Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa

Abstract

The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.

FIG. 1.

Plasmids used for yeast recombinational cloning of the O-antigen region. (A) Genomic organization of the conserved P. aeruginosa genes at the 5′ and 3′ ends of the O-antigen operons. ORFs, which are drawn to scale, are shown as arrows oriented in the directions in which they are transcribed. Five-hundred-base-pair targeting elements used for cloning are shown as stippled boxes. (B) Yeast-E. coli shuttle plasmid used for recombinational cloning. Yeast CEN-ARS and URA3 sequences facilitate plasmid segregation-replication and selection, respectively, in yeast. Plasmid maintenance in E. coli relies on the single-copy mini F′ origin (43), and chloramphenicol resistance (CmR) is used for plasmid selection. (C) Recombinational cloning plasmid used to isolate the himD/ihfB-to-tyrB region from the ATCC O15 strain.

FIG. 2.

Eleven groups of P. aeruginosa O-antigen biosynthetic gene clusters. Genes within the DNA sequences were identified by the GENEMARK algorithm (30) and are represented by arrows drawn to scale. Protein families, identified in queries to the PFAM database (1) and represented three or more times in the overall data set, are represented by single-letter designations. W indicates an ORF with several potential membrane-spanning domains. A detailed description of the families is provided in Table 3. Specific genes described previously in gene clusters O5, O6, and O11 are shown above the clusters. The stippled boxes represent the targeting elements used in recombinational cloning. The C-terminal coding region of wbpM, shown as an open arrow, is presumed to extend rightward of the cloned region, as shown. A denotes orfA, as described in the text. The G+C content of each cluster is shown.

FIG. 3.

Multiple alignment of orfA amino acid sequences (6). Red residues are >90% conserved, blue residues are 50 to 90% conserved, and the exclamation point indicates an I or V residue. orfA has no detectable homolog in the PFAM database.

FIG. 4.

Degeneration of DNA sequence conservation in the O-antigen region. (A) Sequence boundary at the 5′ end of the O-antigen region. Targeting sequences used for cloning are enclosed in a box. Uppercase letters indicate nucleotides that are fully conserved, lowercase letters indicate nucleotides that are 90% conserved, and positions with two or more nucleotide differences are indicated by dots. Sequence conservation degenerates immediately at the end of the himD/ihfB coding region. (B) DNA sequence conservation in the 5′ coding region of wbpM degenerates in the antisense direction. The recombinational targeting sequence is enclosed in a box.