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.

Manuel Montero,Francisco M Codoñer,Javier Pozueta-Romero,Alejandro M Viale,Cristina Bernal,Francisco José Muñoz,Hirotada Mori,Abdellatif Bahaji,Mehdi Rahimpour,Manuel Cánovas,Goizeder Almagro,Ana Belén Lozano,Gustavo Eydallin,Ángel Sevilla,Edurne Baroja-Fernández

doi:10.1371/journal.pone.0106938

Abstract

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 (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT 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 ΔspoT 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 ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT 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 ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT 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.

Highlights

In conditions in which carbon sources are in excess and other nutrients are deficient, Escherichia coli and many bacterial species accumulate glycogen, a branched homopolysaccharide of a-1,4linked glucose subunits with a-1,6-linkages at the branching points [1,2]
In a search for main intracellular signals regulating glycogen metabolism in this model organism, we found that glgBXCAP glycogen gene expression and consequent glycogen accumulation were highly impaired in cells bearing a relA null ((DrelA) allele [7]
We selected KM for the experiments described in this work, reasoning that the use of enriched culture media for E. coli growth should allow a better definition of the threshold ppGpp cellular levels required to trigger glycogen accumulation

Summary

Introduction

In conditions in which carbon sources are in excess and other nutrients are deficient, Escherichia coli and many bacterial species accumulate glycogen, a branched homopolysaccharide of a-1,4linked glucose subunits with a-1,6-linkages at the branching points [1,2]. In a search for main intracellular signals regulating glycogen metabolism in this model organism, we found that glgBXCAP glycogen gene expression and consequent glycogen accumulation were highly impaired in cells bearing a relA null ((DrelA) allele [7]. These results reinforced long proposals that the RelA product ppGpp controls glycogen biosynthesis in E. coli [1,2]. Since ppGpp lies at the top of the network governing global gene expression in response to nutrient availability [9,10,11,12], the overall observations pointed to this nucleotide as a master controller of E. coli glycogen metabolism

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