FIGURE 4.

Control of nosRZDFYLX expression by the regulatory proteins FixK₂ and NnrR. (A) β-Galactosidase activity expressed as Miller units (MU) from the nosR-lacZ transcriptional fusion chromosomally integrated in the B. diazoefficiens WT strain, and ΔnnrR, and ΔfixK₂ strains grown oxically (white bars), under 2% O₂ in the absence (light gray bars) or in the presence of 10 mM KNO₃ (black bars) for 24 h. In the right panel, 10 μM of the NO-scavenger cPTIO was added to a series of cultures containing NO₃^- (dark gray bars). (B) Expression of nosR by qRT-PCR in the WT, and ΔnnrR, and ΔfixK₂ strains. qRT-PCR reactions were performed with cDNA synthesized from three independent RNA samples assayed in triplicate. Fold-change values refer to differences of expression in the ΔnnrR, and ΔfixK₂ mutants relative to the WT. (C) Western-blotted SDS-PAGE gels of the soluble fraction from the WT and ΔnnrR, and ΔfixK₂ strains probed with anti-NosZ antibody from Pa. denitrificans. As control, a B. diazoefficens nosZ mutant was used. The size of B. diazoefficiens NosZ is labeled on the left side. (D) Nitrous oxide reductase (N₂OR) activity in the WT and ΔnnrR, and ΔfixK₂ strains expressed as nmol N₂O consumed × (mg prot^-1) h^-1. In (B–D), cells were grown under 2% O₂ in the absence or in the presence of 10 mM KNO₃ during 24 h. 100 μM of cPTIO was added to some of the cultures containing NO₃^- in (D). In (A,B,D), data shown as means with standard errors from at least two independent cultures, assayed in triplicate.