Nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum

SALAS A.; CABRERA J.J.; LÓPEZ M.F.; TORRES M.J.; LÓPEZ-GARCÍA S.L.; MESA S.; BEDMAR E.J.; RICHARDSON D.J.; GATES A.J.; DELGADO M.J.

León

Congreso; XV National Meeting of the Spanish Society of Nitrogen Fixation (XV SEFIN)- IV Portuguese-Spanish Congress on Nitrogen Fixation; 2015

Sociedad Española de Fijación de Nitrógeno (SEFIN)

Bradyrhizobium japonicum is a soil bacterium that establishes nitrogen-fixing symbiotic associations with soybean plants, and may denitrify under free-living and symbiotic conditions. This bacterium is also able to grow with nitrate (NO3-) or nitrite (NO2-) as the sole nitrogen source. In contrast to other bacteria that assimilate nitrate, the genes nasC and nirA encoding the assimilatory nitrate reductase and nitrite reductase enzymes in B. japonicum are located at distinct loci on the chromosome. Whereas nasC is located in a gene cluster that encodes an ABC-type nitrate transporter, a nitrate/nitrite transporter (NarU), a flavoprotein (Flp) and a single domain haemoglobin (Bjgb), nirA clusters with genes for a nitrate/nitrite responsive regulatory system (NasTS). NasC and NirA are required for NO3- and NO2- assimilation, respectively, and Flp may function as electrondonor to NasC. In addition, bjgb and flp encode a nitric oxide (NO) detoxification system that functions tomitigate cytotoxic NO formed as a by-product of NO3-/NO2- assimilation. NasTS are involved in NO3-/NO2--dependent growth and in nitrate reductase and nitrite reductase activities. In this work, we have demonstrated that narU, bjgb, flp and nasC comprise a transcriptional unit. β-galactosidase activity of a narU-lacZ fusion showed that expression of these genes is repressed by ammonium or glutamate and induced by nitrate and nitric oxide. The involvement of the regulatory proteins NtrBC, NasTS, and NnrR in the expression of the narUbjgb-flp-nasC operon has also been demonstrated. Expression analyses from a nirA-lacZ fusion revealed that while NtrBC and NasTS are involved in nitrate-dependent induction of nirA, NO or NnrR were not involved in nirA expression. Taken together, these results suggest that narU-bjgb-flp-nasC operon involved in nitrate assimilation and NO detoxification is subjected to a co-regulation in response to nitrate and NO, however, expression of nirA involved in nitrite reduction is not.