Investigation of the Wzy alpha and beta O-antigen polymerases in Pseudomonas aeruginosa
B-band, the serotype-specific form of lipopolysaccharide (LPS) produced by 'Pseudomonas aeruginosa' is crucial to the virulence of this bacterial species. The assembly of B-band LPS involves several proteins including the O-antigen polymerase Wzy[alpha] which links the O-antigen repeat units. The peptide sequence of the 'wzy[alpha]' gene of serotypes O2, O5, and O16 were found to be identical; but interestingly, the O-antigen units in serotype O5 are connected by [alpha]-glycosidic linkages, while in O2 and O16 they are connected by [beta]-glycosidic linkages. We hypothesized that a D3 bacteriophage-derived 'wzy[beta]' gene is present in the chromosomes of serotypes O2 and O16 and responsible for their [beta]-linked O antigen structures. Using primers designed from the D3-phag egenome sequence and primer walking, I have amplified, cloned and sequenced 'wzy[beta]' in serotype O2 and O16. The O2 and O16 'wzy[beta]' genes are identical, sharing 87.42% sequence identity with their xenolog in D3. An O16 'wzy [beta]' chromosomal knockout mutant was made and found to lack B-band O antigen. The cloned 'wzy[beta]' polymerase was found to be fully functional 'in vivo' since it could be used to complement the O16 'wzy[beta]' mutant as well as to cross-complement a 'wzy[alpha]' knockout mutant, restoring [beta]-linked O antigen. Results from RT-PCR showed that ' wzy[alpha]' was transcribed in both O2 and O16 serotypes, and both genes were fully functional 'in vivo,' capable of complementing an O5-'wzy[alpha]' mutant. The complemented strain produces LPS with [alpha]-linked O antigen. Since O2 and O16 have [beta]-linked O antigen, a Wzy[alpha] inhibitor is likely present in the chromosomes of these serotypes. Further analysis, such as PCR amplification or genomic DNA sequencing, is predicted to uncover a Wzy [alpha] inhibitor, likely a homologue of Iap, the D3 phage-encoded inhibitor previously characterized by our group. In conclusion, this is the first time that [alpha]- and [beta]- O-antigen polymerases have been discovered in ' P. aeruginosa.' The results from this thesis served to shed light on the diversity and evolution of 0-antigen structure.