Overexpression of the periplasmic nitrate reductase supports anaerobic growth by Ensifer meliloti

The alfalfa endosymbiont Ensifer meliloti strain1021 is known to be an incomplete denitrifier due to its inability to grow anoxically using nitrate as respiratory substrate to produce ATP and grow under anoxic conditions. Although this bacterium contains and expresses the complete set of denitrification genes napEFDABC, nirK, norECBQD and nosRZDFYLX encoding the periplasmic nitrate reductase (Nap), Cu-containing nitrite reductase (NirK), c-type nitric oxide (cNor) and nitrous oxide reductase (Nos), respectively, the reasons of its inability to grow under anoxic conditions are still very poorly understood. In the present study, we have constructed an E. meliloti strain overexpressing napEFDABC genes (Nap) and demonstrated that this strain is able to grow through anaerobic nitrate respiration. Furthermore, Nap showed increased NapC levels as well as Nap, Nir and cNor activities and higher capacity to produce NO and NO compared to wild-type cells. These results suggest that the inability of E. meliloti to grow under anaerobic conditions using nitrate as electron acceptor is attributable to a limitation in the expression of the periplasmic nitrate reductase.This work was supported by Fondo Europeo de Desarrollo Regional (FEDER)-co-financed Grant AGL2013-45087-R from the Ministerio de Economía y Competitividad (Spain). Grant P12-AGR-1968 and support from the Junta de Andalucía to Group BIO-275 are also acknowledged. MT was supported by a contract funded by Grant AGL2013-45087-R.Peer Reviewe