title: 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 the distributed genome hypothesis.
  
  
  METHODS
  High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of approximately 1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with approximately 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 12 components clinical PA isolates. Our sequencing effort ( approximately 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 supra-genome.
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, 320 E. North Avenue, Pittsburgh, PA 15212, United States.
authors.forename: Geza
authors.initials: G
authors.lastname: Erdos
authors.forename: Sameera
authors.initials: S
authors.lastname: Sayeed
authors.forename: Fen Ze
authors.initials: FZ
authors.lastname: Hu
authors.forename: Patricia T
authors.initials: PT
authors.lastname: Antalis
authors.forename: Kai
authors.initials: K
authors.lastname: Shen
authors.forename: Jay D
authors.initials: JD
authors.lastname: Hayes
authors.forename: Azad I
authors.initials: AI
authors.lastname: Ahmed
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Johnson
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Post
authors.forename: Garth D
authors.initials: GD
authors.lastname: Ehrlich
mesh_terms: D002675:Child, Preschool; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D005820:Genetic Testing / Q000379:methods*; D016680:Genome, Bacterial; D015698:Genomic Library; D006801:Humans; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000302:isolation & purification*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial