PilO of Pseudomonas aeruginosa 1244 catalyses the attachment of an O-antigen repeating unit to the b -carbon of the pilin C-terminal residue, a serine. The present study was conducted to locate the regions of this enzyme important in catalysis and to establish the cellular location of the pilin glycosylation reaction. While PilO was not detectable in extracts of P. aeruginosa or Escherichia coli , even under conditions of overexpression, it was found that an intact MalE–PilO fusion protein was produced in significant amounts. This fusion complemented a P. aeruginosa 1244 mutant containing a pilO deletion and targeted to the cytoplasmic membrane of E. coli . Wzy and WaaL, enzymes that also utilize the O-antigen repeating unit as substrate, were found to share a sequence pattern with PilO even though these proteins have little overall sequence similarity. PilO constructs in which portions of this common sequence were deleted or altered by site-directed mutagenesis lacked pilin glycosylating activity. Deletions of segments downstream from the common region also prevented enzyme activity. Topology studies showed that the two PilO regions associated with enzyme activity were located in the periplasm. These results establish regions of this enzyme important for catalysis and present evidence that pilin glycosylation occurs in the periplasmic space of this organism.

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