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Systematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spoT loss and affect glycogen content in Escherichia coli

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dc.citation.titlePLoS ONE
dc.citation.volume9
dc.creatorMontero, Manuel
dc.creatorRahimpour, Mehdi
dc.creatorViale, Alejandro M.
dc.creatorAlmagro, Goizeder
dc.creatorEydallin, Gustavo
dc.creatorSevilla, Ángel
dc.creatorCánovas, Manuel
dc.creatorBernal, Cristina
dc.creatorMuñoz, Francisco José
dc.creatorBaroja Fernández, Edurne
dc.creatorBahaji, Abdellatif
dc.creatorMori, Hirotada
dc.creatorCodoñer, Francisco M.
dc.creatorPozueta Romero, Javier
dc.date.accessioned2024-05-30T16:48:35Z
dc.date.available2024-05-30T16:48:35Z
dc.date.issued2014-09-04
dc.description.abstractIn 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 médium 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.
dc.description.filFil: Montero, Manuel. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Rahimpour, Mehdi. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Viale, Alejandro M. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Consejo Nacional de Investigaciones Científicas y Técnicas Instituto de Biología Molecular y Celular de Rosario. Departamento de Microbiología. (CONICET-IBR); Argentina.
dc.description.filFil: Almagro, Goizeder. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Eydallin, Gustavo. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Sevilla, Ángel. Universidad de Murcia. Facultad de Química. Campus de Excelencia Internacional Regional ‘‘Campus Mare Nostrum’’. Departamento de Bioquímica y Biología Molecular e Inmunología; Spain.
dc.description.filFil: Cánovas, Manuel. Universidad de Murcia. Facultad de Química. Campus de Excelencia Internacional Regional ‘‘Campus Mare Nostrum’’. Departamento de Bioquímica y Biología Molecular e Inmunología; Spain.
dc.description.filFil: Bernal, Cristina. Universidad de Murcia. Facultad de Química. Campus de Excelencia Internacional Regional ‘‘Campus Mare Nostrum’’. Departamento de Bioquímica y Biología Molecular e Inmunología; Spain.
dc.description.filFil: Lozano, Ana Belén. Universidad de Murcia. Facultad de Química. Campus de Excelencia Internacional Regional ‘‘Campus Mare Nostrum’’. Departamento de Bioquímica y Biología Molecular e Inmunología; Spain.
dc.description.filFil: Muñoz, Francisco José. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Baroja Fernández, Edurne. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Bahaji, Abdellatif. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Mori, Hirotada. Nara Institute of Science and Technology. Graduate School of Biological Sciences; Japan.
dc.description.filFil: Pozueta Romero, Javier. Universidad Pública de Navarra. Instituto de Agrobiotecnología; Spain.
dc.description.filFil: Pozueta Romero, Javier. Lifesequencing S.L.; Spain.
dc.description.sponsorshipComisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional:BIO2010-18239, BIO2011-29233-002-01
dc.description.sponsorshipFundación Seneca: 08660/P1/08
dc.description.sponsorshipJapan Society for the Promotion of Science
dc.description.sponsorshipKAKENHI Grant-in-Aid for Scientific Research: 22241050
dc.format.extent1-16
dc.identifier.issn1932-6203
dc.identifier.urihttps://hdl.handle.net/2133/27106
dc.language.isoen
dc.publisherPublic Library of Science
dc.relation.publisherversionhttps://doi.org/10.1371/journal.pone.0106938
dc.relation.publisherversionhttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106938
dc.rightsopenAccess
dc.rights.holderMontero, Manuel
dc.rights.holderRahimpour, Mehdi
dc.rights.holderViale, Alejandro M.
dc.rights.holderAlmagro, Goizeder
dc.rights.holderEydallin, Gustavo
dc.rights.holderSevilla, Ángel
dc.rights.holderCánovas, Manuel
dc.rights.holderBernal, Cristina
dc.rights.holderLozano, Ana Belén
dc.rights.holderMuñoz, Francisco José
dc.rights.holderBaroja Fernández, Edurne
dc.rights.holderBahaji, Abdellatif
dc.rights.holderMori, Hirotada
dc.rights.holderCodoñer, Francisco M.
dc.rights.holderPozueta Romero, Javier
dc.rights.holderUniversidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas
dc.rights.textAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectAlleles
dc.subjectAmino acid sequence
dc.subjectClone cells
dc.subjectEscherichia coli
dc.subjectGene expression regulation
dc.subjectGenetic loci
dc.titleSystematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spoT loss and affect glycogen content in Escherichia coli
dc.typearticulo
dc.type.versionpublishedVersion

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