Dataset Information

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

ABSTRACT: We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

SUBMITTER: Jacobs MA

PROVIDER: S-EPMC283593 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Comprehensive transposon mutant library of Pseudomonas aeruginosa.

Jacobs Michael A MA Alwood Ashley A Thaipisuttikul Iyarit I Spencer David D Haugen Eric E Ernst Stephen S Will Oliver O Kaul Rajinder R Raymond Christopher C Levy Ruth R Chun-Rong Liu L Guenthner Donald D Bovee Donald D Olson Maynard V MV Manoil Colin C

Proceedings of the National Academy of Sciences of the United States of America 20031114 24

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mu ...[more]

PMID: 14617778

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Project description:Klebsiella pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are a major cause of hospital-acquired infections, yet the basis of their success as nosocomial pathogens is poorly understood. To help provide a foundation for genetic analysis of K. pneumoniae, we created an arrayed, sequence-defined transposon mutant library of an isolate from the 2011 outbreak of infections at the U.S. National Institutes of Health Clinical Center. The library is made up of 12,000 individually arrayed mutants of a carbapenemase deletion parent strain and provides coverage of 85% of the predicted genes. The library includes an average of 2.5 mutants per gene, with most insertion locations identified and confirmed in two independent rounds of Sanger sequencing. On the basis of an independent transposon sequencing assay, about half of the genes lacking representatives in this "two-allele" library are essential for growth on nutrient agar. To validate the use of the library for phenotyping, we screened candidate mutants for increased antibiotic sensitivity by using custom phenotypic microarray plates. This screening identified several mutations increasing sensitivity to ?-lactams (in acrB1, mcrB, ompR, phoP1, and slt1) and found that two-component regulator cpxAR mutations increased multiple sensitivities (to an aminoglycoside, a fluoroquinolone, and several ?-lactams). Strains making up the two-allele mutant library are available through a web-based request mechanism.IMPORTANCE K. pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are recognized as a top public health threat by the Centers for Disease Control and Prevention. The analysis of these major nosocomial pathogens has been limited by the experimental resources available for studying them. The work presented here describes a sequence-defined mutant library of a K. pneumoniae strain (KPNIH1) that represents an attractive model for studies of this pathogen because it is a recent isolate of the major sequence type that causes infection, the epidemiology of the outbreak it caused is well characterized, and an annotated genome sequence is available. The ready availability of defined mutants deficient in nearly all of the nonessential genes of the model strain should facilitate the genetic dissection of complex traits like pathogenesis and antibiotic resistance.

Dataset Information

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

Publications

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

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