Specialized shuttle proteins recognize Type IX secretion signals and target effectors to their final destinations in Flavobacterium johnsoniae.

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.