The molecular basis for a temperature-sensitive Mycobacterium smegmatis glgE mutant [article]

Karl Syson, Sibyl F. D. Batey, Steffen Schindler, Rainer Kalscheuer, Stephen Bornemann
2020 bioRxiv   pre-print
Background- 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
more » ... in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. Methods- We reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. Results- The mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature rather than glycogen. 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 primarily due to the lowering of the kcatapp of GlgE, rather than a loss of thermo stability. The reported rescue of the phenotype by GarA appeared not to involve a direct interaction with GlgE. It more likely acts as a decoy to prevent the Ser/Thr protein kinase PknB from phosphorylating GlgE, alleviating the polymerase from negative regulation. Conclusions- We have therefore been able to reconcile apparently contradictory observations and shed light on the molecular basis for the phenotype of the temperature-sensitive mutation. General Significance- This study highlights how perturbing flux through the GlgE pathway without blocking it can affect the growth rate of mycobacteria.
doi:10.1101/2020.04.23.057067 fatcat:khx76bdtcbfolaflkq2hga4ale