Abstract
Members of the phylum Bacteroidota utilize the type IX secretion system (T9SS) to transport diverse substrates into the environment or onto their surface. T9SS substrates feature a Sec-dependent signal peptide for export to the periplasm and a conserved C-terminal domain (CTD), recognized by the T9SS, for translocation across the outer membrane. Following translocation, substrates engage with a shuttle protein, which ensures their final localization. Most CTDs are classified into two major families. Type A CTDs are all recognized by the PorV shuttle. Recognition and transport of Type B CTDs remain less explored. Flavobacterium johnsoniae encodes 12 Type B substrates, often genetically linked to genes encoding PorP/SprF-like shuttle proteins. We demonstrate that two Type B substrates indeed rely on their cognate PorP/SprF specialized shuttle proteins for secretion and identify the shuttle responsible for the secretion of three orphan Type B CTDs. Our findings also reveal that five conserved motifs within Type B CTDs are necessary for secretion but not sufficient for their specific recognition by cognate shuttle proteins. Our results further suggest that CTDs contain a secretion signal, sufficient for secretion of substrates by the T9SS, and a targeting signal, which directs substrates to their final localization.