Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To prov...

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Bibliographic Details
Published in:Journal of Bacteriology 2012-12, Vol.194 (23), p.6387-6389
Main Authors: Held, Kiara, Ramage, Elizabeth, Jacobs, Michael, Gallagher, Larry, Manoil, Colin
Format: Article
Language:English
Subjects:
Alleles
Bacteriology
Biological and medical sciences
DNA Transposable Elements
Fundamental and applied biological sciences. Psychology
Gene Library
Genes
Genetics, Microbial - methods
Genotype & phenotype
Gram-negative bacteria
Microbiology
Miscellaneous
Mutagenesis, Insertional - methods
mutants
Mutation
Pseudomonas aeruginosa
Pseudomonas aeruginosa - genetics
screening
Sequence Analysis, DNA
transposons
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Description
Summary:Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.
ISSN:0021-9193
1098-5530
1067-8832
DOI:10.1128/jb.01479-12