Dual conserved periplasmic loops possess essential charge characteristics that support a catch-and-release mechanism of O-antigen polymerization by Wzy in Pseudomonas aeruginosa PAO1

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.Wzyis 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 RX10G 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 RX10G motifs that are important for Wzy function, of which Arg176, Arg290, and Arg291 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.