A Disulfide Oxidoreductase CHU_1165 is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii.

Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it utilizing a novel cell-contact dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus chu_1165, which encodes a hypothetical protein with a 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that the cells of Delta1165 could not bind to cellulose and many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar Trx-fold containing a Cys-X-X-Cys motif which is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106, 108 were indispensable for the function of CHU_1165. Western blot showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine and the mutation of the cysteine in them did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCE Cytophaga hutchinsonii could rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation through affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies showed that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and the mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate these proteins in the formation of a disulfide bond. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.