Almagro Zabalza, Goizeder
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Almagro Zabalza
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Goizeder
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Instituto de Agrobiotecnología (IdAB)
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Publication Open 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 Bailone, 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 PublikoaIn 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.Publication Open Access A cAMP/CRP-controlled mechanism for the incorporation of extracellular ADP-glucose in Escherichia coli involving NupC and NupG nucleoside transporters(Nature Research, 2018) Almagro Zabalza, Goizeder; Viale Bailone, Alejandro M.; Montero Macarro, Manuel; Muñoz Pérez, Francisco José; Baroja Fernández, Edurne; Mori, Hirotada; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions.Publication Open Access Identification, characterization and evolutionary history of the Escherichia coli glycogen operon(2014) Almagro Zabalza, Goizeder; Pozueta Romero, Javier; Baroja Fernández, Edurne; Producción Agraria; Nekazaritza EkoizpenaRepresenting one of the major storage carbohydrate in many bacteria, glycogen is a branched homopolysaccharide of α-1,4-linked glucose subunits with α-1,6-linked glucose at the branching points that accumulates under conditions of limiting growth when an excess of carbon is available and other nutrients are deficient. The exact role of this polyglucan in bacteria is not as clear-cut as in animal and yeast cells, but some studies have linked glycogen to extended bacterial survival, symbiotic performance, colonization and virulence. It is widely accepted that genes involved in Escherichia coli and Salmonella enterica glycogen metabolism are clustered in two tandemly arranged operons: glgBX (encompassing the genes coding for glycogen branching (GlgB) and debranching (GlgX) enzymes), and glgCAP (encoding the GlgC and GlgA anabolic enzymes, as well as the catabolic glycogen phosphorylase (GlgP)). However, the data regarding regulatory aspects of the expression of glycogen genes in E. coli are contradictory, thus questioning the presence of two operons. To get inshight into the trascriptional organization of glycogen genes, in the first chapter of this work I characterized glg genes transcription using RT (reverse transcriptase)-PCR approach. This analysisW revealed that E. coli cells possess transcripts comprising the five glgBXCAP genes. glg::lacZY expression analyses in cells lacking the region immediately upstream of the glgB gene revealed an almost total abolishment of glgB, glgX and glgC expression and a reduced expression of glgA and glgP. Similar type of analyses showed that glgA and glgP expression was almost totally abolished in cells lacking glgA upstream sequences, including glgC, glgB and the asd-glgB intergenic region upstream of glgB. All the data indicate that the five glgBXCAP genes are transcribed in a single transcriptional unit under the control of promoter sequences upstream of glgB and that an alternative suboperonic promoter driving glgA and glgP expression is located within glgC. Using computer searches for putative bacterial promoters and 5¿ RACE (rapid amplification of cDNA ends) techniques I identified the -35 and -10 sequences of both promoters as well as the trasnscription start sites. Finally, I measured glg::lacZY expression on cells lacking the relA or phoP regulatory genes. These analyses indicated that both glgBXCAP operon and the suboperonic promoter form part of RelA and PhoP-PhoQ regulons. To gain insight into the origin and evolutionary history of E. coli glgBXCAP operon and of its constituent genes, in the second chapter of this work I carried out a detailed comparative analyses of presence, copy number and arrangement of glg genes in 265 gammaproteobacterial species. These analyses revealed the occurrence of large variations in glg homolog copy number and arrangements. Subsequently, I carried out a phylogenetic analysis of glg genes of selected Gammaproteobacteria and I also explored the phylogenetic relationships of gammaproteobacterial glg genes with those of representative species of the main bacterial groups. I found an important discrepancy between the evolution of glg genes and the order of organismal descent, inferred from 16S rRNA analysis, indicating that glg genes have undergone a complex evolutionary history in which horizontal gene transfer have played an important role. However, I detected a notable exception constituted by Enterobacteriales/Pasteurellales (E/P) glg genes which show a strict vertical inheritance. These analyses also revelaed that E/P glg genes are related with glg genes of phylogenetically distant betaproteobacterial species Varivorax paradoxus S110, Thiomonas intermedia K12, Lepthotrix cholodnii SP-6 and Thauera mz1t. The genomic context analysis indicated that the glgBXCAP arrangement is conserved in the E/P group and interestingly, that the above mentioned betaproteobacterial species possess glgBXCAP and/or gene clusters very similar to glgBXCAP. The overall data allowed me tracing the evolutionary origin of glgBXCAP operon in the last common ancestor of the E/P group and suggest a possible horizontal gene transfer event of the glgBXCAP cluster from the E/P group to Betaproteobacteria.Publication Open Access Comparative genomic and phylogenetic analyses of gammaproteobacterial glg genes traced the origin of the Escherichia coli glycogen glgBXCAP operon to the last common ancestor of the sister orders enterobacteriales and pasteurellales(Public Library of Science, 2015) Almagro Zabalza, Goizeder; Viale Bailone, Alejandro M.; Montero Macarro, Manuel; Rahimpour, Mehdi; Muñoz Pérez, Francisco José; Baroja Fernández, Edurne; Bahaji, Abdellatif; Zúñiga, Manuel; González Candelas, Fernando; Pozueta Romero, Javier; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaProduction of branched α-glucan, glycogen-like polymers is widely spread in the Bacteria domain. The glycogen pathway of synthesis and degradation has been fairly well characterized in the model enterobacterial species Escherichia coli (order Enterobacteriales, class Gammaproteobacteria), in which the cognate genes (branching enzyme glgB, debranching enzyme glgX, ADP-glucose pyrophosphorylase glgC, glycogen synthase glgA, and glycogen phosphorylase glgP) are clustered in a glgBXCAP operon arrangement. However, the evolutionary origin of this particular arrangement and of its constituent genes is unknown. Here, by using 265 complete gammaproteobacterial genomes we have carried out a comparative analysis of the presence, copy number and arrangement of glg genes in all lineages of the Gammaproteobacteria. These analyses revealed large variations in glg gene presence, copy number and arrangements among different gammaproteobacterial lineages. However, the glgBXCAP arrangement was remarkably conserved in all glg-possessing species of the orders Enterobacteriales and Pasteurellales (the E/P group). Subsequent phylogenetic analyses of glg genes present in the Gammaproteobacteria and in other main bacterial groups indicated that glg genes have undergone a complex evolutionary history in which horizontal gene transfer may have played an important role. These analyses also revealed that the E/P glgBXCAP genes (a) share a common evolutionary origin, (b) were vertically transmitted within the E/P group, and (c) are closely related to glg genes of some phylogenetically distant betaproteobacterial species. The overall data allowed tracing the origin of the E. coli glgBXCAP operon to the last common ancestor of the E/P group, and also to uncover a likely glgBXCAP transfer event from the E/P group to particular lineages of the Betaproteobacteria.