title: Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden. matthew.francis@umu.se.|Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden. matthew.francis@umu.se.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Francis
authors.affiliation: Department of Molecular Biology, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden.|Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden.|Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Ayad A A
authors.initials: AA
authors.lastname: Amer
authors.affiliation: Department of Molecular Biology, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden.|Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden.|Department of Biological and Environmental Sciences, Troy University, Troy, AL, USA.
authors.forename: Debra L
authors.initials: DL
authors.lastname: Milton
authors.affiliation: Department of Molecular Biology, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden.|Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 901 87, Sweden.|Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London, UK.
authors.forename: Tiago R D
authors.initials: TR
authors.lastname: Costa
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003227:Conjugation, Genetic; D016680:Genome, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics* / Q000378:metabolism*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D025941:Protein Interaction Mapping; D000069296:Type III Secretion Systems / Q000235:genetics* / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism; D015007:Yersinia / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000069296:Type III Secretion Systems; D037521:Virulence Factors
keywords: Genetic-based screens; Mutant libraries; Protein-protein interaction assays; Site-directed mutagenesis; Suicide vector pDM4; Type III secretion systems