Impact of Rap-Phr system abundance on adaptation of Bacillus subtilis

Microbes commonly display great genetic plasticity, which has allowed them to colonize all ecological niches on Earth. The Gram-positive model bacterium Bacillus subtilis is a soil-dwelling organism that can be isolated from a wide variety of environments. An interesting characteristic of this bacterium is its ability to form biofilms that display complex population heterogeneity: individual, clonal cells develop diverse phenotypes in response to different environmental conditions within the biofilm. Here, we scrutinized the impact that the number and variety of the Rap-Phr family of regulators and cell-cell communication modules of B. subtilis has on genetic adaptation and evolution. We examine how the Rap family of phosphatase regulators impacts sporulation in diverse niches using a library of single and double rap-phr mutants in competition under 4 distinct growth conditions. Using specific DNA barcodes and whole-genome sequencing, population dynamics were followed, revealing the impact of individual Rap phosphatases and arising mutations on the adaptability of B. subtilis to thrive, produce spores, and outcompete other strains in various environments. Furthermore, we demonstrate that the used selection regimes can drive the activation of the phi3Ts-SPβ prophage, which contains a Rap homologue and further impacts spore production.