Fig. 3

Development and characterization of a new tool for staining cellulose in biofilms. (A) Design of IMT-CBD-mC. In this probe, CBD of CenA from C. fimi is linked to mCherry through an octa-glycine linker. (B) IMT-CBD-mC (left to the marker) and mCherry (right to the marker) were purified after overexpression in E. coli. Molecular weights on the marker are shown to the left, and the middle lane represents the marker (C). This probe was used to stain cellulose (left tube in the panel) and starch (right tube in the panel). 10 µM of the protein was incubated with 10 mg of cellulose and starch and incubated at 4˚C overnight. The amount of protein bound to the substrate ([B] µmol/g of the substrate) was quantitated using fluorescence from mCherry. A representative image of the IMT-CBD-mC binding to either substrate is shown above the graph (D) Binding of IMT-CBD-mC to chitin and cellulose to increasing concentrations of the protein (left to right). To test if the binding to cellulose is due to CBD and not due to mCherry, cellulose was stained with either of the purified proteins. Only IMT-CBD-mC was observed to bind to cellulose and chitin (i). (E) Purified IMT-CBD-mC was used to stain cellulose and visualized using fluorescence microscopy. The left panel shows the fluorescence of IMT-CBD-mC bound to cellulose, and the right panel shows a differential interference contrast (DIC) image of the stained cellulose crystals. (F) Solution depletion isotherm of IMT-CBD-mC against cellulose indicates that the IMT-CBD-mC probe effectively binds cellulose as its substrate. Y-axis ([B] µmol/g of the substrate) indicates bound protein per gram of cellulose; an X-axis indicates free ([F]) protein in the solution after binding. The Bmax value of the fitted curve is indicated at the top of the graph. A graph with a closed circle represents the actual data, and a line curve shows the mathematical model fitting with the data