Sequence polymorphism in the glycosylation island and flagellins of Pseudomonas aeruginosa

Abstract

A genomic island consisting of 14 open reading frames, orfA to orfN was previously identified in Pseudomonas aeruginosa strain PAK and shown to be essential for glycosylation of flagellin. DNA microarray hybridization analysis of a number of P. aeruginosa strains from diverse origins showed that this island is polymorphic. PCR and sequence analysis confirmed that many P. aeruginosa strains carry an abbreviated version of the island (short island) in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L, and -M are absent. To ascertain whether there was a relationship between the inheritance of the short island and specific flagellin sequence variants, complete or partial nucleotide sequences of flagellin genes from 24 a-type P. aeruginosa strains were determined. Two distinct flagellin subtypes, designated A1 and A2, were apparent. Strains with the complete 14-gene island (long island) were almost exclusively of the A1 type, whereas strains carrying the short island were associated with both A1- and A2-type flagellins. These findings indicate that P. aeruginosa possesses a relatively low number of distinct flagellin types and probably has the capacity to further diversify this antigenic surface protein by glycosylation.

FIG. 1.

Westren blot analysis of P. aeruginosa strains. Whole-cell lysates from different P. aeruginosa strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were subjected to Western blot analysis with FliC-specific antibodies. These antibodies were raised against purified a-type flagellar preparations, and they cross-react with the b-type flagellins. The flagellin type for each strain is indicated at the bottom of the panel. Lane 1 contained the broad-range prestained molecular weight markers which were purchased from Bio-Rad Laboratories, Richmond, Calif.

FIG. 2.

DNA microarray analysis of the GI of different P. aeruginosa strains. The diagram suggests the presence or absence of genes in the GI of different P. aeruginosa strains isolated from CF patients, patients with UTIs, or the blood of infected patients. PAK and PAO1 were used as reference strains. The bottom panel shows the hybridization with the probes for the two flagellin types, a-type or b-type. The genes designated as being present are shown in black, and the genes considered to be absent are shown in light gray. The genes shown in darker gray are designated uncertain. Please note that these results only suggest the presence or absence of genes based on the various degrees of homology with the probes.

FIG. 3.

Schematic diagram showing the structure of GIs of a- and b-type P. aeruginosa strains. The diagram shows the region of the P. aeruginosa chromosome where the GIs are located. The location of the GI insertion is shown to be in the middle of flagellar genes (shown by arrows with gray filling) flgL on the 5′ end and fliC at the 3′ end. Two a-type GIs, long (PAK) with all 14 ORFs and short (JJ692) with a 5.4-kb sequence containing deletions of orfI, -J, -K, -L, and -M, are shown by black filled arrows. The PAO1 GI with 3 ORFs of unknown function and an ORF which had homology to rfbC gene (orfN) is shown by arrows with no fill for comparison. The locations of the primers RER142 and RER144 used for PCR analysis of different P. aeruginosa strains are shown by small arrows.

FIG. 4.

GROWTREE phylogenetic analysis of a-type flagellins. The dendrogram was created with the GROWTREE program of the GCG Wisconsin package. Amino acid sequences of a-type flagellins from 7 P. aeruginosa strains including PAK and two a-type flagellin sequences already existing in the database (accession numbers L81146 and L81147) were used to generate the dendrogram. The two subtypes of a-type flagellins, A1 and A2, are evident.

FIG. 5.

PILEUP analysis of the partial central domains of 27 P. aeruginosa strains. Amino acid (aa) sequences from amino acids 228 to 297 of a-type flagellins of 24 P. aeruginosa strains analyzed in this study, one a-type strain obtained from R. A. Ansorg, and two complete a-type flagellin sequences already present in the database were aligned by using the PILEUP program of the GCG Wisconsin package. Amino acid substitutions at positions 237, 240, 241, 249, 250, 253, 266, 273, 283, 285, 287, 288, and 289 and two small deletions of 3 and 4 amino acids at positions 254 and 279, respectively, are highlighted in different shades of gray. The A2 flagellin of strain DSM 1128 was peculiar in having additional amino acid substitutions (shown in bold italics).