AUTHOR=Hidalgo-García Alba , Torres María J. , Salas Ana , Bedmar Eulogio J. , Girard Lourdes , Delgado María J. 

TITLE=Rhizobium etli Produces Nitrous Oxide by Coupling the Assimilatory and Denitrification Pathways

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00980

DOI=10.3389/fmicb.2019.00980

ISSN=1664-302X

ABSTRACT=<p>More than two-thirds of the powerful greenhouse gas nitrous oxide (N<sub>2</sub>O) emissions from soils can be attributed to microbial denitrification and nitrification processes. Bacterial denitrification reactions are catalyzed by the periplasmic (Nap) or membrane-bound (Nar) nitrate reductases, nitrite reductases (NirK/<italic>cd</italic><sub>1</sub>Nir), nitric oxide reductases (cNor, qNor/ Cu<sub>A</sub>Nor), and nitrous oxide reductase (Nos) encoded by <italic>nap</italic>/<italic>nar</italic>, <italic>nir</italic>, <italic>nor</italic> and <italic>nos</italic> genes, respectively. <italic>Rhizobium etli</italic> CFN42, the microsymbiont of common bean, is unable to respire nitrate under anoxic conditions and to perform a complete denitrification pathway. This bacterium lacks the <italic>nap</italic>, <italic>nar</italic> and <italic>nos</italic> genes but contains genes encoding NirK and cNor. In this work, we demonstrated that <italic>R. etli</italic> is able to grow with nitrate as the sole nitrogen source under aerobic and microoxic conditions. Genetic and functional characterization of a gene located in the <italic>R. etli</italic> chromosome and annotated as <italic>narB</italic> demonstrated that growth under aerobic or microoxic conditions with nitrate as nitrogen source as well as nitrate reductase activity requires NarB. In addition to be involved in nitrate assimilation, NarB is also required for NO and N<sub>2</sub>O production by NirK and cNor, respectively, in cells grown microoxically with nitrate as the only N source. Furthermore, β-glucuronidase activity from <italic>nirK::uidA</italic> and <italic>norC::uidA</italic> fusions, as well as NorC expression and Nir and Nor activities revealed that expression of <italic>nor</italic> genes under microoxic conditions also depends on nitrate reduction by NarB. Our results suggest that nitrite produced by NarB from assimilatory nitrate reduction is detoxified by NirK and cNor denitrifying enzymes that convert nitrite into NO which in turn is reduced to N<sub>2</sub>O, respectively.</p>