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Pseudomonas aeruginosa mexT is an indicator of PAO1 strain integrity.

ABSTRACT: Laboratory research with Pseudomonas aeruginosa commonly involves the prototype strain PAO1. There is continued concern that PAO1 sublines maintained and propagated in the same laboratory or different laboratories exhibit genetic and phenotypic variability that may affect the reproducibility and validity of research. Whole-genome sequencing and other research identified the mexT locus as a mutational hotspot, but the explication of the diverse mutations present in the various sublines and consequences remained rather cursory. Here we present evidence that MexT sequence diversity is a predictor of PAO1 lineage integrity and define the protein's prototype sequence.

SUBMITTER: LoVullo ED

PROVIDER: S-EPMC7363278 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Pseudomonas aeruginosa mexT is an indicator of PAO1 strain integrity.

LoVullo Eric D ED Schweizer Herbert P HP

Journal of medical microbiology 20191218 1

Laboratory research with Pseudomonas aeruginosa commonly involves the prototype strain PAO1. There is continued concern that PAO1 sublines maintained and propagated in the same laboratory or different laboratories exhibit genetic and phenotypic variability that may affect the reproducibility and validity of research. Whole-genome sequencing and other research identified the mexT locus as a mutational hotspot, but the explication of the diverse mutations present in the various subli ...[more]

PMID: 31859619

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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

Pseudomonas aeruginosa mexT is an indicator of PAO1 strain integrity.

Publications

<i>Pseudomonas aeruginosa mexT</i> is an indicator of PAO1 strain integrity.

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