The O antigen (Oag) component of Shigella flexneri lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesized by a Wzy dependent mechanism. S. flexneri Wzy has 12 trans-membrane segments and two large periplasmic loops. Although Wzy was identified 20 years ago, the molecular details regarding the Wzy polymerization still need to be revealed because over-expression and isolation of the purified Wzy has been unsuccessful, and there is lack of knowledge about its functional amino acid residues. A wzy-gfpe expression construct was made; it successfully expressed Wzy-GFPe and complemented a wzy mutant (Δwzy). Random mutagenesis was performed on wzy-gfpe. Mutants were screened by their colicin sensitivity. Mutations were identified by sequencing in the periplasmic loops (PL1, PL2, PL3, PL6), trans-membrane regions, and cytoplasmic loops (CL1, CL2, CL5). No mutation was identified in the largest periplasmic loop PL5. PL3 and PL5 of Wzy contain RX₁₂G motifs (starting from R₁₆₄ and R₂₈₉, respectively) and several Arg residues (R₂₅₀, R₂₅₈, R₂₇₈) are within these motifs. All these Arg residues were mutagenized to Ala by site-directed mutagenesis on wzy-gfpe. Each mutated plasmid from random and site-directed mutagenesis was transformed into a Δwzy for LPS profile analysis and protein expression. The mutations either knocked out or altered the Wzy function as seen by the LPS phenotype of the complemented strain. In-gel fluorescence was used to detect wild type and mutant Wzy-GFPe protein expression in the Δwzy and to compare their relative protein expression level. Mutants were also characterized by their colicin and phage sensitivity. Based on the LPS profile several phenotypic classes were identified, including functionally knocked out, decreased polymerization, reduced Oag chain length, and increased Oag chain length. These phenotypic classes were correlated with their colicin sensitivity, phage susceptibility, and protein expression profiles, providing insight to Wzy structure and function.