Regulation of nitrous oxide reductase genes by NasT-mediated transcription antitermination in Bradyrhizobium diazoefficiens

Abstract

In Bradyrhizobium diazoefficiens, maximal expression of the nitrous oxide reductase gene (nosZ) requires oxygen limitation and the presence of a nitrogen oxide. The putative transcription antiterminator NasT is a positive regulator of nosZ; but in the absence of nitrate, NasT is counteracted by the nitrate sensor NasS. Here, we examined the NasT-mediated mechanism of nosRZDFYLX gene cluster expression. We mapped two transcription start sites of nosR and identified two potential hairpins, H1 and H2, within the 5'-leader of nosR transcripts. Electrophoretic mobility shift assay showed that NasT specifically bound the nosR-leader RNA and deletion of H1 abolished such binding. Under aerobic nitrate-deficient conditions, deletion of H1 or H2 increased the level of nosRZD transcripts. Under denitrifying conditions (anaerobiosis with nitrate supply), the level of nosRZD transcripts was severely impaired in the nasT mutant; in the nasT background, deletions of either hairpin led to increased level of nosRZD transcripts. In contrast to nosRZD coding region, nosR-leader transcript level was not affected by nasS or nasT mutations under aerobic or denitrifying conditions respectively. These results suggest that the two-hairpin RNA structure acts for transcription termination upstream of nosR and the binding of NasT to H1 facilitates read-through transcription to induce nos expression.