Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa

Abstract

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.

FIG. 1.

Histograms of the coverage of the PAO1 genome in high-quality data that could be aligned with the PAO1 reference sequence. (A to C) Data are from strains 1-60 (A), 2-164 (B), and MSH (C). Coverage was estimated by calculating the number of bases with a phred quality score of 20 or higher that could be aligned within 5-kbp windows across the PAO1 genome. (D to L) Inset plots indicate fine-grained views of coverage gaps at the O-antigen biosynthetic locus at PAO1 coordinates 3.53 to 3.55 Mbp (F to H), a 17-kbp deletion common to all three sampled strains relative to PAO1 at coordinate 3.91-mbp (I to K), and a 100-kbp gap in strain 2-164 at coordinate 2.4-mbp (L). (D) Coverage simulation using a random subset of sequencing traces from the PAO1 sequencing project. (E) Coverage histogram obtained in a pure simulation of random sampling to an average coverage of 0.48×.

FIG. 2.

Percent nucleotide mismatches of aligned sequence between PAO1 and strains 1-60 (A), 2-164 (B), and MSH (C) in sliding 5-kbp windows that overlap by 2.5 kbp.

FIG. 3.

SfoI digestions of all P. aeruginosa strains from the patient 1 and patient 2 collections. For each patient, isolates are in historical sequence. Selected lanes are identified by the age (in months) of the patient at the time the isolates were cultured. The exceptional isolate from patient 1 is indicated by *. Additional digestions of PAO1 (a), MSH (b), and three clinical isolates from different CF patients (c to e) are indicated.

FIG. 4.

Gene structure of the pyoverdine locus. (A) PAO1 ORFs 2394 to 2405, and ORFs annotated from the pyoverdine regions of clinical strains 1-60 and 2-164. The shaded boxes indicate the alignable sequence conservation between the PAO1 reference and the pyoverdine synthesis region from strain 1-60. (B) Nucleotide divergence between strains 1- 60 and 2-164 (solid line) and between strains 1-60 and PAO1 (dashed line) in 1,000-bp nonoverlapping windows. Discontinuities in the 1-60/PAO1 comparison reflect the absence of alignable sequences throughout much of the region.

FIG. 5.

O-antigen biosynthetic locus of clinical strains 1-60 and 2-164. Mutational events likely to disrupt lipopolysaccharide biosynthesis are indicated.

FIG. 6.

PCR assay to determine which isolates in the patient 1 collection have an insertion in the O-antigen B-band locus (presence of a 1.8-kbp band). Numbers indicate the approximate age in months of patient 1 at the time selected isolates were cultured. Isolates without the insertion that are not typeable are indicated by *. The outlying isolate, not clonally related to the predominant strain (a), strain 1-60 (b), and a no-DNA control (c) are indicated.