Targeting of Flavobacterium Johnsoniae Proteins for Secretion By the Type IX Secretion System

Abstract

Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to family TIGR04183 (type A CTDs). This thesis focuses on the functional characterization of diverse CTDs for secretion by the F. johnsoniae T9SS. Fusion of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign protein sfGFP that had a signal peptide at the amino terminus (SP-sfGFP) resulted in secretion across the outer membrane. In each case approximately 80 to 100 amino acids from the extreme carboxy-terminus was needed for efficient secretion. Several type A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting secretion of sfGFP by the F. johnsoniae T9SS. The F. johnsoniae adhesin SprB is propelled rapidly along the cell surface resulting in gliding motility. F. johnsoniae SprB requires the T9SS for secretion but lacks a type A CTD. It has a conserved C-terminal domain belonging to family TIGR04131, which we refer to as a type B CTD. Type B CTDs are common in the Bacteroidetes but little is known regarding their roles in secretion. The secretion of the foreign protein sfGFP fused to an N-terminal SP and to C-terminal regions of SprB (SP-sfGFP-CTDSprB) was analyzed. CTDs of 218 AAs or longer resulted in secretion whereas a CTD of 149 AAs did not. sprF, which lies downstream of sprB, is known to be required for SprB secretion. SP-sfGFP-CTDSprB also required SprF for secretion. Efficient secretion only occurred when SP-sfGFP-CTDSprB and SprF were expressed together. Under these conditions CTDs of 218 AAs and 448 AAs resulted in secretion of soluble sfGFP, whereas longer CTDs (663 and 1182 AAs) resulted in attachment of sfGFP to the cell surface. Most F. johnsoniae genes encoding proteins with type B CTDs lie immediately upstream of sprF-like genes. The CTD from one such protein, Fjoh_3952, facilitated secretion of sfGFP only when it was coexpressed with its cognate SprF-like protein, Fjoh_3951. Secretion did not occur when SP-sfGFP-CTDFjoh_3952 was expressed with SprF, or when SP-sfGFP-CTDSprB was expressed with Fjoh_3951. The results highlight the need for extended regions of type B CTDs for secretion and cell-surface localization, and the requirement for the appropriate SprF-like protein for secretion. Since type B CTD-containing proteins and associated SprF-like proteins are common among members of the phylum Bacteroidetes the unique features required for secretion of these proteins may have broad implications.