Cytophaga hutchinsonii chu_2177, encoding the O-antigen ligase, is essential for cellulose degradation

Cytophaga hutchinsonii can efficiently degrade crystalline cellulose, in which the cell surface cellulases secreted by the type IX secretion system (T9SS) play important roles, but the degradation mechanism remains unclear, and the anchor mechanism of cellulases on the outer membrane in C. hutchinsonii has not been studied. Here, chu_2177 was identified by transposon mutagenesis and was proved to be indispensable for cellulose utilization in C. hutchinsonii. Disruption of chu_2177 resulted in O-antigen deficiency and chu_177 could confer O-antigen ligase activity upon an Escherichia coli waal mutant, indicating that chu_2177 encoded the O-ntigen ligase. Moreover, deletion of chu_2177 caused defects in cellulose utilization, cell motility, biofilm formation, and stress resistance. Further study showed that the endoglucanase activity was markedly decreased in the outer membrane but was increased in the culture fluid without chu_2177. Western blot proved that endoglucanase CHU_1336 was not located on the outer membrane but was released in the culture fluid of the Δ2177 mutant. Further proteomics analysis showed that many cargo proteins of T9SS were missing in the outer membrane of the Δ2177 mutant. Our study revealed that the deletion of chu_2177 affected the localization of many T9SS cargo proteins including cellulases on the outer membrane of C. hutchinsonii.