PilO of Pseudomonas aeruginosa 1244: subcellular location and domain assignment

Abstract

SummaryPilO 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.

Type IV Pilin Proteins: Versatile Molecular Modules

“…A stem-loop transcriptional terminator is present between the genes and likely functions to attenuate the level of tfpO expression from the strong pilA promoter (84). The very low abundance of TfpO required concentration of membranes before the protein could be detected by Western blotting (324). Together, these data suggest that the resulting stoichiometry would be such that a single TfpO enzyme could glycosylate multiple pilins.…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…The same phenomenon could also be linked to the PglB N-OST of C. jejuni, the only requirement of which is the presence of a C-2 acetamido group in the nonreducing sugar (53,323). The PilO O-OST is also promiscuous but to a lesser extent (see above) (392). An example of the promiscuity of a non-GT is the fact that the PglK transporter of C. jejuni can complement a Wzx defect (321).…”

“…This O-OST has a sequence pattern that is also found in WaaL (LPS ligase). The two enzymes compete for the same substrate, namely, lipid-linked monomeric O antigens, so it is no surprise that they are part of the same Pfam Wzy_C family (51,392). Nevertheless, the active site of PilO differs from that of the other enzyme.…”

“…N-OSTs, like the PglB N-OST of C. jejuni, seem to be unrelated and of different evolutionary origins (see above) (13). These enzymes remove the glycan from its lipid carrier by cleaving the phosphate-sugar bond and form a new linkage with the substrate (51,150,392). Nevertheless, it is currently very difficult to predict the substrate specificity, i.e., the glycoconjugate biosynthesis pathways in which these enzymes are involved, merely from sequence information.…”

“…Nevertheless, the active site of PilO differs from that of the other enzyme. The Wzy domain of PilO and the C-terminally proximal hydrophilic portion of PilO are the regions linked to enzyme activity (392). PilO has a relaxed glycan specificity for short oligosaccharides, which prevents the addition of polysaccharides that would impede pilus assembly (67).…”