Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Biochem Biophys Res Commun ; 503(4): 2386-2392, 2018 09 18.
Article in English | MEDLINE | ID: mdl-29966652

ABSTRACT

Bacterial conjugation, such as that mediated by the E. coli F plasmid, is a main mechanism driving bacterial evolution. Two important proteins required for F-pilus assembly and DNA transfer proficiency are TraW and TrbC. As members of a larger complex, these proteins assemble into a type IV secretion system and are essential components of pore formation and mating pair stabilization between the donor and the recipient cells. In the current report, we demonstrate the physical interaction of TraW and TrbC, show that TraW preferentially interacts with the N-terminal domain of TrbC, and that this interaction is important in restoring conjugation in traW/trbC knockouts.


Subject(s)
Conjugation, Genetic , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , F Factor/genetics , Protein Interaction Maps , Amino Acid Sequence , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , F Factor/metabolism , Gene Knockout Techniques , Protein Interaction Domains and Motifs , Sequence Alignment
2.
J Vis Exp ; (119)2017 01 04.
Article in English | MEDLINE | ID: mdl-28117821

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.


Subject(s)
Conjugation, Genetic/genetics , Escherichia coli/genetics , Plasmids/metabolism , Bacterial Secretion Systems/genetics , Bacterial Secretion Systems/metabolism , Chloramphenicol O-Acetyltransferase/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Drug Resistance, Bacterial/genetics , Homologous Recombination/genetics , Mutation , Plasmids/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...