Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Eliana Drenkard; Rhianna M. Hibbler; D. Alina Gutu; Alexander D. Eaton; Amy L. Silverio; Frederick M. Ausubel; Bryan P. Hurley; Lael M. Yonker

doi:10.3791/57298

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Method Article

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

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DOI:

10.3791/57298

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May 4th, 2018

Eliana Drenkard^*¹ , Rhianna M. Hibbler^*¹ , D. Alina Gutu² , Alexander D. Eaton¹ , Amy L. Silverio¹ , Frederick M. Ausubel²^,³ , Bryan P. Hurley¹^,⁴ , Lael M. Yonker¹^,⁴

¹Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, ²Department of Molecular Biology, Massachusetts General Hospital, ³Department of Genetics, Harvard Medical School, ⁴Department of Pediatrics, Harvard Medical School

^* These authors contributed equally

Summary

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

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.

Introduction

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium present in soil, water, and most human environments, as well as skin microflora. Compared to many bacterial species, P. aeruginosa has a relatively large genome of 5.5-7 Mbp with high G+C content (65-67%). Furthermore, a significant proportion of its genes are involved in metabolic adaptability and are part of regulatory networks, allowing for great flexibility in response to environmental stress¹. P. aeruginosa expresses a plethora of virulence factors, exhibits proclivity to form biofilms, possesses the ability....