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, ϳ10% 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.

Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

“…The overrepresentation of transitions (83 %) and synonymous substitutions (80 %) is similar to that found for evolutionarily neutral SNPs in other loci (Kiewitz & Tümmler, 2000) or in the bulk genome (Spencer et al, 2003). The 61 SNPs segregated into 18 mucABD genotypes, one of which split into two variants (Table 2, Figs 1 and 2).…”

“…Sequencing of the mucA, mucB and mucD genes in 37 sequential P. aeruginosa strains from 10 CF patients (Table 2) and in nine environmental isolates revealed 61 SNPs (Table 3) corresponding to 2 % sequence diversity at this locus, which is significantly higher than the mean sequence diversity of 0?5 % in the P. aeruginosa genome (Spencer et al, 2003). The overrepresentation of transitions (83 %) and synonymous substitutions (80 %) is similar to that found for evolutionarily neutral SNPs in other loci (Kiewitz & Tümmler, 2000) or in the bulk genome (Spencer et al, 2003).…”

“…In living patients, the late isolates were collected after 13-15 years of continuous chronic airway colonization, and in deceased patients, the last isolates prior to death were studied. Sequence analysis of sequential isolates has so far only been performed on strains from a young CF patient who acquired P. aeruginosa during infancy, and who was monitored for a period of 8 years thereafter (Spencer et al, 2003;Smith et al, 2006).…”

Development of a Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Pseudomonas aeruginosa

“…This degree of variation has important implications for the methods used to study the evolution and epidemiology of this organism. Recent work suggests that more than 80% of the genome of the sequenced strain (strain PAO1) is shared (with only 0.5% nucleotide divergence) by CF and environmental strains (26). Denamur et al (5) and Picard et al (16) considered that the species had a panmictic population structure, but Kiewitz and Tummler (13) proposed a net-like structure characterized by high frequencies of recombination.…”

Isolation and Detection of Genes Responsible for Pyoverdines Biosynthesis in Pseudomonas putida KNUK9

“…Pyoverdines (PVDs) are organic compounds produced by the fluorescent Pseudomonads. The importance of these high-affinity siderophores for rhizosphere colonization by P. putida has been previously reported (Nelson et al, 2002;Spencer et al, 2003). However, little is known about the molecular mechanisms of pyoverdines secretion and regulation in plant growth promoting bacteria.…”