Exploring the influence of bacterial warfare on horizontal gene transfer and evolution of B. subtilis

Background: Natural genetic transformation is the ability of bacteria to take up extracellular DNA and integrate it into their genomes. Natural transformation has a vital impact on adaptation and evolution, despite existing mechanisms that reduce successful DNA integration. Our model microorganism Bacillus subtilis exhibits sexual isolation, in which the transformation frequency decreases dramatically with sequence divergence between the donor DNA and recipient genome. However, recent findings suggest that intra-species antagonism may promote the gene flow (acquisition of antibiotic resistances) and diversification. Problem description: the proposed project will focus on the evolutionary role of Kin discrimination (KD) is a process in which bacteria recognize less related non-kin strains and react with an antagonistic response (killing). This in turn up-regulates the natural ability to take up DNA and could provide a mechanism for bacterial intra-species diversification. The key question of this study is: how do bacterial interactions in relation to relatedness, influence gene transfer and evolution of B. subtilis and related species? The aim of the study: The project aims to determine how KD influences horizontal gene transfer within B. subtilis and between related species and to determine whether the distribution of KD genes correlates with the phylogenetic distance of strains. Furthermore, we will test whether KD overpowers sexual isolation during bacterial interactions and test at which relatedness KD-related DNA transfer between two interacting bacteria is the highest. In addition, we will perform experimental evolution, and test whether increased DNA transfer between non-kin strains further promotes the formation of new KD types and determine gene alterations that lead to new KD types in kin and non-kin encounters. Originality of the results: A few microbial species have been found to be capable of KD based on specific genes (e.g. toxins), but there is very little evidence of phylogeny-based KD in bacteria and even less is known about the consequences of such interactions. The antagonism in B. subtilis, which is reflected in increased DNA exchange most likely influences genome diversification, however, this hypothesis has not yet been tested. The proposed project is therefore very original in that it addresses a previously undiscovered evolutionary mechanism based on antagonism-driven diversification and could pave the way for many scientists to observe this diversification mechanism in other bacterial species, especially in medicine where multiresistant bacteria pose a serious threat. Methods: we have a set of 40 wt B. subtilis strains isolated from a 1 cm3 soil sample and approximately 500 genomes from the NCBI database, which will be compared. We will perform additional genome sequencing and bioinformatics analyses to perform genome comparisons. In vitro evolution will be performed on swarm agar plates and methodoloy for cell separation via cell sorter will be developed. Newly evolved strains will be sequenced and sequences analysed to find gene alterations that lead to new KD types. Results potential and relevancy: Only a few microbial species are able to distinguish kin from non-kin and we were the first to find this in B. subtilis. The proposed project is highly original and knowledge obtained will 1) support the development of bioinformatics tools to detect and predict new KD types 2) allow deeper understanding of the role of KD on the evolution of this species, 3) demonstrate the potential impact of this phenomenon on the ecology and sociality of this species and 4) improve understanding of inter- and intra-species interactions in the context of horizontal gene transfer as a consequence of social interactions. The results will be novel and exciting and we will be able to publish our work in prestigious scientific journals, which will increase the international recognisability of all involved research groups.