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
Fecha
2014Autor
Versión
Acceso abierto / Sarbide irekia
Tipo
Artículo / Artikulua
Versión
Versión publicada / Argitaratu den bertsioa
Impacto
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10.1371/journal.pone.0106938
Resumen
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. Iodi ...
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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. [--]
Materias
Escherichia coli,
relA locus,
spoT,
Glycogen
Editor
Public Library of Science
Publicado en
Plos One, 9(9):e106938
Departamento
Universidad Pública de Navarra/Nafarroako Unibertsitate Publikoa. IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Versión del editor
Entidades Financiadoras
This research was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) [grant numbers BIO2010-18239 and BIO2011-29233-002-01], the Fundación Séneca [grant number 08660/P1/08] and JSPS (Japan Society for the Promotion of Science) KAKENHI Grant-in-Aid for Scientific Research (A) [grant number 22241050]. GA and GE acknowledge fellowships from the Public University of Navarra. MR acknowledges a pre-doctoral JAE fellowship from the Consejo Superior de Investigaciones Cientı´ficas. AMV is grateful to the funding of the Programa Campus Ibericus de Excelencia Internacional, Ministerio de Educación, Spain.