A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation

Abstract

Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in Pseudomonas aeruginosa PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30 P. aeruginosa isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.

Figure 1

Genetic organization of the polymorphic chromosomal region of P. aeruginosa strains PAO1 and PAK involved in the biosynthesis and assembly of flagellar components. Indicated are segments of DNA that are inserted at specific locations within individual genomes, including a large segment of DNA (≈16 kb) containing a cluster of 14 ORFs that are associated with glycosylation of flagellin, present in PAK, and an apparent duplication of the fliS gene. The genome of PAO1 has a ≈6-kb DNA segment containing three genes of unknown function followed by PA1091, a homologue of the rfbC gene that is 35% identical to the PAK orfN.

Figure 2

(A) Glycosylation status of the flagellin protein from PAKorfN mutant and the complemented strains assessed by Western blots. The wild-type PAK flagellin migrates as a diffuse band indicating glycosylation of flagellin (lane 2). The flagellin from the PAKorfN mutant migrates much faster, suggesting loss of glycosylation (lane 3). Introduction of the complete orfN gene on a plasmid (pMMB194H) complements the glycosylation defect of the PAKorfN mutant (lane 5), whereas the vector control (pMMB66) remains nonglycosylated (lane 4). Size markers (lane 1) are in kDa. (B) Glycosylation status of the flagellins from PAKorfA mutant and the complemented strains assessed by Western blots. The wild-type PAK flagellin migrates as a diffuse band indicating glycosylation of flagellin (lane 2). The flagellin from the PAKorfA mutant migrates faster than the wild-type flagellin, suggesting loss of glycosylation (lane 3). Introduction of the complete orfA gene on a plasmid (pSP329Gm10EH) restores flagellin glycosylation in the PAKorfA mutant (lane 5) but not in mutants carrying the vector control (pSP329Gm) (lane 4). Lane 1 contains the size markers. (C) Effect of chemical deglycosylation on flagellin mobility during SDS/PAGE. Lane 1, PAK flagellin; lane 2, deglycosylated PAK flagellin; lane 3, PAKorfA mutant flagellin; lane 4, PAO1 flagellin; lane 5, deglycosylated PAO1 flagellin; lane 6, horseradish peroxidase; lane 7, deglycosylated horseradish peroxidase; and lane 8, molecular mass markers in kDa.

Figure 3

Flagellin specificity of the glycosylation apparatus. Lane 2, wild-type strain PAK (a-type); lane 3, PAKfliC mutant; lane 4, PAKfliC mutant carrying a plasmid pPT244 expressing the a-type flagellin; lane 5, PAK flagellin mutant carrying a plasmid pDN186970 expressing the b-type flagellin; lane 6, wild-type strain PAO1 (b-type); lane 7, PAO1fliC mutant; lane 8, PAO1fliC mutant carrying a plasmid pDN186970 expressing the b-type flagellin; lane 9, PAO1fliC mutant carrying a plasmid pPT244 expressing the a-type flagellin; and lane 10, wild-type strain PAK (a-type). Size markers, in kDa, are in lane 1.

Figure 4

The glycosylation island contains all of the missing requirements for flagellin glycosylation in the b-type strain PAO1. Lanes 1 and 7 contain molecular size markers. Lane 2, wild-type strain PAK; lane 3, PAO1 flagellin mutant carrying the vector (pMMB66); lane 4, PAO1 flagellin mutant carrying the plasmid pMMB194H containing incomplete glycosylation island and the a-type flagellin; lane 5, PAO1 flagellin mutant carrying the vector control (pSP329Gm); lane 6, PAO1 flagellin mutant carrying the plasmid pSP329Gm10EH8 containing the complete glycosylation island along with the a-type flagellin gene.