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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/29425
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oai:ri.conicet.gov.ar:11336/29425 |
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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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1846781853974921216 |
score |
13.229304 |