Genome Structure of Bacillus cereus tsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

Figure 4

Characterization of cellulase gene and enzymatic activity of Bacillus cereus tsu1. (a) Cellulase genes annotated in the Bacillus cereus tsu1 genome. (b) Congo red plate assay of cellulase activity. Bacteria were cultured on the double-layered carboxymethylcellulose sodium salt- (CMC-Na-) containing plates of which the bottom M9 minimal salt (11%; wt/vol) agar medium was overlaid with soft-agar containing 1% (wt/vol) CMC-Na (Sigma, St. Louis, MO). Plates from two-day incubation at 37°C were stained with 0.1% Congo red. The yellowish halo around the bacterial colony indicates degradation of CMC. Plate 1, E. coli showing no CMC degradation activity (negative control); Plate 2, B. cereus tsu1; Plate 3, Paenibacillus polymyxa with CMC degradation activity (a positive control of cellulolytic bacterial strain from the Bacillus Genetic Stock Center, Columbus, OH). (c) Gel permeation assay of CMC derived products after incubation with B. cereus tsu1 for 2 days and 6 days. The right shifts of the peaks indicate that the CMC derived molecules after the digestion with B. cereus tsu1 were smaller in size and therefore they were eluted at a delayed time-frame than the original CMC. These results confirmed the extracellular cellulase activity of the bacterial strain.

(a) Gene sequences

(b) Congo Red staining assay on CMC plates. Plate 1, Escherichia coli (negative control); Plate 2, Bacillus cereus tsu1; Plate 3, Paenibacillus polymyxa (positive control)

(c) Gel permeation chromatography assay of products derived from CMC cultured with Bacillus cereus tsu1