Spontaneous suppressor mutations can compensate the total absence of SpoT in Escherichia coli

Póster presentado en el XXIII Congreso Nacional de Microbiología (SEM), celebrado en Salamanca del 11 al 14 de julio de 2011.Peer Reviewe

GlgS, previously described as a glycogen synthesis control protein, is a major negative regulator of flagellar synthesis and motility, type 1 fimbriae adhesins and biofilm polysaccharides production in Escherichia coli

Trabajo presentado en la 9th Warsaw International Medical Congress for Young Scientists (WIMC), celebrada en Varsovia del 9 al 12 de mayo de 2013.Peer Reviewe

GlgS, previously described as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli

Trabajo presentado en el XXIV Congreso de Microbiología SEM (Sociedad Española de Microbiología), celebrado en L´Hospitalet del 10 al 13 de julio de 2013.Peer Reviewe

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

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 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; Españ

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

Pozueta Romero, Javier et al.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.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 Científicas. AMV is grateful to the funding of the Programa Campus Ibericus de Excelencia Internacional, Ministerio de Educación, Spain. His 2-months visit (January–March 2014) to the Institute of Agrobiotechnology, Public University of Navarra, Pamplona, Spain, was included into the Proyecto financiado por el Ministerio de Educación en el marco del Programa Campus de Excelencia Internacional.Peer Reviewe

Regulation of glycogen metabolism in yeast and bacteria

34 p., 10 figures and bibliographyMicroorganisms have the capacity to utilize a variety of nutrients and adapt to continuously changing environmental conditions. Many microorganisms, including yeast and bacteria, accumulate carbon and energy reserves to cope with the starvation conditions temporarily present in the environment. Glycogen biosynthesis is a main strategy for such metabolic storage, and a variety of sensing and signaling mechanisms have evolved in evolutionarily distant species to ensure the production of this homopolysaccharide. At the most fundamental level, the processes of glycogen synthesis and degradation in yeast and bacteria share certain broad similarities. However, the regulation of these processes is sometimes quite distinct, indicating that they have evolved separately to respond optimally to the habitat conditions of each species. This review aims to highlight the mechanisms, both at the transcriptional and at the post-transcriptional level, that regulate glycogen metabolism in yeast and bacteria, focusing on selected areas where the greatest increase in knowledge has occurred during the last few years. In the yeast system, we focus particularly on the various signaling pathways that control the activity of the enzymes of glycogen storage. We also discuss our recent understanding of the important role played by the vacuole in glycogen metabolism. In the case of bacterial glycogen, special emphasis is placed on aspects related to the genetic regulation of glycogen metabolism and its connection with other biological processes.This research was partially supported by the grant BIO2007-63915 from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain), and by Iden Biotechnology S.L. M.M. acknowledges a postdoctoral contract from the I3P program of Consejo Superior de Investigaciones Científicas. The work of W.A.W. was supported in part by grant GM081810 from the National Institutes of Health, and work in the laboratory of P.J.R. was supported by grants DK27221 and DK42576, also from the National Institutes of Health

Occurrence of more than one important source of ADPglucose linked to glycogen biosynthesis in Escherichia coli and Salmonella

AbstractTo explore the possible occurrence of sources, other than GlgC, of ADPglucose linked to bacterial glycogen biosynthesis we characterized Escherichia coli and Salmonella ΔglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery. These mutants displayed the expected glycogen-less phenotype but accumulated ADPglucose. Importantly, ΔglgCAP cells expressing the glycogen synthase encoding glgA gene accumulated glycogen. Protein chromatographic separation of crude extracts of ΔglgCAP mutants and subsequent activity measurement analyses revealed that these cells possess various proteins catalyzing the conversion of glucose-1-phosphate into ADPglucose. Collectively these findings show that enterobacteria possess more than one important source of ADPglucose linked to glycogen biosynthesis