Abstract
Assembly of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the wzy(alpha) product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are alpha-glycosidically linked, while those in O2 and O16 are beta-linked. We hypothesized that a derivative of the D3 bacteriophage wzy(beta) is present in the chromosomes of O2 and O16 and that this gene is responsible for the beta-linkage. By a combination of PCR and primer walking, wzy(beta) genes of both serotypes have been amplified and cloned. They are identical but share only 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16 wzy(beta) was made, and it produces semirough LPS devoid of B-band O antigen. The cloned wzy(beta) is capable of complementing the O16 wzy(beta) mutant, as well as cross-complementing a wzy(alpha) knockout mutant. However, in the latter case, the restored O antigen was beta-linked. Using reverse transcription-PCR, we showed that wzy(alpha) was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the wzy(alpha) mutant of O5. With the coexistence of wzy(alpha) and wzy(beta) in O2 and O16 and the B-band O polysaccharides in these being beta-linked, we hypothesized that iap, an inhibitor of the alpha-polymerase gene, must be present in these serotypes. Indeed, through PCR, TOPO-cloning, and nucleotide-sequencing results, we verified the presence of iap in both O2 and O16 serotypes.