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

Abstract

Objective-To 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 for testing the distributed genome hypothesis.Methods-High molecular weight genomic DNAs from twelve clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of ~1.5Kb. 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.Results-Restriction 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 4.5x coverage for each of the genomes from the twelve component 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.Conclusions-Our 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 supragenome.

Pseudomonasgenomes: diverse and adaptable

“…(2006) generated a large clone genomic library from 12 strains of P. aeruginosa from which 348 (approximately 11%) of a set of randomly chosen clones harboured sequences distinct from those found in PAO1. In a similar approach, 1300 random clones from a large library derived from 12 clinical P. aeruginosa strains were examined; 13% contained sequences not found in the PAO1 genome or GenBank (Erdos et al ., 2006). There is clearly a greater degree of variation among P. aeruginosa isolates than generally considered, which will become more apparent with increased numbers of sequenced genomes.…”

Beyond the pan-genome: current perspectives on the functional and practical outcomes of the distributed genome hypothesis

“…Based on the DGH we developed a comparative and functional genomics program that has provided the data establishing the veracity of the postulate that the genetic determinants of virulence and antibiotic resistance are unique to each strain for multiple pathogenic bacterial species (reviewed in [48]). These include: the Gram-negative pathogens, H. influenzae [14,19,62,65,[70][71][72][73], M. catarrhalis [30,31], P. aeruginosa [12,27,28], and Burkholderia cenocepacia [68]; and the Gram-positive pathogens S. pneumoniae [19][20]29,[74][75][76][77], S. aureus [19], and Gardnerella vaginalis [16]. This information can be exploited for the development of targeted prevention and treatment strategies [62,65,71].…”

“…Bacterial genomic plasticity is characterized by each strain in a population possessing a unique set of distributed/accessory genes from the population supragenome [ 1 , 7 , 13 , 15 ]. We have demonstrated high degrees of genomic plasticity within multiple species that underlies the observation that these species have both pathogenic and commensal strains, these include: Haemophilus influenzae [ 10 , 14 , 19 ], Pseudomonas aeruginosa [ 12 , 27 , 28 ], Streptococcus pneumoniae [ 11 , 18–20 , 29 ], Moraxella catarrhalis [ 30 , 31 ] among others. Genomic diversity results from a balance between genome expansion (addition of genes) and genome reduction (deletion of genes) [ 14–15 ].…”