Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates

Autores
Brambilla, Silvina Maricel; Soto, Gabriela; Odorizzi, Ariel; Arolfo, Valeria; McCormick, Wayne; Primo, Emiliano; Giordano, Walter; Jozefkowicz, Cintia; Ayub, Nicolás Daniel
Año de publicación
2020
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1–6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (−ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4–6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.
Instituto de Biotecnología
Fil: Brambilla, Silvina Maricel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.
Fil: Soto, Gabriela Cinthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.
Fil: Odorizzi, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina
Fil: Arolfo, Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina
Fil: McCormick, Wayne. Ottawa Research and Development Centre; Canadá
Fil: Primo, Emiliano. Universidad Nacional de Río Cuarto. Departamento de Biología Molecular; Argentina
Fil: Giordano, Walter. Universidad Nacional de Río Cuarto. Departamento de Biología Molecular; Argentina
Fil: Jozefkowicz, Cintia . Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.
Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentin
Fuente
Microbial Ecology 79 : 1044–1053 (2020)
Materia
Medicago sativa
Óxido Nitroso
Inoculación
Nitrato Reductasa
Rhizobiaceae
Factores Climáticos
Nitrous Oxide
Inoculation
Nitrate Reductase
Genes
Climatic Factors
Alfalfa
Sinorhizobium meliloti
Lucerne
Nivel de accesibilidad
acceso restringido
Condiciones de uso
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/7665
Acceder
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oai_identifier_str oai:localhost:20.500.12123/7665
network_acronym_str INTADig
repository_id_str l
network_name_str INTA Digital (INTA)
spelling Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different ClimatesBrambilla, Silvina MaricelSoto, GabrielaOdorizzi, ArielArolfo, ValeriaMcCormick, WaynePrimo, EmilianoGiordano, WalterJozefkowicz, CintiaAyub, Nicolás DanielMedicago sativaÓxido NitrosoInoculaciónNitrato ReductasaRhizobiaceaeFactores ClimáticosNitrous OxideInoculationNitrate ReductaseGenesClimatic FactorsAlfalfaSinorhizobium melilotiLucerneWe have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1–6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (−ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4–6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.Instituto de BiotecnologíaFil: Brambilla, Silvina Maricel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.Fil: Soto, Gabriela Cinthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.Fil: Odorizzi, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; ArgentinaFil: Arolfo, Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; ArgentinaFil: McCormick, Wayne. Ottawa Research and Development Centre; CanadáFil: Primo, Emiliano. Universidad Nacional de Río Cuarto. Departamento de Biología Molecular; ArgentinaFil: Giordano, Walter. Universidad Nacional de Río Cuarto. Departamento de Biología Molecular; ArgentinaFil: Jozefkowicz, Cintia . Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; ArgentinSpringer2020-08-04T11:39:43Z2020-08-04T11:39:43Z2020info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/7665https://link.springer.com/article/10.1007/s00248-019-01473-w0095-36281432-184Xhttps://doi.org/10.1007/s00248-019-01473-wMicrobial Ecology 79 : 1044–1053 (2020)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccess2025-09-29T13:44:59Zoai:localhost:20.500.12123/7665instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:45:00.101INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
title Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
spellingShingle Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
Brambilla, Silvina Maricel
Medicago sativa
Óxido Nitroso
Inoculación
Nitrato Reductasa
Rhizobiaceae
Factores Climáticos
Nitrous Oxide
Inoculation
Nitrate Reductase
Genes
Climatic Factors
Alfalfa
Sinorhizobium meliloti
Lucerne
title_short Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
title_full Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
title_fullStr Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
title_full_unstemmed Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
title_sort Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates
dc.creator.none.fl_str_mv Brambilla, Silvina Maricel
Soto, Gabriela
Odorizzi, Ariel
Arolfo, Valeria
McCormick, Wayne
Primo, Emiliano
Giordano, Walter
Jozefkowicz, Cintia
Ayub, Nicolás Daniel
author Brambilla, Silvina Maricel
author_facet Brambilla, Silvina Maricel
Soto, Gabriela
Odorizzi, Ariel
Arolfo, Valeria
McCormick, Wayne
Primo, Emiliano
Giordano, Walter
Jozefkowicz, Cintia
Ayub, Nicolás Daniel
author_role author
author2 Soto, Gabriela
Odorizzi, Ariel
Arolfo, Valeria
McCormick, Wayne
Primo, Emiliano
Giordano, Walter
Jozefkowicz, Cintia
Ayub, Nicolás Daniel
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Medicago sativa
Óxido Nitroso
Inoculación
Nitrato Reductasa
Rhizobiaceae
Factores Climáticos
Nitrous Oxide
Inoculation
Nitrate Reductase
Genes
Climatic Factors
Alfalfa
Sinorhizobium meliloti
Lucerne
topic Medicago sativa
Óxido Nitroso
Inoculación
Nitrato Reductasa
Rhizobiaceae
Factores Climáticos
Nitrous Oxide
Inoculation
Nitrate Reductase
Genes
Climatic Factors
Alfalfa
Sinorhizobium meliloti
Lucerne
dc.description.none.fl_txt_mv We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1–6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (−ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4–6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.
Instituto de Biotecnología
Fil: Brambilla, Silvina Maricel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.
Fil: Soto, Gabriela Cinthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.
Fil: Odorizzi, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina
Fil: Arolfo, Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina
Fil: McCormick, Wayne. Ottawa Research and Development Centre; Canadá
Fil: Primo, Emiliano. Universidad Nacional de Río Cuarto. Departamento de Biología Molecular; Argentina
Fil: Giordano, Walter. Universidad Nacional de Río Cuarto. Departamento de Biología Molecular; Argentina
Fil: Jozefkowicz, Cintia . Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentina.
Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular e Instituto de Genética; Argentin
description We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1–6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (−ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4–6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.
publishDate 2020
dc.date.none.fl_str_mv 2020-08-04T11:39:43Z
2020-08-04T11:39:43Z
2020
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/7665
https://link.springer.com/article/10.1007/s00248-019-01473-w
0095-3628
1432-184X
https://doi.org/10.1007/s00248-019-01473-w
url http://hdl.handle.net/20.500.12123/7665
https://link.springer.com/article/10.1007/s00248-019-01473-w
https://doi.org/10.1007/s00248-019-01473-w
identifier_str_mv 0095-3628
1432-184X
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
eu_rights_str_mv restrictedAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv Microbial Ecology 79 : 1044–1053 (2020)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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