Understanding the role of two conserved, but hypothetical bacteriophage genes, in controlling the biology of their host cells

Abstract

Stx phages convert their bacterial hosts, providing them with shigatoxigenic potential. All E. coli carrying Stx phage are known as Shiga toxin-encoding Escherichia coli (STEC) and have become a global challenge to food safety. Around 74% of the genes carried by the model Stx phage, ϕ24B (vB_EcoP_24B), are annotated as hypothetical, and we have been ascribing function to many of these genes. The expression of some hypothetical genes has been shown to be uncoupled from viral replication, but function to aid the lysogen in surviving environmental stresses such as antibiotic and acid exposures. Two genes are examined in this study, gene 21 (vB _24B_21) and gene 48 (vB _24B_48). The former is expressed only at the end of the lytic cycle and the latter only during the lysogenic cycle. We have created a series of isogenic mutants to establish whether P21 impacts upon phage release. Though, the crystal structure of P21 was recently solved and we demonstrated that this protein has the previously unknown ability to modify peptidoglycan and to bind to it. Work is currently ongoing to better understand if and how the action of P21 controls phage release. Why the phage would do this is left to speculation, but it could be a mechanism to hold on to the phage due to the significant fitness advantage provided to the lysogen when the Stx phage exists as a resident prophage. The initial data presented in this work has demonstred that an induced lysogen carrying a phage producing P21 releases fewer progeny phages than an induced lysogen that does not produce P21, even though the same number of phages are made intracellularly. We examined whether the difference in phage release happened at the cell level or at the population level. We demonstrated that there were differences in cell morphology and different live and dead cell ratios showing that phage release was impacted across the population. The ϕ24B lysogen has been shown phenotypic microarray to confer a multitude of low level resistances to its bacterial host cell. Gene 48 is an enormous hypothetical gene that is predicted to encode a protein of 2808 a.a. It is located at the right end of the integrated prophage ϕ24B. However, this protein has many of the conserved features of the bacterial “giant genes” that usually encode a surface protein associated in bacterial fitness. It was hypothesised that this gene might be behind some not all of these resistant phenotypes. A synthetic plasmid carrying gene 48 was constructed and E.coli naiive MC1061 cells were transformed and compared with naiive and lysogen cells in the six antimicrobial agents resistance assays. The bacterial cells that carry the plasmid possessing gene 48 showed more resistance to 8-hydroxyquinoline than naïve cells in an arabinose inducible manner, indicating that gene 48 is involved in this resistance. As a result, it may explain which gene that is encoded by ϕ24B lysogen modulates the phage-mediated resistance to 8-hydroxyquinoline that has been previously described.