Iodine staining. Cell patches were grown overnight on M9 medium plates supplemented with glucose. The plates were then flooded with an iodine solution (4% I₂, 0.4% KI). (A) Cell patches 1 to 4 correspond to RR1, IK5, BWX1, and the transduced strain containing the IK5 mutation in the BW25113 genetic background, respectively. (B) Three different glgX mutant strains (IK5, BWX1, and BWX2 containing an empty pUC19) are at the top (cell patches 5, 6, and 7, respectively), and the same strains carrying the complementation plasmid (pX) are at the bottom (cell patches 8, 9, and 10, respectively). The M9 medium plate used for this test was supplemented with 100 μg of ampicillin per ml and 5 mM IPTG.

Debranching activity of E. coli extracts with various substrates. Crude extracts of RR1 (solid bars) and IK5 (open bars) were prepared from log-phase cells grown in LB medium. The phosphorylase-limit dextrin of glycogen (GPLD) was produced by exhaustive incubation of glycogen with excess phosphorylase in the presence of phosphate buffer until no further release in G1P from glycogen in the presence of P_i and active enzyme was obtained. (A) Activity determined by a glucan solubilization assay procedure. The activities are expressed in nanomoles of glucose equivalents liberated per minute per milligram of protein. (B) Debranching activity measured by the branch linkage assay. The assay conditions used are described in Materials and Methods. The activities are expressed in nanomoles of equivalent reducing ends produced per minute per milligram of protein. (C) Debranching activity on GPLD assayed with wild-type strains RR1 and BW25113 and the IK5, BWX1, and BWX2 glgX-deficient strains. The solid bars indicate the activities of the original strains (in nanomoles of glucose equivalents liberated per minute per milligram of protein), and the open bars indicate the activities of the same strains containing the pX plasmid and grown in the presence of 100 μg of ampicillin per ml.

Analysis of GlgX activity by ¹H-NMR spectroscopy. The proton NMR spectrum of phosphorylase-limit dextrin (A) is compared to the spectra of the phosphorylase-limit dextrin cleavage products induced by wild-type (C) and mutant (D) extracts and the phosphorylase-limit dextrin cleavage products generated by pure commercial isoamylase (B). The chemical shifts for the protons of α-(1,4) linkages and α-(1,6) linkages are at 5.4 and 5.0 ppm, respectively. The chemical shift of the α-anomer of the reducing ends produced is at 5.27 ppm.

Glycogen accumulation by IK5 (○), MS201 (▴), RR1 (•), BW25113 (▪), BWX1 (×), and BWX2 (□). Strains were grown in M9 medium containing 4 g of glucose per liter (and 50 μg of kanamycin per ml for IK5 and BWX1). (A) Ratio of glycogen to protein accumulation over time. (B) Cell density, as measured by A₆₀₀ (600 nm OD).

Chain length distribution of oligosaccharides determined by capillary electrophoresis of the isoamylase-debranched glycogen produced by RR1 (•) and IK5 (○). (A) Chain length distributions expressed as the percentage of total chains, expressed on a molar basis. (B) Difference plot generated by subtracting the molar percentage for IK5 at each chain length from the corresponding molar percentage for RR1 at the corresponding chain length.

Products of GlgX incubation with glycogen: detection of the oligosaccharides produced by incubation of purified GST-GlgX (shaded bars) and purified six-His-tagged GlgX (open bars) with rabbit liver glycogen by capillary electrophoresis. The results are expressed as percentages of the DP liberated. The x axis indicates the degree of polymerization. The results were obtained with two different enzyme preparations for each tagged protein.