Prokaryotic division interactome: setup of an assay for protein–protein interaction mutant selection

• Published in: Research in microbiology Vol. 161; no. 2; pp. 118 - 126
• Main Authors: Barbati, S., Grenga, L., Luzi, G., Paolozzi, L., Ghelardini, P.
• Format: Journal Article
• Language: English
• Published: Issy-les-Moulineaux Elsevier SAS 01-03-2010; Elsevier Masson
• Subjects: Bacteriology; Bacteriophages - genetics; Biological and medical sciences; Chimera; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli K12 - genetics; Escherichia coli K12 - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fts division proteins; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Microbiology; Miscellaneous; Mutant Proteins - genetics; Mutant Proteins - metabolism; Protein Binding; Protein Interaction Mapping; Protein–protein interaction; Selection, Genetic; Two-hybrid assay; Two-Hybrid System Techniques; Fts division proteins; Two-hybrid assay; Escherichia coli; Protein–protein interaction; Interactome; Bacteria; Protein—protein interaction; Mutation; Protein; Enterobacteriaceae
• ISSN: 0923-2508; 1769-7123
• DOI: 10.1016/j.resmic.2010.01.001

A method which enables selection of protein mutants impaired in their ability to interact with their normal protein partners is presented here. The method is the two-phage two-hybrid assay adapted for mutant selection. In the two-phage assay, the interaction between two proteins enables the formation of a functional hybrid lambdoid repressor that shuts down expression of a reporter gene governed by a chimeric promoter/operator region. To adapt the assay to interaction mutant selection, antibiotic resistance was used as a reporter gene. In this case, the interaction between the two proteins resulted in antibiotic sensitivity, whereas the loss of interaction conferred resistance to the bacterial strain. Therefore, turning on reporter gene expression highlights the loss of interaction due to a mutation in one of the genes for the two protein partners, and leads to direct selection of the mutants regardless of the mutant phenotype. In this paper, application of this method to isolation of interaction mutants in proteins involved in
Escherichia coli K12 cytokinesis is reported.