Nitric oxide metabolism in Neisseria meningitidis

Abstract

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.

FIG. 1.

Effect of nitrite on growth of N. meningitidis. Growth curves for wild-type (A and D), cycP (B and E), and norB (C and F) strains grown aerobically (A, B, and C) and microaerobically (D, E, and F) in the presence (triangles) and absence (squares) of 5 mM nitrite are shown. Results are means from at least four experiments ± standard deviations. OD₆₀₀, optical density at 600 nm

FIG. 2.

Nitrite utilization by N. meningitidis. Time courses of nitrite concentration in wild-type (filled squares), cycP (open squares), and norB (filled triangles) cultures grown aerobically (A) or microaerobically (B) in the presence of 5 mM nitrite are shown. Results are means from at least four experiments ± standard deviations.

FIG. 3.

Metabolism of nitrite and NO by N. meningitidis. NO concentration was measured with an iso-NO electrode (World Precision Instruments). Suspensions containing 3 ml of N. meningitidis at a concentration of 0.1 mg of protein ml⁻¹ in MHB were sparged with nitrogen gas until anaerobic in a 7-ml water-jacketed chamber kept at 30°C. The cell suspension was stirred with a magnetic flea, and the chamber was kept anaerobic with a rubber septum through which the NO electrode probe was inserted. Traces show the effects of 1 mM nitrite on wild-type (A) and norB (B) strains and the effect of 8 μM NO on cycP (C) and norB (D) strains.

FIG. 4.

Evidence for expression of holocytochrome c′. Samples were run on SDS-10% PAGE and stained for proteins containing covalently bound hemes. Lane 1, 1 μg of cytochrome c′ purified from R. capsulatus used as a control hemoprotein; lane 2, 25 μg of total soluble extract from wild-type N. meningitidis (cytochrome c′ is marked by an arrow); lane 3, 25 μg of total soluble extract from N. meningitidis cycP.

FIG. 5.

Oxygen and NO measurements in MC58, MA1, and MA2. Oxygen disappearance was measured with a Clark electrode (solid lines) while NO was measured simultaneously (dashed lines). The chamber of the oxygen electrode contained 3 ml of wild-type (A), cycP (B), or norB (C) N. meningitidis strains at a concentration of 0.5 mg protein ml⁻¹ in MHB. The NO electrode was inserted into the chamber stopper such that there was no headspace within the reaction vessel. Arrows mark the additions of 9.3 μM NO at an oxygen concentration of 120 μM.

FIG. 6.

Lack of effect of oxygen concentration on the period of inhibition by NO. The effects of NO on respiration rates of wild-type N. meningitidis are shown. Arrows indicate the point at which NO (9.3 μM) was added to the oxygen electrode chamber. The reaction chamber contained 3 ml of wild-type N. meningitidis at a concentration of 0.5 mg of protein ml⁻¹ in MHB. Oxygen concentration had no effect on the period of inhibition by NO.