Coexistence of Two Distinct Versions of O-Antigen Polymerase, Wzy-Alpha and Wzy-Beta, in Pseudomonas aeruginosa Serogroup O2 and Their Contributions to Cell Surface Diversity

Abstract

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 α product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are α-glycosidically linked, while those in O2 and O16 are β-linked. We hypothesized that a derivative of the D3 bacteriophage wzy β is present in the chromosomes of O2 and O16 and that this gene is responsible for the β-linkage. By a combination of PCR and primer walking, wzy β 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 β was made, and it produces semirough LPS devoid of B-band O antigen. The cloned wzy β is capable of complementing the O16 wzy β mutant, as well as cross-complementing a wzy α knockout mutant. However, in the latter case, the restored O antigen was β-linked. Using reverse transcription-PCR, we showed that wzy α was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the wzy α mutant of O5. With the coexistence of wzy α and wzy β in O2 and O16 and the B-band O polysaccharides in these being β-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.