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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

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received 12 February 2007/ Accepted 16 March 2007

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

Published ahead of print on 23 March 2007.

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