Bringing to light the dialogue between bacteria and prophage – BacPhageChat

Bacterial genomes are extremely diverse. Part of this diversity is due to prophages, which are viral genomes integrated into the ones of bacteria. Most prophage genes are silent, but those that are expressed can provide new properties to their host. In turn, bacterial regulators can control genes from prophage origin. Therefore a regulatory dialogue takes place between prophages and bacteria.
RpoS, a sigma subunit of the RNA polymerase, is the major regulator of the general stress response in gamma-proteobacteria. It is involved in stress adaptation, biofilm formation, antibiotic resistance, bacterial virulence and persistence. RpoS regulation is complex and occurs mainly at the post-translational level via changes in protein stability. Indeed, in favorable growth conditions, RpoS is addressed to the ClpXP protease by the adaptor protein RssB to be degraded. Under certain stress conditions, small anti-adaptor proteins called Ira (Inhibitor of RssB Activity) titrate RssB and therefore block this degradation pathway and stabilize RpoS.
Strikingly, I have found that a protein from phage origin, AppY, directly interacts with RssB to stabilize RpoS. AppY is a transcriptional regulator from the AraC family and to my knowledge, it is the first time that a protein from this family is involved in a regulation independent of transcription.
The goal of the BacPhage Chat project is to understand the dialogue existing between bacteria and phage.
My proposal is organized in three tasks. First, I want to demonstrate that AppY is a new Ira protein, the only one to have a dual function in the cell. In this part of the project, I aim at elucidating the molecular mechanism leading to RssB titration and want to identify the physiological conditions leading to the stabilization of RpoS by AppY. In the second part of the project, I want to define the AppY regulon. Indeed, although only two AppY targets have been defined so far, our preliminary results from RNA-Seq experiments suggest that the AppY regulon is broader than expected. Using transcriptional gfp fusions, but also ChIP-Seq assays and gel shift experiments, I want to identify direct AppY targets and characterize their regulations as well as their conditions of induction. Finally, the last part of the project will focus on RpoS. Indeed, it has been shown that an E. coli strain deleted of its 9 prophages present phenotypes similar to a delta rpoS strain. Moreover, the RpoS regulon as well as proteins involved in its regulation are extremely diverse, even between closely related bacteria. Therefore, I propose that part of this plasticity could be due to prophage encoded proteins. In this last part, I want to identify proteins from prophage involved in RpoS regulation as well as prophage-encoded genes under RpoS control.
The identification of the AppY regulon as well as the definition of the regulatory network involving RpoS and proteins from phage will allow to measure the extent of the dialogue between prophages and bacteria. The BacPhage Chat project is innovative in considering that proteins from foreign origin can shape the host physiology and contribute to stress adaptation by taking control of global bacterial regulators. I am confident that my study will undoubtedly lead to breakthrough in the stress response field and will give a better understanding of how bacteria and phage can communicate.