Kin discrimination modifies fitness, spatial segregation and matrix sharing between strains with low relatedness in Bacillus subtilis biofilms

Microorganisms in nature form multicellular groups called biofilms. In biofilms bacteria embedded in a matrix of extracellular polymeric substances (EPS) interact intensely, due to their proximity to each other. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (non-kin) phylogenetic relatedness, reflected in merging (kin) and boundaries (non-kin) between swarms. However, it is not clear how kinship between interacting strains affects their fitness, the genotype distribution, and the EPS sharing in floating biofilms (pellicles). To address this gap in knowledge we cultivate B. subtilis strains as mixtures of kin and non-kin strains in static cultures, allowing them to form pellicles. We show here that in non-kin pellicles only one strains fitness was reduced; at the same time, strains segregated into larger patches and exhibited decreased matrix sharing, as compared to kin and isogenic pellicles, in which both strains had comparable colony forming units (CFU) counts and more homogenous cell mixing. Overall, our results emphasize kin discrimination (KD) as a social behavior that shapes fitness, spatial segregation and sharing of the extracellular matrix in genetically heterogenous biofilms of B. subtilis. IMPORTANCEBiofilm communities have both beneficial and harmful effects on human societies in natural, medical and industrial environments. Bacillus subtilis, a Gram-positive and biotechnologically important bacterium, serves as a model for studying biofilms. Recent studies have shown that this species engages in kin discriminatory behavior during swarming, which may have implications for community assembly, thus being of fundamental importance. Effects of KD on fitness, genotype segregation and matrix sharing in biofilms is not well understood. By using environmental strains with determined kin types and integrated fluorescent reporters we provide evidence that KD involves antagonism of the dominant strain against non-kin, which has important implications for genotype segregation and sharing of matrix polysaccharides between producers and non-producers. Our results reveal novel consequences of KD and are important for advancing our fundamental understanding of microbial sociality, and its role in the assembly of multicellular groups and in the shaping of microbial diversity.