain of N. meningitidis
In order to investigate further the components of the respiratory chain in N. meningitidis, we used spectroscopy of intact cells and cell extracts. N. meningitidis was grown in liquid cultures in which the medium was supplemented with glucose so that this carbon source could be used as a physiological electron donor into the respiratory chain following the catabolic production of reducing equivalents (NADH etc.). Under anaerobic conditions, cell suspensions supplemented with glucose rapidly became reduced, giving rise to UV–visible spectral features characteristic of b- and c-type cytochromes (Figure 2A). Following the addition of oxygen to these cell suspensions, the spectral features due to reduced α- and β-bands disappear and the Soret peak is blue-shifted, indicating oxidation of cytochromes. Difference spectra (Figure 2B) show that the oxygen-dependent oxidation is characterized by the disappearance of a major α-feature at 552 nm. This feature is presumably made up of a number of c-type cytochromes that are found in the electron transport pathway to oxygen, and fewer b-type cytochromes as indicated by the slight shoulder on the α-band around 560 nm.
Spectroscopic analysis of redox changes in response to respiratory electron acceptors in N. meningitidis
With aerobically cultured N. meningitidis, nitrite has no effect on the spectra of cell suspensions, but following culturing under conditions to allow expression of denitrification machinery, the oxidation by nitrite causes predominantly the oxidation of b-type cytochromes (Figure 2c). This is most likely to be due to the oxidation of reduced NorB by the nitric oxide generated by nitrite reduction, and this is confirmed by oxidizing intact cells with nitric oxide by treating cells with 1 mM DEA-NONOate [2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt] as a source of NO (Figure 2d). We conclude that there are c-type cytochromes in the respiratory chain of the meningococcus that are oxidized by oxygen but not nitrite. It is not yet clear whether these oxygen-specific cytochromes are part of the oxidase itself or are also c-type cytochromes that are involved in shuttling electrons to the oxidase. Following oxidation of cells by oxygen, it is possible to obtain a further oxidation of c-type cytochromes using the oxidizing agent ammonium persulphate (Figure 2e). The ammonium persulphate-oxidizable cytochrome is distinct from the cytochrome oxidized by oxygen (note the shifted Soret band).
In order to characterize further the impacts of the oxidants oxygen and nitrite on the respiratory components, we investigated the effect of oxygen plus nitrite on the spectra of cytochromes in intact cells. It was found that the degree of oxidation by nitrite+oxygen was intermediate between the oxidations by nitrite alone and oxygen alone (Figure 2f). We initially considered that this would be due to the production of nitric oxide from nitrite, which in turn partially inhibits oxidase activity under these experimental conditions. However, we found that the inhibition by nitrite is also observed in a mutant deficient in the nitrite reductase, which is unable to synthesize NO from nitrite (results not shown). This indicates that nitrite may have a direct inhibitory effect on oxygen metabolism in this bacterium, rather than just inhibiting via the production the nitric oxide radical.
The 11th Nitrogen Cycle Meeting 2005: Independent Meeting held at Estación Experimental del Zaidín, Granada, Spain, 15–17 September 2005. Organized and Edited by E.J. Bedmar (Granada, Spain), M.J. Delgado (Granada, Spain) and C. Moreno-Vivián (Córdoba, Spain).
J.M. is grateful to the Wellcome Trust for funding (grant 070268/Z/03/Z). M.D. is supported by a studentship from the Royal Thai Government.