Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

Abstract

The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.

Importance: The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.