title: Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation.
abstract: The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.
authors.affiliation: Department of Chemistry, York University.
authors.forename: Fettah
authors.initials: F
authors.lastname: Erdogan
authors.affiliation: Department of Chemistry, York University.
authors.forename: Cristina
authors.initials: C
authors.lastname: Lento
authors.affiliation: Department of Chemistry, York University.
authors.forename: Ayat
authors.initials: A
authors.lastname: Yaseen
authors.affiliation: Department of Chemistry, York University.
authors.forename: Roksana
authors.initials: R
authors.lastname: Nowroozi-Dayeni
authors.affiliation: Department of Chemistry, York University.
authors.forename: Sasha
authors.initials: S
authors.lastname: Kheyson
authors.affiliation: Department of Chemistry, York University; The Centre for Research on Biomolecular Interactions, York University; audette@yorku.ca.
authors.forename: Gerald F
authors.initials: GF
authors.lastname: Audette
mesh_terms: D058947:Bacterial Secretion Systems / Q000235:genetics / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003227:Conjugation, Genetic / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D059765:Homologous Recombination / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*
chemical_list: D058947:Bacterial Secretion Systems; D004269:DNA, Bacterial; D015500:Chloramphenicol O-Acetyltransferase