Role of the Escherichia coli glgX Gene in Glycogen Metabolism

David DauvilléE,Michael S Samuel,Lynette Rampling,Zhongyi Li,Behjat Kosar-Hashemi,Isabelle S Kinderf,Steven Ball,Matthew K Morell

doi:10.1128/jb.187.4.1465-1473.2005

Abstract

A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.

Highlights

The synthesis of glycogen in Escherichia coli occurs when carbon is abundant but another nutrient required for growth is limiting
In E. coli these genes are located in a gene cluster which includes the glgC, glgA, and glgB genes and two other genes thought to be primarily involved in glycogen degradation, glgX and glgP, encoding a putative glycogen debranching enzyme (EC 3.2.1.-) and glycogen phosphorylase (EC 1.4.1.1), respectively [30]
Previous studies had shown that the glgX gene was not absolutely required for synthesis of linear ␣-1,4-glucosylglucan [30], no E. coli strain lacking the glgX gene alone was available prior to this study

Summary

Introduction

The synthesis of glycogen in Escherichia coli occurs when carbon is abundant but another nutrient required for growth is limiting. Class A mutants were devoid of iodine-staining polysaccharide, class B mutants displayed blue staining that revealed the presence of moderately branched polysaccharides, and class C mutants displayed darker staining than the wild type owing to glycogen overproduction [7] These genetic and biochemical studies demonstrated that two activities are absolutely required for the synthesis of ␣-1,4-glucosyl-glucan, ADPglucose pyrophosphorylase (EC 2.7.7.17; encoded by glgC) and glycogen synthase (EC 2.4.1.21; encoded by glgA). Examples of type 1 mutants are the sugary-1 maize mutant, whose mutation is best known as the mutation underpinning sweet corn; the sugary rice mutant, which has a similar phenotype [24]; and the sta isoamylasedeficient mutant of Chlamydomonas reinhardtii [22], which lacks granular starch but produces a phytoglycogen-like molecule Analysis of these mutants led to the formulation of a hypothesis which suggests that debranching activities are required to modify the structure of the growing amylopectin. We describe the creation by homologous recombination of E. coli strains having defined mutations in glgX, and we describe the accumulation and structure of the glycogen produced by these mutants and relate these properties to the properties of the purified recombinant enzyme

Methods

Results

Conclusion