title: Glycosylation of b-Type flagellin of Pseudomonas aeruginosa: structural and genetic basis.
abstract: The flagellin of Pseudomonas aeruginosa can be classified into two major types-a-type or b-type-which can be distinguished on the basis of molecular weight and reactivity with type-specific antisera. Flagellin from the a-type strain PAK was shown to be glycosylated with a heterogeneous O-linked glycan attached to Thr189 and Ser260. Here we show that b-type flagellin from strain PAO1 is also posttranslationally modified with an excess mass of up to 700 Da, which cannot be explained through phosphorylation. Two serine residues at positions 191 and 195 were found to be modified. Each site had a deoxyhexose to which is linked a unique modification of 209 Da containing a phosphate moiety. In comparison to strain PAK, which has an extensive flagellar glycosylation island of 14 genes in its genome, the equivalent locus in PAO1 comprises of only four genes. PCR analysis and sequence information suggested that there are few or no polymorphisms among the islands of the b-type strains. Mutations were made in each of the genes, PA1088 to PA1091, and the flagellin from these isogenic mutants was examined by mass spectrometry to determine whether they were involved in posttranslational modification of the type-b flagellin. While mutation of PA1088, PA1089, and PA1090 genes altered the composition of the flagellin glycan, only unmodified flagellin was produced by the PA1091 mutant strain. There were no changes in motility or lipopolysaccharide banding in the mutants, implying a role that is limited to glycosylation.
authors.affiliation: Department of Medicine/Infectious Diseases, P.O. Box 100277, JHMHC, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Amrisha
authors.initials: A
authors.lastname: Verma
authors.forename: Michael
authors.initials: M
authors.lastname: Schirm
authors.forename: Shiwani K
authors.initials: SK
authors.lastname: Arora
authors.forename: P
authors.initials: P
authors.lastname: Thibault
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
authors.forename: Reuben
authors.initials: R
authors.lastname: Ramphal
mesh_terms: D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008970:Molecular Weight; D016366:Open Reading Frames / Q000502:physiology; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012694:Serine
chemical_list: D011134:Polysaccharides; D005408:Flagellin; D012694:Serine