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.hole-genome sequences provide the foundation for the creation of relatively complete collections of strains carrying defined mutations in individual genes. Such libraries should facilitate the comprehensive identification of genes required for a wide range of biological processes. A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach (1). Other projects, also following a strategy of gene-by-gene disruption, are underway for Escherichia coli (E. coli genome project, www. genome.wisc.edu͞functional͞tnmutagenesis.htm), and have recently been completed for Bacillus subtilis (2).An alternative strategy for generating mutant libraries consists of ''random'' whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites. The approach is cost-effective and applicable to a wide variety of microbes (3, 4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5). Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth, although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined (6)(7)(8). In this report, we describe the generation and initial phenotypic analysis of a near-saturation library of transposon insertion mutants of the opportunistic pathogen Pseudomonas aeruginos...

Negative regulation of bacterial quorum sensing tunes public goods cooperation

“…Mutant strains of P. aeruginosa PAO1 were obtained from a nonredundant transposon library at the University of Washington (Jacobs et al, 2003). The insertions are in individual genes rather than in operons eliminating the possibility of polar effects, they contain a suitable antibiotic resistance marker to distinguish strains in coculture, eliminating the need for an additional genetic manipulation, and they are out-of-frame such that translational lacZ fusions are not generated.…”

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…Traditionally, transposon insertion mutagenesis has been used to identify gene functions in many different bacteria (Glass et al, 2006; Hensel et al, 1995; Jacobs et al, 2003; Mack et al, 1994), including P. putida KT2440 (de Lorenzo & Timmis, 1994; Herrero, Lorenzo, & Timmis, 1990; Molina‐Henares et al, 2010). However, genome wide screening and identification of all genes involved in tolerance has been a limiting factor, which in the last years have been overcome through the use of next generation sequencing (NGS) techniques.…”

Siderophore production and biofilm formation as linked social traits

“…One clone was an isogenic transposon-bearing PA01 mutant (Jacobs et al, 2003) with an insertion in pvdF, a gene required for synthesis of the primary siderophore pyoverdine (Visca et al, 2007). The remaining clones evolved from the methionine auxotroph PA06049 (a transposon mutant of PA01: Rella et al, 1985)) under selection for siderophore cheats (Harrison and Buckling, 2005; Supplementary information).…”