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.
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: Kai Shen; John Gladitz; Patricia Antalis; Bethany Dice; Benjamin Janto; Randy Keefe; Jay Hayes; Azad Ahmed; Richard Dopico; Nathan Ehrlich; Jennifer Jocz; Laura Kropp; Shujun Yu; Laura Nistico; David P Greenberg; Karen Barbadora; Robert A Preston; J Christopher Post; Garth D Ehrlich; Fen Z Hu Journal: Infect Immun Date: 2006-01 Impact factor: 3.441
Authors: Deborah A Lewis; Andrew Jones; Julian Parkhill; David P Speert; John R W Govan; John J Lipuma; Stephen Lory; A Kevin Webb; Eshwar Mahenthiralingam Journal: Am J Respir Cell Mol Biol Date: 2005-04-15 Impact factor: 6.914
Authors: Kai Shen; Patricia Antalis; John Gladitz; Sameera Sayeed; Azad Ahmed; Shujun Yu; Jay Hayes; Sandra Johnson; Bethany Dice; Richard Dopico; Randy Keefe; Benjamin Janto; William Chong; Joseph Goodwin; Robert M Wadowsky; Geza Erdos; J Christopher Post; Garth D Ehrlich; Fen Z Hu Journal: Infect Immun Date: 2005-06 Impact factor: 3.441
Authors: C K Stover; X Q Pham; A L Erwin; S D Mizoguchi; P Warrener; M J Hickey; F S Brinkman; W O Hufnagle; D J Kowalik; M Lagrou; R L Garber; L Goltry; E Tolentino; S Westbrock-Wadman; Y Yuan; L L Brody; S N Coulter; K R Folger; A Kas; K Larbig; R Lim; K Smith; D Spencer; G K Wong; Z Wu; I T Paulsen; J Reizer; M H Saier; R E Hancock; S Lory; M V Olson Journal: Nature Date: 2000-08-31 Impact factor: 49.962