A Type IX Secretion System Substrate Involved in Crystalline Cellulose Degradation by Affecting Crucial Cellulose Binding Proteins in Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an abundant soil cellulolytic bacterium that uses a unique cellulose degradation mechanism different from those that involve free cellulases or cellulosomes. Though several proteins have been identified as important for cellulose degradation, the mechanism used by to digest crystalline cellulose remains a mystery. In this study, was identified by insertional mutation and gene deletion as an important gene locus indispensable for crystalline cellulose utilization. Deletion of resulted in defects in crystalline cellulose utilization. The Δ mutant completely lost the ability to grow on crystalline cellulose, even with extended incubation, and selectively utilized the amorphous region of cellulose, leading to increased crystallinity. As a protein secreted by the type IX secretion system (T9SS), CHU_0922 was found to be located on the outer membrane, and the outer membrane localization of CHU_0922 relied on the T9SS. Comparative analysis of the outer membrane proteins revealed that the abundance of several cellulose-binding proteins, including CHU_1276, CHU_1277, and CHU_1279, was reduced in the Δ mutant. Further study showed that CHU_0922 is crucial for the full expression of the gene cluster containing , , , , and (), which is essential for cellulose utilization. Moreover, CHU_0922 is required for the cell surface localization of CHU_3220, a cellulose-binding protein that is essential for crystalline cellulose utilization. Our study provides insights into the complex system that uses to degrade crystalline cellulose. The widespread aerobic cellulolytic bacterium Cytophaga hutchinsonii, belonging to the phylum , utilizes a novel mechanism to degrade crystalline cellulose. No genes encoding proteins specialized in loosening or disruption the crystalline structure of cellulose were identified in the genome of , except for and . The crystalline cellulose degradation mechanism remains enigmatic. This study identified a new gene locus, , encoding a typical T9SS substrate that is essential for crystalline cellulose degradation. Notably, CHU_0922 is crucial for the normal transcription of , , , , and (), which play important roles in the degradation of cellulose. Moreover, CHU_0922 participates in the cell surface localization of CHU_3220. These results demonstrated that CHU_0922 plays a key role in the crystalline cellulose degradation network. Our study will promote the uncovering of the novel cellulose utilization mechanism of