Development of a Method for Characterization of Bacterial Host DNA Degradation During Nucleolytic Phage Infection – Investigation of Preferential Degradation of Host genomic DNA Using T7 Bacteriophage Infection of Escherichia coli K-12

09/01/2016

Edrick Lin, Olivia Sawatsky, Amanda Wong, Fangwen Zhao​

Volume 20
Fall 2015 / Winter 2016

The T7 bacteriophage lytic life cycle has been thoroughly characterized, however, the possibility of site-specific cleavage of host genomic DNA by T7 nucleases has not been well-investigated and could provide a novel method of probing chromatin structures in bacterial genomic DNA. Previous studies have shown that T7 endonuclease gp3 cleaves branched cruciform DNA structures, the migration of which are known to be blocked by nucleoid proteins composing nucleosomes in the Escherichia coli genome. Here we describe a method for investigating the stability of DNA regions known to bind heat unstable (HU) nucleoid and integration host factor (IHF) proteins. Host DNA was sampled over the course of E. coli K-12 MG1655 infection by T7 bacteriophage and visualized by pulsed-field gel electrophoresis. Potential protected regions were then probed by quantitative polymerase chain reaction. The resulting data indicates a possible persistence of these nucleoid-associated regions during phage infection, which may be confirmed by further optimization of PFGE and qPCR protocols. This experimental approach represents a possible novel means of probing bacterial chromatin structure.