FIGURE 5.

Nitric oxide and nitrite production by S. meliloti WT and mutant strains. S. meliloti strains were grown exponentially at 28°C in VMM containing either NH₄Cl or glutamate (OD₆₀₀= 0.2–0.3). To remove NH₄Cl or glutamate from the medium, cells were collected by centrifugation and resuspended in VMM without NH₄Cl or glutamate. When needed, KNO₃ (20 mM) was added to the cultures. Cultures were either kept at 28°C under agitation (aerobic conditions, gray bars) or left without agitation under 2% oxygen atmosphere (microaerobic conditions, dark bars) for 1.5 h. Samples were collected to measure: (A) NO production : NO produced by the bacteria and released in the culture medium was quantified using the fluorescent non-permeable probe DAF-2. Fluorescence was measured using a microplate spectrofluorimeter (excitation wavelength 485 nm/emission wavelength 520 nm). NO produced is expressed as relative fluorescence unit (RFU)/OD_{600 nm}. (B) Nitrite concentration in the medium: 100 μl of culture medium was incubated for 30 min with the Griess reagent. Absorbance was measured at 540 nm. NO₂^– concentration was calculated from a calibration curve using NaNO₂ (from 0 to 50 μM). The means and standard errors of the mean obtained from 4 (NO production) or 5 (Nitrite concentration) independent experiments are presented on the Figure. Statistical analysis was performed by means of a one-way ANOVA followed by a Bonferroni statistical test. ^*, ^∗∗, and ^∗∗∗ indicate significant difference (P < 0.05, P < 0.01, and P < 0.001, respectively) when compared with the wild type strain (WT).