Identification of a gene responsible for higher N2O reductase activity in Bradyrhizobium japonicum. Bradyrhizobium diazoefficiens USDA 110

Nitrous oxide (N2O) is a powerful greenhouse gas and the major ozone depleting substance in the stratosphere. In soybean ecosystems N2O is emitted from the degradation of the root nodules at the late growth period. Inside root nodules, soybean hosts the nitrogen-fixing bacterium Bradyrhizobium japonicum that is able to scavenge N2O through N2O reductase enzyme (Nos), contributing to mitigation of N2O emissions from soybean fields. B. japonicum is a complete denitrifier carrying nap, nirK, nor and nos genes, which encode the reductases for nitrate, nitrite, nitric oxide and N2O, respectively. We isolated Nos++ strains (mutants with increased Nos activity) of B. japonicum USDA110 through the impairment of proofreading activity and the use of enrichment culture under selection pressure for N2O respiration. In order to identify the mutated genes responsible for the higher Nos activity in Nos++ strains we compared the genome sequences of six strains. We founded only one gene (bll4572) that was mutated in all six strains. A B. japonicum mutant in bll4572 exhibited same values of Nos activity as the reference Nos++ strain 5M09. Sequence analysis revealed that bll4572 and bll4573 (the gene located upstream bll4572) encode NasST proteins, a two-component regulatory system which is involved in the regulation of nitrate assimilation genes in other bacteria. Transcriptional analysis showed that regulation of nos and also nap genes is dependent on the nasT gene product and that the nasS gene product plays a negative regulatory role. Thus here we show that NasST is regulating denitrification genes in B. japonicum USDA110.