Bacillus subtilis biofilm formation and evolution on Arabidopsis thaliana roots

Plant growth-promoting rhizobacteria (PGPR) can enhance plant growth and protect plants against disease through various mechanisms. The use of these bacteria within agriculture thereby represents a sustainable alternative or supplement to the use of chemical fertilizers and pesticides to improve crop yields. Among these PGPR is the Gram positive bacterium Bacillus subtilis that belongs to the Bacillus genus and is commonly found in association with plants and their roots. While commercial products containing this bacterium are applied within agriculture, the interactions between B. subtilis and the plant root as well as the soil environment have only started to be unraveled. The purpose of this PhD project was to study how B. subtilis interacts with and adapts to different environments with a special emphasis on the plant root.

Previous studies have shown that B. subtilis isolates with variation in number and combination of Rap-Phr cell-cell signaling systems display different sporulation timing, suggesting that the precise combination of these systems in a certain strain matches the specific sporulation needs of its ecological niche. Using an experimental competition approach containing all combinations of single and double ∆rap-phr mutants of a B. subtilis strain analyzed with high-throughput barcode sequencing, we demonstrated that variability in Rap-Phr systems fine-tunes the ability of the strains to compete in different sporulation-requiring environments, thereby supporting the importance of Rap-Phr systems in ecological adaptation. By examining the single ∆rap-phr mutants for root colonization, we could further show that certain Rap-Phr systems affect root colonization of the model plant Arabidopsis thaliana, revealing the importance of these cell-cell signaling systems in the fitness of B. subtilis also in this ecologically relevant environment.

To investigate the adaptation of B. subtilis to A. thaliana roots we employed an experimental evolution approach. We found that B. subtilis rapidly adapted to the plant root as evidenced by improved root colonizers observed already after 12 successive transfers. Moreover, we observed that selected evolved isolates were improved only in colonization of A. thaliana roots, but not on tomato, which might indicate a plant species-specific adaptation under the applied selective conditions. Investigation of selected evolved isolates for alterations in bacterial traits compared to the ancestral strain revealed that several evolved isolates displayed biofilms with increased robustness and reduced motility, supporting the existence of an evolutionary trade-off between these two traits in B. subtilis. We detected mutations within or in the surrounding regions of the sinR gene, encoding a biofilm repressor, in evolved isolates with robust biofilm development, suggesting that sinR is a target for selection during adaptation to A. thaliana roots. We further found that evolved isolates outcompeted the ancestral strain during root colonization and that a selected evolved isolate displayed increased root colonization also in the presence of a fourmembered community containing resident soil bacteria. These experiments demonstrate that B. subtilis became highly adapted to the plant root environment during the employed experimental evolution. However, two selected evolved isolates suffered a fitness disadvantage compared to the ancestral strain in a non-selective environment, revealing an evolutionary cost associated with adaptation to plant roots.

In conclusion, this PhD project has contributed to the understanding of how B. subtilis interacts with plant roots, demonstrating a role of cell-cell signaling systems in root colonization, and providing insights into the evolutionary adaptation of B. subtilis to A. thaliana roots. This insight can direct future studies of B. subtilis-plant interactions and aid in the development of B. subtilis strains as efficient biocontrol agents to support a sustainable agricultural production.