Periodic Reporting for period 1 - SNiB-cdG-P (Global mapping of second messenger c-di-GMP signaling networks in bacteria using proteomics)
Reporting period: 2021-03-01 to 2023-02-28
Bacteria are masters in sensing environmental cues and in processing this information to adequately adjust their behavior. A key element of bacterial signal transduction is the nucleotide second messenger cyclic di-guanosine-monophosphate (c-di-GMP). This powerful signalling molecule acts in minute amounts to control important cellular functions, such as motility, biofilm formation, or bacterial virulence and persistence. Despite of its global importance in regulating bacterial growth and behavior, studies systematically investigating the c-di-GMP network in bacteria are missing. To address this, the project aimed to employ recently developed quantitative proteomics techniques to identify and characterize proteins involved in c-di-GMP signaling pathways in bacterial model organisms.
For this project, we adapted and optimised a quantitative proteomics method called thermal proteome profiling (TPP) to study c-di-GMP signalling in two bacterial model organisms, Escherichia coli and Caulobacter crescentus. Recording differential thermal stability of proteins at different intracellular concentrations of c-di-GMP levels not only uncovered known c-di-GMP pathway proteins but also generated datasets with novel candidate proteins and pathways regulated by c-di-GMP. The generated new hypothesis include the involvement of c-di-GMP in biological processes important for bacterial survival, such as bacterial surface attachment and stress response.
We are currently in the process of publishing these findings and depositing the data in an open access format.
We are currently in the process of publishing these findings and depositing the data in an open access format.
In this project we have successfully adapted TTP technology to the investigation of a small molecule-based signaling network in different bacteria in vivo. By establishing a robust technology to globally investigate the interaction of proteins with their small-molecule ligands, we have provided a highly useful experimental tool for researchers in similar fields. We are confident that this technique will have an impact and will be used by other scientists in the field of microbiology and beyond. At the same time, we have generated high-quality datasets of proteins directly or indirectly interacting with c-di-GMP. This is a highly valuable resource for the field and provides a direct entry point into mechanistic follow-up studies that will help uncover this prototypical bacterial signaling systems and by that contribute to a better understanding of bacterial growth and growth control.