Glycosylation of Pilin and Nonpilin Protein Constructs byPseudomonas aeruginosa1244

Abstract

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the ␤ carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus with Escherichia coli alkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO. E. coli alkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.

Glycosylation Regulates Specific Induction of Rice Immune Responses by Acidovorax avenae Flagellin

“…In Gram-negative bacteria, examples of surface-associated glycoproteins include the pilins of P. aeruginosa and Neisseria spp. (29,30), the adhesins TibA and Aida-1 of E. coli (31,32) and HMW1 of Haemophilus influenza (33), and the flagellins of A. avenae and many other pathogenic bacteria (7,34,35). Although the full significance of the glycosylation of these proteins has not been determined, a number of reports have shown that these modifications are involved in virulence and motility.…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…Their small size and ability to mediate motility or transduce electrons highlight the potential for their exploitation for industrial purposes, including formation of nanotubes with emergent properties (25,27), movement of nanoscale cargo (410), and powering of bacterial batteries (261). Their ability to tolerate bulky modifications such as sugars (55,73,131,323,405) or peptides (400) makes them amenable to synthetic biology approaches (272) for development of completely novel uses, once a compre-hensive understanding of their functional logic is available. The use of filamentous phages that have T4P-like assembly systems, such as M13, has revolutionized many aspects of molecular biology (406), and the type IV pilins are equally suited to take us in new directions.…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…The factors determining this remarkable preference for short oligosaccharides remain to be established. The only substrate specificity that could be found for PilO is the presence of a C-terminal serine or threonine residue and the above-mentioned minimum distance from the pilin surface and compatible surface charge (393).…”

“…Commonalities in glycosylation pathways reduce the arsenal of precursors and enzymes needed and are thus energy saving. Bacteria also need a way to discriminate between these bifurcated biosynthesis pathways and have evolved several solutions to do so: they can express only a subset of the glycoconjugate repertoire (e.g., H. pylori [322]), use downstream enzymes with different specificities (e.g., P. aeruginosa [393]), and have both pathways compete (e.g., the same precursors for LOS, CPS, and N-glycoprotein biosynthesis in C. jejuni [14]), and their expression can be dependent on environmental stimuli (e.g., expression of colonic acid in E. coli [65]). …”