title: A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries.
abstract: High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Skurnik
authors.forename: Damien
authors.initials: D
authors.lastname: Roux
authors.forename: Hugues
authors.initials: H
authors.lastname: Aschard
authors.forename: Vincent
authors.initials: V
authors.lastname: Cattoir
authors.forename: Deborah
authors.initials: D
authors.lastname: Yoder-Himes
authors.forename: Stephen
authors.initials: S
authors.lastname: Lory
authors.forename: Gerald B
authors.initials: GB
authors.lastname: Pier
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial / Q000502:physiology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D037521:Virulence Factors
references.citation: J Immunol. 2008 Jul 1;181(1):586-92
references.pmid: 18566425
references.citation: J Infect Dis. 1985 Jul;152(1):99-103
references.pmid: 3925032
references.citation: Environ Microbiol. 2012 Aug;14(8):1805-16
references.pmid: 22187957
references.citation: Infect Immun. 2003 Mar;71(3):1453-61
references.pmid: 12595463
references.citation: Science. 1995 Jun 30;268(5219):1899-902
references.pmid: 7604262
references.citation: J Bacteriol. 2000 May;182(9):2363-9
references.pmid: 10762233
references.citation: Antimicrob Agents Chemother. 1993 Nov;37(11):2385-90
references.pmid: 8285622
references.citation: Infect Immun. 2011 Mar;79(3):1289-99
references.pmid: 21149583
references.citation: PLoS One. 2011;6(12):e29113
references.pmid: 22216178
references.citation: Plasmid. 2000 Jan;43(1):59-72
references.pmid: 10610820
references.citation: Dev Cell. 2004 Nov;7(5):745-54
references.pmid: 15525535
references.citation: Infect Immun. 1983 Oct;42(1):170-7
references.pmid: 6413410
references.citation: Infect Immun. 2005 Apr;73(4):2262-72
references.pmid: 15784570
references.citation: Annu Rev Microbiol. 2012;66:493-520
references.pmid: 22746331
references.citation: Mol Microbiol. 2003 Apr;48(1):77-84
references.pmid: 12657046
references.citation: Cell Host Microbe. 2009 Sep 17;6(3):279-89
references.pmid: 19748469
references.citation: Mol Microbiol. 1994 Sep;13(6):1079-91
references.pmid: 7854122
references.citation: PLoS Pathog. 2012 Sep;8(9):e1002945
references.pmid: 23028334
references.citation: J Bacteriol. 2006 Jul;188(13):4851-60
references.pmid: 16788194
references.citation: FEMS Microbiol Lett. 2002 Apr 23;210(1):73-80
references.pmid: 12023080
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2833-8
references.pmid: 16477005
references.citation: Mol Microbiol. 2003 Aug;49(4):905-18
references.pmid: 12890017
references.citation: Arch Intern Med. 2000 Feb 28;160(4):501-9
references.pmid: 10695690
references.citation: Proc Natl Acad Sci U S A. 2001 Oct 23;98(22):12712-7
references.pmid: 11606763
references.citation: Front Microbiol. 2011 Aug 22;2:167
references.pmid: 21991261
references.citation: Mol Microbiol. 1994 Jan;11(1):137-53
references.pmid: 7908398
references.citation: J Infect Dis. 2012 Jun;205(11):1709-18
references.pmid: 22448004
references.citation: mBio. 2011 Jan 18;2(1):e00315-10
references.pmid: 21253457
references.citation: Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10191-6
references.pmid: 10963681
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14339-44
references.pmid: 14617778
references.citation: Genome Biol. 2006;7(10):R90
references.pmid: 17038190
references.citation: J Bacteriol. 2010 Jul;192(13):3249-58
references.pmid: 20363934
references.citation: Environ Microbiol. 2003 Dec;5(12):1294-308
references.pmid: 14641575
references.citation: J Bacteriol. 2008 Apr;190(8):2739-58
references.pmid: 18203836
references.citation: Appl Environ Microbiol. 2012 May;78(9):3098-107
references.pmid: 22367088
references.citation: J Bacteriol. 2010 Feb;192(4):994-1010
references.pmid: 20008072
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12487-92
references.pmid: 16891416
references.citation: Genome Res. 2012 Dec;22(12):2541-51
references.pmid: 22826510
references.citation: Nat Immunol. 2003 Dec;4(12):1247-53
references.pmid: 14625549
references.citation: Genome Res. 2009 Jan;19(1):12-23
references.pmid: 19047519
references.citation: New Microbiol. 2003 Oct;26(4):353-61
references.pmid: 14596346
references.citation: PLoS One. 2010 Dec 10;5(12):e15131
references.pmid: 21170272
references.citation: J Bacteriol. 1988 Jun;170(6):2575-83
references.pmid: 2836362
references.citation: Infect Immun. 2006 Feb;74(2):975-83
references.pmid: 16428743
references.citation: J Bacteriol. 2008 Nov;190(21):6961-9
references.pmid: 18776008
references.citation: Curr Opin Microbiol. 2007 Dec;10(6):644-8
references.pmid: 17981495
references.citation: Mol Microbiol. 1995 Feb;15(4):703-17
references.pmid: 7783642
references.citation: Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16422-7
references.pmid: 19805314
references.citation: Haematologica. 2006 Aug;91(8):1134-8
references.pmid: 16885056
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D596-600
references.pmid: 20929876
references.citation: Mol Microbiol. 1997 Oct;26(2):399-407
references.pmid: 9383163
references.citation: J Bacteriol. 2011 Sep;193(18):4859-68
references.pmid: 21764924