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 Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm. Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S. flexneri Wzz (Wzz(SF)) and Salmonella typhimurium Wzz (Wzz(ST)). These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination, Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of Wzz(SF) Some of the Wzz(SF) variants resulted 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 indicate that amino acids throughout the length of the Wzz(SF) protein are important in determination of O-antigen modal chain length. In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins. 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 able to oligomerize, indicating that the aminoterminal region is sufficient for the Wzz-Wzz interaction observed. Shortened Wzz(SF) proteins having internal deletions in the amino-terminal region were also able to oligomerize, suggesting that residues 59-194 are not essential for oligomerization. Crosslinking 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 TM2 segment affects Wzz(SF)-Wzz(SF) dimer mobility in SDS polyacrylamide gels. These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis.