Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen C...

Autores
Montero, Manuel; Rahimpour, Mehdi; Viale, Alejandro Miguel; Almagro, Goizeder; Eydallin, Gustavo; Sevilla, Angel; Canovas, Manuel; Bernal, Cristina; Lozano, Ana Belen; Muñoz, Francisco Jose; Bora Fernandez, Edurne; Bahaji, Abdellatif; Mori, Hirotada; Codoñer, Francisco M.; Potueza Romeo, Javier
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (DspoT) mutants obtained by transducing a DspoT allele from DrelADspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 DspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of DspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 DspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the DspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the DspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase
Fil: Montero, Manuel. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Rahimpour, Mehdi. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Viale, Alejandro Miguel. Gobierno de Navarra. Instituto de Agrobiotecnología; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Almagro, Goizeder. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Eydallin, Gustavo. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Sevilla, Angel. Universidad de Murcia; España
Fil: Canovas, Manuel. Universidad de Murcia; España
Fil: Bernal, Cristina. Universidad de Murcia; España
Fil: Lozano, Ana Belen. Universidad de Murcia; España
Fil: Muñoz, Francisco Jose. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Bora Fernandez, Edurne. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Bahaji, Abdellatif. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Mori, Hirotada. Nara Institute of Science and Technology. Graduate School of Biological Sciences; Japón
Fil: Codoñer, Francisco M.. Lifesequencing SL. Valencia; España
Fil: Potueza Romeo, Javier. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Materia
stringent response
glycogen metabolism
RelA
Escherichia coli
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/29425

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network_name_str CONICET Digital (CONICET)
spelling Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coliMontero, ManuelRahimpour, MehdiViale, Alejandro MiguelAlmagro, GoizederEydallin, GustavoSevilla, AngelCanovas, ManuelBernal, CristinaLozano, Ana BelenMuñoz, Francisco JoseBora Fernandez, EdurneBahaji, AbdellatifMori, HirotadaCodoñer, Francisco M.Potueza Romeo, Javierstringent responseglycogen metabolismRelAEscherichia colihttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (DspoT) mutants obtained by transducing a DspoT allele from DrelADspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 DspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of DspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 DspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the DspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the DspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetaseFil: Montero, Manuel. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Rahimpour, Mehdi. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Viale, Alejandro Miguel. Gobierno de Navarra. Instituto de Agrobiotecnología; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Almagro, Goizeder. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Eydallin, Gustavo. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Sevilla, Angel. Universidad de Murcia; EspañaFil: Canovas, Manuel. Universidad de Murcia; EspañaFil: Bernal, Cristina. Universidad de Murcia; EspañaFil: Lozano, Ana Belen. Universidad de Murcia; EspañaFil: Muñoz, Francisco Jose. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Bora Fernandez, Edurne. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Bahaji, Abdellatif. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Mori, Hirotada. Nara Institute of Science and Technology. Graduate School of Biological Sciences; JapónFil: Codoñer, Francisco M.. Lifesequencing SL. Valencia; EspañaFil: Potueza Romeo, Javier. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaPublic Library Of Science2014-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/29425Montero, Manuel; Rahimpour, Mehdi; Viale, Alejandro Miguel; Almagro, Goizeder; Eydallin, Gustavo; et al.; Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli; Public Library Of Science; Plos One; 9; 9; 9-2014; 1-161932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:28:01Zoai:ri.conicet.gov.ar:11336/29425instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:28:01.363CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
title Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
spellingShingle Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
Montero, Manuel
stringent response
glycogen metabolism
RelA
Escherichia coli
title_short Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
title_full Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
title_fullStr Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
title_full_unstemmed Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
title_sort Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
dc.creator.none.fl_str_mv Montero, Manuel
Rahimpour, Mehdi
Viale, Alejandro Miguel
Almagro, Goizeder
Eydallin, Gustavo
Sevilla, Angel
Canovas, Manuel
Bernal, Cristina
Lozano, Ana Belen
Muñoz, Francisco Jose
Bora Fernandez, Edurne
Bahaji, Abdellatif
Mori, Hirotada
Codoñer, Francisco M.
Potueza Romeo, Javier
author Montero, Manuel
author_facet Montero, Manuel
Rahimpour, Mehdi
Viale, Alejandro Miguel
Almagro, Goizeder
Eydallin, Gustavo
Sevilla, Angel
Canovas, Manuel
Bernal, Cristina
Lozano, Ana Belen
Muñoz, Francisco Jose
Bora Fernandez, Edurne
Bahaji, Abdellatif
Mori, Hirotada
Codoñer, Francisco M.
Potueza Romeo, Javier
author_role author
author2 Rahimpour, Mehdi
Viale, Alejandro Miguel
Almagro, Goizeder
Eydallin, Gustavo
Sevilla, Angel
Canovas, Manuel
Bernal, Cristina
Lozano, Ana Belen
Muñoz, Francisco Jose
Bora Fernandez, Edurne
Bahaji, Abdellatif
Mori, Hirotada
Codoñer, Francisco M.
Potueza Romeo, Javier
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv stringent response
glycogen metabolism
RelA
Escherichia coli
topic stringent response
glycogen metabolism
RelA
Escherichia coli
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (DspoT) mutants obtained by transducing a DspoT allele from DrelADspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 DspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of DspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 DspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the DspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the DspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase
Fil: Montero, Manuel. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Rahimpour, Mehdi. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Viale, Alejandro Miguel. Gobierno de Navarra. Instituto de Agrobiotecnología; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Almagro, Goizeder. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Eydallin, Gustavo. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Sevilla, Angel. Universidad de Murcia; España
Fil: Canovas, Manuel. Universidad de Murcia; España
Fil: Bernal, Cristina. Universidad de Murcia; España
Fil: Lozano, Ana Belen. Universidad de Murcia; España
Fil: Muñoz, Francisco Jose. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Bora Fernandez, Edurne. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Bahaji, Abdellatif. Gobierno de Navarra. Instituto de Agrobiotecnología; España
Fil: Mori, Hirotada. Nara Institute of Science and Technology. Graduate School of Biological Sciences; Japón
Fil: Codoñer, Francisco M.. Lifesequencing SL. Valencia; España
Fil: Potueza Romeo, Javier. Gobierno de Navarra. Instituto de Agrobiotecnología; España
description In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (DspoT) mutants obtained by transducing a DspoT allele from DrelADspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 DspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of DspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 DspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the DspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the DspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase
publishDate 2014
dc.date.none.fl_str_mv 2014-09
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/11336/29425
Montero, Manuel; Rahimpour, Mehdi; Viale, Alejandro Miguel; Almagro, Goizeder; Eydallin, Gustavo; et al.; Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli; Public Library Of Science; Plos One; 9; 9; 9-2014; 1-16
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/29425
identifier_str_mv Montero, Manuel; Rahimpour, Mehdi; Viale, Alejandro Miguel; Almagro, Goizeder; Eydallin, Gustavo; et al.; Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli; Public Library Of Science; Plos One; 9; 9; 9-2014; 1-16
1932-6203
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library Of Science
publisher.none.fl_str_mv Public Library Of Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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