Poster Presentation 24th Annual Lorne Proteomics Symposium 2019

Exploring the bacteriophage φX174 infection response (#133)

Bradley W Wright 1 , Dominic Y Logel 1 , Paul R Jaschke 1 , Mark P Molloy 1 2
  1. Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. Northern Clinical School, Sydney University, Sydney, NSW, Australia

We have investigated the transcriptomic and proteomic response of Escherichia coli C122 to φX174 infection and found that there is a large and rapid cellular response present during the later stages of the 60-minute infection cycle. Using TMT-labelling and mass spectrometry we quantified approximately 46% of the E. coli proteome and observed large fold-change inductions of bacterial proteins involved in membrane biogenesis, in particular the Sec protein translocation and lipoprotein processing and trafficking pathways. The cell’s heat shock response and protein folding machinery were largely upregulated. “Holdases” IbpA and IbpB had fold-changes equivalent to φX174 proteins indicating their significance during infection. Separate Crispr-Cas9 mediated knockouts of these two genes led to no observable changes in infection dynamics, however, double knockouts resulted in lower efficiency of plating and significantly larger plaques. Interestingly, ΔIbpA/B also resulted in a slower growth phenotype at a wide range of temperatures, relative to the wild-type and single knockout strains, and plasmid complementation with the native genes only partially resolved the defect. These preliminary results are in contrast to gene disruptions in the K12 strain, and will require further exploration.

A further experiment aims to understand the significance of φX174’s highly compressed genome. We have a genome decompressed variant which we have observed to have the same lysis timing as the wild-type, but displays fitness defects in burst size and plaque formation. We endeavour to investigate the effects of genome decompression on protein expression using the targeted mass spectrometry method of parallel reaction monitoring on the wild-type phage and decompressed variant and will present preliminary results.