Heteropolymeric B-band lipopolysaccharide in Pseudomonas aeruginosa PAO1 is synthesized via the so-called Wzy-dependent pathway, requiring a functional Wzy for polymerization of O-antigen repeat units in the periplasm. Wzy is an integral inner membrane protein for which the detailed topology has been mapped in a recent investigation (Islam, S. T., Taylor, V. L., Qi, M., and Lam, J. S. (2010) mBio 1, e00189-10), revealing two principal periplasmic loops (PL), PL3 and PL5, each containing an RX(10)G motif. Despite considerable sequence conservation between the two loops, the isoelectric point for each peptide displayed marked differences, with PL3 exhibiting a net-positive charge and PL5 showing a net-negative charge. Data from site-directed mutagenesis of amino acids in each PL have led to the identification of several key Arg residues within the two RX(10)G motifs that are important for Wzy function, of which Arg(176), Arg(290), and Arg(291) could not be functionally substituted with Lys. These observations support the proposed role of each PL in a catch-and-release mechanism for Wzy-mediated O-antigen polymerization.