Experimental evolution of bacterial virulence and plant beneficial traits

Bacillus cereus sensu lato is a group of bacteria that encompasses three major species Bacillus cereus, Bacillus thuringiensis, and Bacillus anthracis, which contains both beneficial and harmful species in a variety of habitats and has a highly controversial taxonomy. For example, the capacity of B. thuringiensis to attack and kill insects has acquired it a remarkable reputation as a microbial pesticide. However, current knowledge of its natural ecosystem is inadequate. While not considered as a major niche of B. thuringiensis, plant rhizosphere has been reported to promote bacterial sustainability by derived nutrient compounds. On the other hand, B. cereus is becoming more recognized as a human pathogen capable of causing infections by emetic and hemolysin toxins. Due to the breadth of phenotypic characteristics, B. cereus may infiltrate food manufacturing and processing chains at various stages, posing several challenges to the food industry. The nature and degree of ecological and host spectrum difference within the B. cereus group has been extensively debated, and closely intertwined to concerns over the safety issues and taxonomy classification into species.

To study how pathogens evolve and adapt to distant hosts or niches, most studies apply high throughput tool such as comparative genomics and multi-locus sequence typing. In this study, nevertheless, experimental evolution has been applied to address the question of whether adaptation of B. thuringiensis to certain environmental niches affect important phenotypes such as differentiation and virulence pathways. Experimental evolution serves as a powerful method to unravel geno- and phenotypic associations in microbial communities and only recently, it has been applied in B. subtilis to study bacterial interactions in biofilms. In this work, the experimental evolution outcome of B. thuringiensis biofilms both associating with plant roots (Arabidopsis thaliana) and nylon beads has bee explored. Combing with traditional methods such as molecular cloning and sequencing techniques, evolutionary genomics behind phenotypic changes have been also addressed.

The results showed that, when evolving isolates interacting with plant roots, bacteria acquired mutations in particular metabolic pathways, enhancing their fitness during plant colonization. Certain evolved populations mutated in transcriptional termination factor Rho, leading to promoted adapted responses to a continuously changing conditions and effective root re-colonization. Surprisingly, nonsense mutation in rho resulted in increased toxicity. Importantly, results also indicated that carbohydrate metabolism is critical for effective root colonization, which was regulated by Rho. It was suggested that the plant polysaccharides acted as a driving factor during the experimental evolution. The other model, associating with nylon beads, was presented as the first complete study to evaluate B. thuringiensis evolutionary diversity in abiotic biofilm populations. The results offered additional insights on the previously unrecognized adaptation strategy. Specifically, due to the larger populations and ecological opportunities provided, morphological diversification was identified by a novel isolate, Fuzzy spreader (FS), which mutated in a guanylyltransferase-coding gene. The gene, related to capsular polysaccharide synthesis, has been disrupted by a transposon insertion element, leading to higher production of carbohydrates and elevated biofilm formation.

In conclusion, this PhD project has contributed to the understanding of previously unknown capability of laboratory evolution in B. cereus species. As far as we know, it also provided the first sight into the biofilm evolutionary pathways of this group in the lab scale, which should direct the future studies of B. cereus group interacting with plants in field trail and evaluation of the potential risks of virulence lifestyle during the adaption to certain niche.