Evolutionary landscape of bacterial adaptation to diverse environmental niches

Numerous species of the Bacillus genus are used in green biotechnology to improve plant growth. An important plant-growth-promoting rhizobacterium (PGPR) is Bacillus subtilis that is commonly isolated in association with plants and their rhizosphere. Likewise, Bacillus thuringiensis is commonly used as a biological pesticide. Both species are prevalent biofilm formers in diverse environments. Biofilms are matrix-enclosed microbial communities that adhere to biotic or abiotic surfaces. Experimental evolution has been previously utilized to study how these two Bacillus species adapt to different environmental niches, e.g., floating or plastic bead attached biofilms and during plant root colonization. However, the full understanding of the dynamic mutational landscapes was lacking over the full experimental evolution setup. The purpose of this PhD project was to unveil the evolutionary landscape how Bacillus adapts to diverse environmental niches using whole-population genome sequencing.

By comparing the genetic adaptive mechanisms between B. subtilis and B. thuringiensis when these two species were adapted to the same environment and when one species is adapted to two distinct environments, we detected higher number of mutations, greater genotypic diversity, and higher evolvability in the populations evolved in biotic conditions. Our results also uncovered the influence of insertion sequences on the adaptation of B. thuringiensis. Finally, the higher degree of genetic diversification observed in biotic selective environments suggests an increased spatial niche heterogeneity and a nutritional source difference created by the plant host that provided a strong selection during the adaptation process.

Subsequently, we investigated how B. subtilis evolves on Arabidopsis thaliana and tomato seedlings, as well as in an alternating host regime of the two plants. We identified parallel evolution across multiple levels of biological organization in all conditions. We also observed species-specific adaptation at genetic level, which was potentially provoked by specific host plant-imposed selection, either due to root exudates or certain stress conditions. Additionally, motility-biofilm trade-off was also revealed in the mutational landscape of related genes, confirming the experimentally observed reduced motility of evolved isolates. Lastly, we identified both condition-specific and shared list of mutated genes of B. subtilis when evolved in different biofilm environments.

In the final approach, a simple Bacterial-Fungal Interactions (BFIs) system was explored to reveal the effects of a long-term cultivation of B. subtilis in the presence of Aspergillus niger with focus on the evolution of B. subtilis. In specific populations, B. subtilis was selected with enhanced surfactin production and spreading behavior, which was mimicked by recreation of specific mutations in genes encoding DegS-DegU two-component system. Increased surfactin production and niche colonization by the bacterium dismantled fungal expansion and acidification of the medium, in addition to introduction of cell wall stress in A. niger.

In conclusion, this PhD project has contributed to the understanding the genetic mechanisms on how two Bacillus species adapt to different environmental niches, and deepen our understanding of bacterial interactions with plants and fungi, which will likely to support strain improvements for sustainable agriculture in the future.