JB Accepts, published online ahead of print on 23 March 2007

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J. Bacteriol. doi:10.1128/JB.00237-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The Co-existence of Two Distinct Versions of O-Antigen Polymerase, Wzy-Alpha and Wzy-Beta, in Pseudomonas aeruginosa Serogroup O2 and its Contributions to Cell Surface Diversity

Katarina Kaluzny, Priyanka D. Abeyrathne, and Joseph S. Lam^*

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

^* To whom correspondence should be addressed. Email: jlam{at}uoguelph.ca.

Assembly of B-band 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 -glycosidic-linked, while those in O2 and O16 are -linked. We hypothesized that a derivative of the D3 bacteriophage wzy is present in the chromosomes of O2 and O16,, and that this gene is responsible for the -linkage. By a combination of PCR and primer walking, wzy of both serotypes have been amplified and cloned. Both are identical, but only share 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16 wzy was made and it produces rough LPS devoid of B-band O antigen. The cloned wzy is capable of complementing the O16 wzy mutant, as well as cross-complementing a wzy knockout mutant. However, in the latter case, the restored O-antigen was -linked. Using RT-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 co-existence of wzy and wzy in O2 and O16 and the B-band O polysaccharides in these being -linked, we hypothesized that iap, an inhibitor of 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.

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