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Viale, Alejandro M.

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Viale

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Alejandro M.

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Instituto de Agrobiotecnología (IdAB)

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2006 - 200912010 - 20141
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Author: Eydallin, Gustavo × Subject: Escherichia coli ×

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Now showing 1 - 2 of 2
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    PublicationOpen Access
    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
    (Public Library of Science, 2014) Montero Macarro, Manuel; Rahimpour, Mehdi; Viale, Alejandro M.; Almagro Zabalza, Goizeder; Eydallin, Gustavo; Sevilla, Ángel; Cánovas, Manuel; Bernal, Cristina; Lozano, Ana Belén; Muñoz Pérez, Francisco José; Baroja Fernández, Edurne; Bahaji, Abdellatif; Mori, Hirotada; Codoñer, Francisco M.; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    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.
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    PublicationOpen Access
    Glycogen phosphorylase, the product of the glgP Gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli
    (American Society for Microbiology, 2006) Alonso Casajús, Nora; Dauvillee, David; Viale, Alejandro M.; Muñoz Pérez, Francisco José; Baroja Fernández, Edurne; Morán Zorzano, María Teresa; Eydallin, Gustavo; Ball, Steven; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    To understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (ΔglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in ΔglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by ΔglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.