Kin discrimination, broadly defined as differential treatment of conspecifics according to their relatedness, could help biological systems direct cooperative behavior toward their relatives. Here we investigated the ability of the soil bacterium Bacillus subtilis to discriminate kin from nonkin in the context of swarming, a cooperative multicellular behavior. We tested a collection of sympatric conspecifics from soil in pairwise combinations and found that despite their history of coexistence, the vast majority formed distinct boundaries when the swarms met. Some swarms did merge, and most interestingly, this behavior was only seen in the most highly related strain pairs. Overall the swarm interaction phenotype strongly correlated with phylogenetic relatedness, indicative of kin discrimination. Using a subset of strains, we examined cocolonization patterns on plant roots. Pairs of kin strains were able to cocolonize roots and formed a mixed-strain biofilm. In contrast, inoculating roots with pairs of nonkin strains resulted in biofilms consisting primarily of one strain, suggestive of an antagonistic interaction among nonkin strains. This study firmly establishes kin discrimination in a bacterial multicellular setting and suggests its potential effect on ecological interactions.swarming | biofilm | social evolution | kin recognition | antagonism

SUMMARY Multicellularity inherently involves a number of cooperative behaviors that are potentially susceptible to exploitation but can be protected by mechanisms such as kin discrimination. Discrimination of kin from non-kin has been observed in swarms of the bacterium Bacillus subtilis, but the underlying molecular mechanism has been unknown. We used genetic, transcriptomic, and bioinformatic analyses to uncover kin recognition factors in this organism. Our results identified many molecules involved in cell surface modification and antimicrobial production and response. These genes varied significantly in expression level and mutation phenotype among B. subtilis strains, suggesting interstrain variation in the exact kin discrimination mechanism used. Genome analyses revealed a substantial diversity of antimicrobial genes present in unique combinations in different strains, with many likely acquired by horizontal gene transfer. The dynamic combinatorial effect derived from this plethora of kin discrimination genes creates a tight relatedness cutoff for cooperation that has likely led to rapid diversification within the species. Our data suggest that genes likely originally selected for competitive purposes also generate preferential interactions among kin, thus stabilizing multicellular lifestyles.

Bacillus subtilis strains communicate through the comQXPA quorum sensing (QS) system, which regulates genes expressed during early stationary phase. A high polymorphism of comQXP loci was found in closely related strains isolated from desert soil samples separated by distances ranging from meters to kilometers. The observed polymorphism comprised four communication groups (pherotypes), such that strains belonging to the same pherotype exchanged information efficiently but strains from different pherotypes failed to communicate. To determine whether the same level of polymorphism in the comQXP QS system could be detected at microscale, B. subtilis isolates were obtained from two separate 1-cm 3 soil samples, which were progressively divided into smaller sections. Cross-activation studies using pherotype-responsive reporter strains indicated the same number of communication pherotypes at microscale as previously determined at macroscale. Sequencing of the housekeeping gene gyrA and the QS comQ gene confirmed different evolutionary rates of these genes. Furthermore, an asymmetric communication response was detected inside the two pherotype clusters, suggesting continuous evolution of the QS system and possible development of new languages. To our knowledge, this is the first microscale study demonstrating the presence of different QS languages among isolates of one species, and the implications of this microscale diversity for microbial interactions are discussed.Quorum sensing (QS), a widespread phenomenon in the bacterial world, controls a wide range of cell density-dependent behaviors. Bacillus subtilis uses QS to control production of antimicrobial peptides, bacteriocins, and antibiotics (20) but also to alternate between two cell types during stationary phase: competent cells, able to take in DNA from the environment, and dormant spores, able to survive harsh environmental conditions (9,12,24). Development of genetic competence in B. subtilis is controlled by a QS system encoded by the comQXPA operon (2, 53, 54). This involves the ComX pheromone that accumulates during exponential growth (25,46,47) and is initially synthesized as a 55-residue protein that is processed, modified, and released into the extracellular medium as a 5-to 10-amino-acid peptide. The isoprenoidal modification on the tryptophan residue of this peptide is catalyzed by the ComQ protein (2,25,34,35,42,52). Upon reaching the threshold concentration, processed and modified ComX binds to the membrane-associated, histidine protein kinase ComP and triggers the QS response, linking autophosphorylation of ComP and transfer of phosphate to the response regulator ComA (59). The level of phosphorylated ComA is also controlled by dephosphorylation, which is dependent on a separate QS system involving competence sporulation factor (CSF) and the RapC phosphatase (3, 59). Phosphorylated ComA directly controls expression of various genes (6, 33), including the srfAB operon that contains the comS gene (15, 41), required for development of competence (55).P...

No abstract

Members of the family Bacillaceae are among the most robust bacteria on Earth, which is mainly due to their ability to form resistant endospores. This trait is believed to be the key factor determining the ecology of these bacteria. However, they also perform fundamental roles in soil ecology (i.e., the cycling of organic matter) and in plant health and growth stimulation (e.g., via suppression of plant pathogens and phosphate solubilization). In this review, we describe the high functional and genetic diversity that is found within the Bacillaceae (a family of low-G+C% Gram-positive spore-forming bacteria), their roles in ecology and in applied sciences related to agriculture. We then pose questions with respect to their ecological behavior, zooming in on the intricate social behavior that is becoming increasingly well characterized for some members of Bacillaceae. Such social behavior, which includes cell-to-cell signaling via quorum sensing or other mechanisms (e.g., the production of extracellular hydrolytic enzymes, toxins, antibiotics and/or surfactants) is a key determinant of their lifestyle and is also believed to drive diversification processes. It is only with a deeper understanding of cell-to-cell interactions that we will be able to understand the ecological and diversification processes of natural populations within the family Bacillaceae. Ultimately, the resulting improvements in understanding will benefit practical efforts to apply representatives of these bacteria in promoting plant growth as well as biological control of plant pathogens.