Dataset Information

Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1.

ABSTRACT: 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.

SUBMITTER: Held K

PROVIDER: S-EPMC3497512 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1.

Held Kiara K Ramage Elizabeth E Jacobs Michael M Gallagher Larry L Manoil Colin C

Journal of bacteriology 20120914 23

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. ...[more]

PMID: 22984262

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Project description:The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

Dataset Information

Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1.

Publications

Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1.

Similar Datasets

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