Social interactions and territoriality of Bacillus subtilis kin types in biofilmsBolješić, Maja (Avtor) Mandić Mulec, Ines (Mentor) Bacillus subtilisbiofilmkin discriminationincorporation of extracellular matrix polysaccharide mutantBacteria in natural environments mostly live in multicellular groups called biofilms. The biofilm-dwelling cells embedded in the extracellular matrix (ECM) due to their proximity engage intensely in social interactions. As biofilms are usually studied in monocultures, there is a lack of knowledge on genetically heterogeneous biofilms. Recently, it has been discovered that Gram-positive model bacterium, Bacillus subtilis, has the ability to discriminate between strains of high (kin) and low (nonkin) genetic relatedness evident as merging (kin) or boundary formation (nonkin) between swarms. However, it is not well understood how genetic relatedness between two strains mixed in a floating biofilm (pellicle) affects their fitness, the spatial cell assortment and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into the pellicles. Additionally, the role of the contact-dependent inhibition (CDI) toxin WapA in intraspecies competition is unclear. Thus, we tackled this question by cocultivation of B. subtilis strains of different genetic relatedness in the biofilm-promoting minimal medium (MSgg) under static conditions, allowing them to form pellicles. We show that in nonkin floating biofilms one strain prevails over the other by strongly reducing the frequency of its competitor, while in kin and isogenic combinations both strains have comparable relative frequencies. Nonkin strains segregate into larger cell patches relative to genetically more similar pairs which mix more homogenously. Furthermore, the invasion of the EpsA-O-deficient mutants into pellicles of the nonkin EpsA-O-producer is restricted. The wapAI mutant shows decreased fitness when cocultured with the nonkin strain in swarms and pellicles, whereas the parental strain reduces fitness of the toxin-sensitive strain only in swarming assay. However, the cell patches formed by the wapAI mutant when mixed with the parental strain are larger relative to pellicle containing two isogenic strains suggesting impaired mixing with the parental strain. In nonkin pellicle, however, the mutant lacking wapAI is almost completely excluded. In total, our results emphasize kin discrimination as a social behavior that influences strain fitness, spatial distribution, and restricts mixing and ECM sharing with the nonkin EpsA-O mutants. It also confirms the role of epsA-O and the wapAI loci as kin discrimination loci and provides evidence for the increased sensitivity of the toxin-sensitive mutant to nonkin attack. Our results importantly broaden the knowledge of bacterial kin discrimination.[M. Bolješić]20242024-09-26 07:16:53Doktorsko delo/naloga162619sl