Replication of the Ordered, Nonredundant Library of <em>Pseudomonas aeruginosa</em> strain PA14 Transposon Insertion Mutants

Abstract

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320

“…However, this method has historically been limited by the relative difficulty in determining the precise genomic location of transposon insertions and the capacity of screening experiments to handle large numbers of mutants (e.g., laborious screens or screens with bottleneck effects). The combination of random mutagenesis with modern high-throughput sequencing has facilitated the development of ordered transposon libraries where most nonessential genes in an organism are available as single-insertion mutants in an array of 96-well plates (for example, see references 5 – 12 ). The resulting ordered library allows the selection of specific mutants, and any interesting mutants obtained from a screen using an ordered library can be easily gathered for follow-up studies.…”

Escherichia coli CFT073 Fitness Factors during Urinary Tract Infection: Identification Using an Ordered Transposon Library

“…Random mutant libraries are the most common genetic approach due to the simplicity of their construction; however, limitations include a large gene bias and loss of information due to intergenic transposon insertions. Construction of a defined mutant library allows for optimization within the composition of a given inoculum, avoiding transposon insertions in intergenic regions and reducing gene size bias (15,16). For our studies, we chose to generate a defined mutant library for a more thorough and optimized characterization of genes involved in UTI fitness.…”

A group B Streptococcus indexed transposon mutant library to accelerate genetic research on an important perinatal pathogen

“…Assaying for biofilm formation in plastic, flat-bottomed multi-sample cell culture plates revealed three mutants with significant differences in biofilm formation. Wild-type A909 is a relatively poor biofilm-producing strain ( 58 ), and we were interested to see that the mutants we identified in our screen with biofilm alterations produced significantly more substantial biofilms than the wild-type control ( Fig. 5A ).…”