Analysis of Shigella flexneri Wzz (RoI) function by mutagenesis and cross-linking: Wzz is able to oligomerize

The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein. The Wz z protein is anchored into the cytoplasmic membrane by two transmembrane do mains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loo p of the protein located in the periplasm. Plasmids were constructed encodi ng hybrid Wzz proteins consisting of regions of S. flexneri Wzz (Wzz(SF)) a nd Salmonella typhimurium Wzz (Wzz(ST)). These imparted O-antigen modal cha in lengths that implied that the carboxy-terminal region of Wzz was involve d in chain length determination, Site-directed mutagenesis was undertaken t o investigate the functional significance of highly conserved residues in a mino-/carboxy-terminal domains of Wzz(SF) Some of the Wzz(SF) variants resu lted in O-antigen modal chain lengths much shorter than those of wild-type Wzz(SF), whereas other mutants inactivated Wzz(SF) function entirely and a third class had a longer O-antigen chain length distribution. The data indi cate that amino acids throughout the length of the Wzz(SF) protein are impo rtant in determination of O-antigen modal chain length. In vivo cross-linki ng experiments were performed to investigate the interactions between Wzz p roteins. The experiments indicated that the Wzz(SF) protein is able to form dimers and oligomers of at least six Wzz(SF) proteins. A carboxy-terminal- truncated Wzz(SF) protein having the amino terminal 194 amino acids was abl e to oligomerize, indicating that the aminoterminal region is sufficient fo r the Wzz-Wzz interaction observed. Shortened Wzz(SF) proteins having inter nal deletions in the amino-terminal region were also able to oligomerize, s uggesting that residues 59-194 are not essential for oligomerization. Cross linking of Wzz(SF) proteins with mutationally altered residues showed that loss of Wzz(SF) function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the T M2 segment affects Wzz(SF)-Wzz(SF) dimer mobility in SDS polyacrylamide gel s. These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis.