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The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. We reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. The mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the k cat of GlgE as well as a loss of stability 42 °C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. We have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. This study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria. Unlabelled Image • A single amino acid substitution in GlgE leads to loss of activity and stability. • Temperature-sensitivity leads to the accumulation of GlgE's substrate in vivo. • Reduced bacterial growth can be attributed to the accumulation of this substrate. • It was shown how a glycogen assay can give a false positive with this substrate. • This reconciles apparently contradictory observations published previously. [ABSTRACT FROM AUTHOR]