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.

FixK2 Is the Main Transcriptional Activator of Bradyrhizobium diazoefficiens nosRZDYFLX Genes in Response to Low Oxygen

“…In this work, we have identified two nosR TSS, i.e., TSS 1 and TSS 2 , positioned at +40.5 and +67.5 bp, respectively, from the axis of symmetry of the FixK-like binding site (TTGATCCAGCGCAA). Similarly, a TSS at +40.5 from the axis of symmetry of the FixK box has been recently identified by Sánchez et al (2017) . In contrast to our results, the TSS at +67.5 bp was not identified in the latter studies.…”

“…The capacity of B. diazoefficiens to couple N 2 O reduction to growth as well as a role for the NasST regulatory system on modulation of nosZ gene transcription has also been reported ( Sánchez et al, 2013 , 2014 ). Furthermore, recent studies have demonstrated the capacity of NasT to interact with B. diazoefficiens nosR 5′-leader RNA ( Sánchez et al, 2017 ).…”

“…Interestingly, the highest transcriptional activity was derived from the DNA region upstream of the nosR gene compared to that detected from the nosZ gene, and no transcription was observed from the 5′ DNA region of the nosD gene. The presence of a transcriptionally active promoter upstream of the nosZ gene was previously demonstrated by using a nosZ-lacZ transcriptional fusion ( Velasco et al, 2004 ) and by performing 5′-RACE ( Sánchez et al, 2017 ). However, since a binding motif for FixK-type regulators was only present within the promoter region of nosR , we suggest that this promoter plays the major role in B. diazoefficiens nosRZDFYLX regulation.…”

“…In contrast to our results, the TSS at +67.5 bp was not identified in the latter studies. This discrepancy could be due to the different growth conditions used by Sánchez et al (2017) where cells were cultured in HMM medium ( Sameshima-Saito et al, 2006 ) under anoxic conditions (replacement of O 2 by N 2 in the gas phase). FixK 2 -like boxes are present within the promoters of the B. diazoefficiens napEDABC (TTGATCCAGATCAA), nirK (TTGTTGCAGCGCAA), and norCBDQD (TTGCGCCCTGACAA) genes ( Velasco et al, 2001 ; Mesa et al, 2002 ; Delgado et al, 2003 ; Supplementary Figure S1 ).…”

Nitrogen Cycling in Soybean Rhizosphere: Sources and Sinks of Nitrous Oxide (N2O)

“…The nos ++ mutants retained higher nos expression and N 2 OR activity in both free-living and symbiotic cells than the wild-type nos + strains (Itakura et al, 2013; Sánchez et al, 2014). Comparative analysis of the nos ++ mutant genomes revealed the mechanism underlying the nos ++ phenotype, a point mutation in nasS gene encoding the NO 3 – sensor of the two-component NasST regulatory system (Sánchez et al, 2014, 2017), which will be discussed later.…”