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Open Access Research article

Genetic basis for denitrification in Ensifer meliloti

Maria J Torres1, Maria I Rubia1, Teodoro Coba de la Peña2, José J Pueyo2, Eulogio J Bedmar1 and María J Delgado1*

Author Affiliations

1 Estación Experimental del Zaidin, Consejo Superior de Investigaciones Científicas (CSIC), P.O. Box 419, 18080 Granada, Spain

2 Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Científicas (CSIC), Serrano 115-bis, 28006 Madrid, Spain

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BMC Microbiology 2014, 14:142  doi:10.1186/1471-2180-14-142

Published: 2 June 2014

Abstract

Background

Denitrification is defined as the dissimilatory reduction of nitrate or nitrite to nitric oxide (NO), nitrous oxide (N2O), or dinitrogen gas (N2). N2O is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Legume crops might contribute to N2O production by providing nitrogen-rich residues for decomposition or by associating with rhizobia that are able to denitrify under free-living and symbiotic conditions. However, there are limited direct empirical data concerning N2O production by endosymbiotic bacteria associated with legume crops. Analysis of the Ensifer meliloti 1021 genome sequence revealed the presence of the napEFDABC, nirK, norECBQD and nosRZDFYLX denitrification genes. It was recently reported that this bacterium is able to grow using nitrate respiration when cells are incubated with an initial O2 concentration of 2%; however, these cells were unable to use nitrate respiration when initially incubated anoxically. The involvement of the nap, nirK, nor and nos genes in E. meliloti denitrification has not been reported.

Results

E. meliloti nap, nirK and norC mutant strains exhibited defects in their ability to grow using nitrate as a respiratory substrate. However, E meliloti nosZ was not essential for growth under these conditions. The E. meliloti napA, nirK, norC and nosZ genes encode corresponding nitrate, nitrite, nitric oxide and nitrous oxide reductases, respectively. The NorC component of the E. meliloti nitric oxide reductase has been identified as a c-type cytochrome that is 16 kDa in size. Herein, we also show that maximal expression of the E. meliloti napA, nirK, norC and nosZ genes occurred when cells were initially incubated anoxically with nitrate.

Conclusion

The E. meliloti napA, nirK, norC and nosZ genes are involved in nitrate respiration and in the expression of denitrification enzymes in this bacterium. Our findings expand the short list of rhizobia for which denitrification gene function has been demonstrated. The inability of E. meliloti to grow when cells are initially subjected to anoxic conditions is not attributable to defects in the expression of the napA, nirK, norC and nosZ denitrification genes.

Keywords:
Cu-containing nitrite reductase; Nitrate respiration; Nitric oxide reductase; Nitrous oxide reductase; Periplasmic nitrate reductase