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Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens

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Abstract

Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ54, was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.

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Abbreviations

Bjgb:

Bradyrhizobium japonicum haemoglobin

BN3:

Bergersen minimal medium-nitrate

C:

Carbon

CFU:

Colony formation units

Flp:

Flavoprotein

MU:

Miller units

MV-NiR:

Methyl viologen-dependent nitrite reductase

MV-NR:

Methyl viologen-dependent nitrate reductase

N:

Nitrogen

NarK:

Nitrate/nitrite transporter

NasC:

Assimilatory nitrate reductase

NirA:

Assimilatory nitrite reductase

NO:

Nitric oxide

NtrB:

Two-component system kinase

NtrC:

Two-component system response regulator

OD500 :

Optical density-500 nm

PSY:

Peptone–salts–yeast extract

RpoN:

Alternative sigma factor

WT:

Wild-type

YEM:

Yeast-extract-mannitol

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Acknowledgements

We thank Dr. Hans-Martin Fisher (ETH Zürich, Institute of Microbiology, Zürich, Switzerland) for kindly providing the B. japonicum rpoN 1/2 mutant. We are also grateful to Paula Giménez, Silvana Tongiani, Abel Bortolameotti (members of CPA CONICET at IBBM), Ruben Bustos from UNLP, and Alba Hidalgo-García for their excellent technical assistance (EEZ CSIC). Dr. Donald F. Haggerty edited the final version of the manuscript. This work was supported by the Agencia Nacional de Promoción de la Investigación Científica y Tecnológica (ANPCyT) project PICT 2013-2864, Consejo Nacional de Investigaciones Científicas y Técnicas—CONICET and Secyt-UNLP, Argentina. MJD received financial support from the European Regional Development Fund (ERDF) cofinanced Grants AGL2013-45087-R from the Ministerio de Economía y Competitividad (Spain) and PE2012-AGR1968 from the Junta de Andalucía. Continuous support from Junta de Andalucía to group BIO275 is also acknowledged. MFL was supported by fellowships from CONICET and by a short-stay fellowship from EMBO. SLLG is researcher at CONICET.

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  1. Instituto de Biotecnología y Biología Molecular (IBBM), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata y CCT La Plata-CONICET, Calles 47 y 115, B1900AJL, La Plata, Argentina

    María F. López & Silvina L. López-García

  2. Estación Experimental del Zaidín, CSIC, PO Box 419, 18080, Granada, Spain

    Juan J. Cabrera, Ana Salas & María J. Delgado

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  1. María F. López
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  2. Juan J. Cabrera
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  3. Ana Salas
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  4. María J. Delgado
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  5. Silvina L. López-García
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Correspondence to María J. Delgado or Silvina L. López-García.

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López, M.F., Cabrera, J.J., Salas, A. et al. Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens . Antonie van Leeuwenhoek 110, 531–542 (2017). https://doi.org/10.1007/s10482-016-0821-3

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