Journal Article

Exploiting a Phage-Bacterium Interaction System as a Molecular Switch to Decipher Macromolecular Interactions in the Living Cell

Vértessy, Beáta, Tarjányi, Szilvia, Surányi, Éva, Nyíri, Kinga, Kőhegyi, Bianka, Tóth, Judit, Hírmondó, Rita

2018

Overview

Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular tools. The Stl repressor from this protein family has been recently investigated and although the binding site of Stl on DNA was recently discovered, there is a lack of knowledge on the specific protein segments involved in this interaction. Here, we develop a generally applicable system to reveal the mechanism of the interaction between Stl and its cognate DNA within the cellular environment. Our unbiased approach combines random mutagenesis with high-throughput analysis based on the lac operon to create a well-characterized gene expression system. Our results clearly indicate that, in addition to a previously implicated helix-turn-helix segment, other protein moieties also play decisive roles in the DNA binding capability of Stl. Structural model-based investigations provided a detailed understanding of Stl:DNA complex formation. The robustness and reliability of our novel test system were confirmed by several mutated Stl constructs, as well as by demonstrating the interaction between Stl and dUTPase from the Staphylococcal ϕ11 phage. Our system may be applied to high-throughput studies of protein:DNA and protein:protein interactions.

• Publisher: MDPI AG,MDPI
• Subject: Bacteria / Bacteria - virology / Bacteriophages - physiology / Binding Sites / Cloning / Deoxyribonucleic acid / DNA / dUTP pyrophosphatase / E coli / Gene Expression / Gene Expression Regulation / Gene Order / Genes, Reporter / Genetic Vectors - genetics / Host-Pathogen Interactions / Islands / Lactose operon / macromolecular interactions / Macromolecules / molecular probe / Mutagenesis / Mutation / Mycobacterium smegmatis - physiology / Mycobacterium smegmatis - virology / Pathogenicity / Pathogenicity islands / phage-host interaction / Phages / Plasmids / Promoter Regions, Genetic / Protein Binding / Protein expression / Protein interaction / Proteins / Random mutagenesis / Repressor Proteins - chemistry / Repressor Proteins - metabolism / Structure-Activity Relationship / Transcription factors