Linked Expressions of nap and nos Genes in a Bradyrhizobium japonicum Mutant with Increased N ₂ O Reductase Activity

Abstract

To understand the mechanisms underlying the increased N 2 O reductase activity in the Bradyrhizobium japonicum 5M09 mutant from enrichment culture under N 2 O respiration, we analyzed the expression of genes encoding denitrification reductases and regulators. Our results suggest a common regulation of nap (encoding periplasmic nitrate reductase) and nos (encoding N 2 O reductase). N itrous oxide (N 2 O) is a key greenhouse gas that also damages the ozone layer. Soybean nodules emit N 2 O under field conditions in the late growth period (1-3). While N 2 O generation is due to a diversity of microbial enzymes, N 2 O consumption appears to be due exclusively to N 2 O reductase (Nos) (4-6). Nos catalyzes the two-electron reduction of N 2 O to N 2 . In strain USDA110 of the soybean symbiont Bradyrhizobium japonicum, the complete reduction of nitrate to N 2 depends on napEDABC (which encode periplasmic nitrate reductase [Nap]), nirK (copper-containing nitrite reductase [NirK]), norCBQD (c-type nitric oxide reductase [cNor]), and nosRZDYFLX (Nos) (7).A B. japonicum mutant, 5M09, with increased N 2 O reductase activity (Nos ϩϩ ) was isolated through the impairment of proofreading activity and the use of enrichment culture under selection pressure for N 2 O respiration (8). Recently, it was shown that postharvest N 2 O emissions from soybean fields can be mitigated by inoculation with 5M09 at the field scale (9). Because the mechanisms underlying the increased Nos activity in 5M09 remain unclear, the main objective of this work was to evaluate the expression of genes encoding denitrification reductases and regulators in 5M09. A B. japonicum mutant (PRNOS) that overexpresses the nos genes as controlled by the rRNA gene promoter (9) was used as a Nos ϩϩ reference. B. japonicum USDA110 (United States Department of Agriculture, Beltsville, MD) and the mutant derivative strains 5M09 (8) and PRNOS (9) were used in this study. The bacterial strains and plasmids used in this work are listed in Table S1 in the supplemental material. Cells were routinely cultured at 30°C in HM salt medium (10) supplemented with 0.1% arabinose, 0.025% (wt/vol) yeast extract, and trace metals (HMM medium) (11). For ␤-galactosidase assays, methyl viologen (MV)-dependent nitrate reductase assays, and RNA isolation, aerobically grown cells were collected and washed twice. They were then adjusted to an optical density at 660 nm of about 0.1 by the addition of HMM medium. Cells (10 ml) were incubated for 24 h in 120-ml airtight vials with air (aerobic conditions) or N 2 (anaerobic conditions). Where appropriate, either N 2 O (1%, 5%, 20%, or 25% [vol/vol]) or KNO 3 (10 or 20 mM) was added.For ␤-galactosidase assays, we used chromosomally integrated transcriptional lacZ fusions with the napE, nirK, norC, and nosZ promoters. The plasmids pBG0614 (12), pRJ2498 (13), pRJ2499 (13), and pNOSLZch were integrated by homologous recombination into the chromosome of each of USDA110 and 5M09. ␤-Galactosidase activity was determined with permeabilized cells as descri...

The nitrate‐sensing NasST system regulates nitrous oxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum

“…The induction of napE expression depended on the nasT product since β‐galactosidase activity in the nasST mutant was not significantly different from that in USDA110 (Table ). The β‐galactosidase activities by the P nirK ‐ lacZ and P norC ‐ lacZ fusions in the nasS and nasST mutants did not differ significantly from that in USDA110 (Table ), in line with the absence of nirK and nor induction in 5M09 reported earlier (Sánchez et al ., ). This suggests that the regulation of transcription of denitrification genes by NasST is specific to the nos and nap genes, and occurs under aerobic and anaerobic conditions.…”

“…Inoculation of soybeans with the Nos ++ strain 5M09 mitigated N 2 O emission in both pot and field experiments (Itakura et al ., ). We have also shown that in strain 5M09, transcription of nosZ , nosR and nosD (genes from the N 2 O reduction cluster), and napA (encoding the catalytic subunit of the periplasmic nitrate reductase), was higher than in USDA110 (Sánchez et al ., ). The induced transcription of nos and nap genes did not depend on the FixLJ‐FixK 2 ‐NnrR and RegSR‐NifA regulatory cascades (Sánchez et al ., ).…”

“…We have also shown that in strain 5M09, transcription of nosZ , nosR and nosD (genes from the N 2 O reduction cluster), and napA (encoding the catalytic subunit of the periplasmic nitrate reductase), was higher than in USDA110 (Sánchez et al ., ). The induced transcription of nos and nap genes did not depend on the FixLJ‐FixK 2 ‐NnrR and RegSR‐NifA regulatory cascades (Sánchez et al ., ). In this paper, we describe the causal gene for the Nos ++ phenotype and the underlying mechanism.…”

Soil biochar amendment affects the diversity of nosZ transcripts: Implications for N2O formation

“…The calculated production of typical and atypical nosZ transcripts in all other relevant microbial species we identified based on the sequencing of typical and atypical nosZ transcripts was not significantly affected by biochar addition. Recent studies revealed that well-studied strains of some of these species ( Ensifer meliloti 1021 and Bradyrhizobium diazoefficiens USDA 110) are able to grow with externally supplied N 2 O as sole electron acceptor 68, 69 . Thus, these strains take up N 2 O from the environment and further reduce it to N 2 .…”

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

“…Denitrification can be considered as a modular process and each nitrogen oxide reductase appears to provide a selectable benefit independent of other denitrification enzymes (Graf et al ., ; Roco et al ., ). In this respect, B. diazoefficiens is able to grow with N 2 O as the sole electron acceptor (Sánchez et al ., ). In this bacterium, the nosRZDFYLX cluster encodes Nos (NosZ) and several ancillary proteins required for its expression, maturation and maintenance.…”