Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis
Fig 1
Evidence for an alternative route to M1P in M. smegmatis independent of TreS-Pep2 and trehalose.
(A) Heterologous expression of the M. tuberculosis glgC gene (Rv1213) restores M1P accumulation in the M. smegmatis ΔglgE Δpep2 double mutant harboring a spontaneous IS1096 element insertion in the endogenous glgC locus (i.e. M. smegmatis ΔglgE Δpep2 glgC:IS1096). Equivalent quantities of crude extracts of M. smegmatis strains were analyzed using 1H NMR spectroscopy. The assignment of the peaks was based on our previous studies [16, 18]. (B) Hot water extracts from 1 ml culture aliquots of M. smegmatis strains (normalized to OD600 nm = 0.5) were analyzed by TLC, demonstrating M1P accumulation in the M. smegmatis ΔglgE Δpep2 glgC:IS1096 strain expressing the M. tuberculosis glgC gene (Rv1213). M1P and trehalose (5 μg each) were used as standards. (C) M. smegmatis Δpep2(u) ΔglgE and ΔtreS(u) ΔglgE double mutants accumulate M1P. TLC analysis was performed as described in (B). (D) The M. smegmatis Δpep2(u) ΔglgE mutant is trehalose resistant despite accumulating M1P, indicating trehalose-independent M1P formation. Strains were grown on Middlebrook 7H10 agar plates with or without 1 mM trehalose and incubated at 37°C for 3 days. (E) Conditional silencing of the glgE gene in M. smegmatis mutant strains reveals the requirement of GlgC and GlgA for the alternative route to M1P synthesis. Cells of the indicated conditional c-glgE-tet-off mutant strains were cultivated for 24 h with or without 1 μg ml-1 ATc as indicated, and hot water extracts from 1 ml culture aliquots (normalized to OD600 nm = 0.5) were analyzed by TLC.