Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, Wzy(Sf). Oag chain length is regulated by chromosomally encoded Wzz(Sf) and pHS-2 plasmid-encoded Wzz(pHS2). To identify functionally important amino acid residues in Wzy(Sf), random mutagenesis was performed on the wzy(Sf) gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzy(Sf) proteins in the wzy(Sf)-deficient (Delta wzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of Wzy(Sf) and Wzz(Sf) was investigated by transforming these mutated wzy(Sf) plasmids into a wzy(Sf)- and wzz(Sf)-deficient (Delta wzy Delta wzz) strain. Comparison of the LPS profiles in the Delta wzy and Delta wzy Delta wzz backgrounds identified Wzy(Sf) mutants whose polymerization activities were Wzz(Sf) dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the Wzy(Sf)-GFP mutants in the Delta wzy and Delta wzy Delta wzz backgrounds identified a role for Wzz(Sf) in Wzy(Sf) stability. Hence, in addition to its role in regulating Oag modal chain length, Wzz(Sf) also affects Wzy(Sf) activity and stability.