Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq.
We describe a deep-sequencing procedure for tracking large numbers of transposon mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq methodology based on the generation and amplification of single-strand circles carrying transposon junction sequences (the Tn-seq circle method), a method which can be used with virtually any transposon. The procedure reliably identified more than 100,000 transposon insertions in a single experiment, providing near-saturation coverage of the genome. To test the effectiveness of the procedure for mutant identification, we screened for mutations reducing intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic tobramycin resistance had been previously analyzed at genome scale using mutant-by-mutant screening and thus provided a benchmark for evaluating the new method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the majority of which were then verified with individual mutants. The group of genes with the strongest mutant phenotypes included nearly all (13 of 14) of those with strong mutant phenotypes identified in the previous screening, as well as a nearly equal number of new genes. The results thus show the effectiveness of the Tn-seq method in defining the genetic basis of a complex resistance trait of P. aeruginosa and indicate that it can be used to analyze a variety of growth-related processes.
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Larry A
LA
Gallagher
Jay
J
Shendure
Colin
C
Manoil
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial; D016680:Genome, Bacterial; D016254:Mutagenesis, Insertional / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000502:physiology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements
Biochim Biophys Acta. 2004 Nov 11;1694(1-3):121-34
15546662
Genome Res. 2009 Dec;19(12):2308-16
19826075
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14339-44
14617778
Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14570-5
19706543
Genome Res. 2008 Nov;18(11):1851-8
18714091
Trends Biotechnol. 2007 Dec;25(12):547-55
17997179
Cell Host Microbe. 2009 Sep 17;6(3):279-89
19748469
EMBO J. 1998 Jul 15;17(14):3968-80
9670013
J Bacteriol. 2002 Dec;184(23):6472-80
12426334
J Bacteriol. 2010 Jun;192(12):3001-10
20348252
Biosci Biotechnol Biochem. 2009 Dec;73(12):2698-704
19966467
Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2833-8
16477005
Nucleic Acids Res. 2007;35(7):e47
17317684
Nat Rev Microbiol. 2006 May;4(5):383-94
16715050
Mol Microbiol. 2009 Apr;72(1):183-201
19226327
Annu Rev Microbiol. 1999;53:129-54
10547688
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16422-7
19805314
Nat Methods. 2009 Oct;6(10):767-72
19767758
Gene. 1998 May 28;212(1):77-86
9661666
FEMS Microbiol Rev. 2005 Sep;29(4):703-17
16102599
J Hosp Infect. 2009 Dec;73(4):338-44
19699552