Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: Identification of Wzz-dependent Wzy mutants

Abstract

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 plasmidencoded 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 wzySf-deficient (Δwzy) 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 (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz 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 Δwzy and Δwzy Δwzz 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.