Construction and characterization of a highly redundant Pseudomonas aeruginosa genomic library prepared from 12 clinical isolates: Application to studies of gene distribution among populations

SummaryObjectiveTo create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis.MethodsHigh molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of ∼1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with ∼250,000 clones that was arrayed and subjected to quality control analyses.ResultsRestriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5 kb with the desired mean insert size of 1.4 kb. Thus, this library provides better than 4.5× coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort (∼1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01.ConclusionsOur data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome.