Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1531; p. 11
• Main Authors: Francis, Matthew S, Amer, Ayad A A, Milton, Debra L, Costa, Tiago R D
• Format: Journal Article
• Language: English
• Published: United States 01.01.2017
• Subjects: Alleles; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Conjugation, Genetic; Genome, Bacterial; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - metabolism; Gram-Negative Bacterial Infections - microbiology; Mutagenesis, Site-Directed; Mutation; Phenotype; Plasmids - genetics; Polymerase Chain Reaction; Protein Binding; Protein Interaction Mapping; Type III Secretion Systems - genetics; Type III Secretion Systems - metabolism; Virulence Factors - genetics; Virulence Factors - metabolism; Yersinia - genetics; Yersinia - metabolism; Type III secretion systems; Genetic-based screens; Suicide vector pDM4; Protein-protein interaction assays; Mutant libraries; Site-directed mutagenesis
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-6649-3_2

Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections-unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.