IDENTIFICATION AND FUNCTIONAL-CHARACTERIZATION OF AN ABC TRANSPORT-SYSTEM INVOLVED IN POLYSACCHARIDE EXPORT OF A-BAND LIPOPOLYSACCHARIDE INPSEUDOMONAS-AERUGINOSA

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LP S) molecules known as A band and B band. B band is the serospecific LP S, while A band is the common LPS antigen composed of a D-rhamnose O-p olysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been ident ified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterizati on of two genes within the cluster, designated wzm and wzt. The Wzm an d Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively , and are homologous to a number of proteins that comprise ABC (ATP-bi nding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-bin ding protein containing a highly conserved ATP-binding motif. Chromoso mal wzm and wzt mutants were generated by using a gene replacement str ategy in P. aeruginosa PAO1 (serotype O5). Western blot analysis and i mmunoelectron microscopy using A-band- and B band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synt hesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band pol ysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rath er, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. Thes e results demonstrate that A-band and B-band O-antigen assembly proces ses follow two distinct pathways, with the former requiring an ABC tra nsport system for cell surface expression.