-Glycosylation of a Cargo Protein C-Terminal Domain Recognized by the Type IX Secretion System in Cytophaga Hutchinsonii Affects Protein Secretion and Localization

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum . It digests crystalline cellulose with an unknown mechanism and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of the CTD in the secretion and localization of T9SS substrates in were studied by fusing the green fluorescent protein (GFP) with the CTD from CHU_2708. The CTD is necessary for the secretion of GFP by T9SS. The GFP-CTD fusion protein was found to be glycosylated in the periplasm, with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide--glycosidase F, which can hydrolyze -linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTD suggested that -glycosylation occurred on the CTD. CTD glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in . Glycosyltransferase-encoding gene , a homologous gene of Campylobacter jejuni , was found to participate in the -glycosylation of . Deletion of affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided evidence that the CTD as the signal of T9SS was -glycosylated in the periplasm of . The bacterial -glycosylation system has previously been found only in several species of and , and the role of -linked glycans in bacteria is still not fully understood. has a unique cell contact cellulose degradation mechanism, and many cell surface proteins, including cellulases, are secreted by the T9SS. In this study, we found that , a member of the phylum , has an -glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the -glycosylation of proteins and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, -glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of the CTD appears to play an important role in affecting T9SS substrate transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of -glycosylation in bacteria.