Fig. 6

Analyses of cellulosomes and cellulose-bound proteins synthesized by the various R. cellulolyticum recombinant strains. Cellulose-bound proteins and cellulosomes were isolated by filtration of 6-day cultures of the various recombinant strains in basal rich medium containing 5 g/L of microcrystalline cellulose (Sigmacell 20). The left panel shows the size exclusion profile of the cellulose-bound fraction produced by the control strain (top), the strain carrying pCel9A (middle) and the strain harboring pCel9Ac (bottom). The ordinate “mAU” designates milli Absorbance Unit at 280 nm. The fractions analyzed by SDS-PAGE and western blots in each chromatogram are indicated. The other panel shows from left to right SDS-PAGE (stained using Coomassie blue), and western blot analyses using fluorescent Cel5A (for detection of the scaffoldin CipC), anti-His tag, anti-Cel48F, anti-Cel9G, and biotinylated hybrid scaffoldin Scaf4 (for detection of dockerin-containing proteins), respectively. The staining procedure or probes used for western blot analyses are indicated at the bottom of each gel. Lane C designates the cellulosomes; lanes 38 (observed for strain carrying pCel9Ac), 42 (observed for strain carrying pCel9Ac), 45 (observed for strain carrying pCel9A) and 49 (observed for all strains) designate the corresponding peaks generated during the size exclusion chromatography (left panel). For each series of three pictures, the top photo corresponds to the control strain, the middle photo designates the strain carrying pCel9A, and the bottom picture designates the strain harboring pCel9Ac.