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

A quorum-sensing mechanism involving the pheromone ComX and the ComP-ComA two-component system controls natural competence in Bacillus subtilis. ComX is expressed as a cytoplasmic inactive precursor that is released into the extracellular medium as a cleaved, modified decapeptide. This process requires the product of comQ. In the presence of ComX, the membrane-localized ComP histidine kinase activates the response regulator ComA. We compared the sequences of the quorum-sensing genes from four closely related bacilli, and we report extensive genetic polymorphism extending through comQ, comX, and the 5 two-thirds of comP. This part of ComP encodes the membrane-localized and linker domains of the sensor protein. We also determined the sequences of the comX genes of four additional wild-type bacilli and tested the in vivo activities of all eight pheromones on isogenic strains containing four different ComP receptor proteins. A striking pattern of specificity was discovered, providing strong evidence that the pheromone contacts ComP directly. Furthermore, we show that coexpression of comQ and comX in Escherichia coli leads to the production of active pheromone in the medium, demonstrating that comQ is the only dedicated protein required for the processing, modification, and release of active competence pheromone. Some of the implications of these findings for the evolution and the mechanism of the quorum-sensing system are discussed.Under certain growth conditions, Bacillus subtilis expresses a set of proteins involved in the uptake and integration of extracellular DNA (reviewed in reference 3). The expression of these proteins in the competent state is tightly controlled and initially regulated by a quorum-sensing (QS) system involving the ComX pheromone (14-17, 30, 31) and the ComP-ComA two-component system (38, 40). The membrane-localized histidine kinase ComP, in the presence of ComX, activates the response regulator ComA, permitting the expression of downstream regulatory genes (4). This signal transduction cascade culminates with the expression of the competence transcription factor ComK (35,36), resulting in the synthesis of the DNA uptake and recombination proteins. A convergent pathway utilizes a second pheromone, the competence-stimulating factor (CSF) peptide, to modulate this response (16,31). This second pathway, which exerts a relatively minor effect, probably acts by inhibiting a phosphatase specific for ComA-ComP (22, 23). The two pathways thus appear to converge, both controlling the level of ComA phosphorylation (14,15,30).The ComX pheromone is translated as an inactive propeptide, which is then cleaved and released in the extracellular medium as a modified decapeptide, with the addition of an uncharacterized lipid moiety to a tryptophan residue (17). At least one other gene, comQ (39), is required for the production of modified, active ComX (17), but the precise role of ComQ is not understood.In the laboratory strains of B. subtilis, derived from strain 168, the comQ, comX, comP, and comA genes are...

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.

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens group by horizontal transmission.ComK is the major transcription factor driving the development of competence for transformation in Bacillus subtilis (37). rok encodes a direct repressor of comK transcription (18) and thereby plays an important, but incompletely understood, role in the complex regulation that takes place at the promoter of comK. Five proteins are known to bind to PcomK (reviewed in reference 9): ComK itself, DegU, Rok, AbrB, and CodY, the last three of which exert negative effects.The use of a rok-lacZ fusion construct revealed no major change in rok transcription during growth in the population as a whole (18). Given this, it is surprising that the regulation of rok transcription is complex; rok is repressed by Rok itself, by ComK, and by the transition state regulators SinR and AbrB, although only ComK and Rok have been shown to bind directly to Prok (18). As cultures approach the end of exponential growth, the concentrations of active AbrB and SinR decrease (reviewed in references 31 and 32), leading to the expectation that rok transcription would increase. However, since ComK acts negatively on Prok, the increased synthesis of ComK in competent cells after the cessation of exponential growth would tend to place a limit on increased rok transcription. In addition, negative autoregulation at Prok would be expected to maintain a constant time-averaged level of rok expression. Transient changes in the concentration of Rok may play a role in the timing of competence expression, in the selection of which cells will develop competence, and in limiting the final level of comK expression in the competent subpopulation. Although transient changes have not been detected, it may be that our experiments lacked sufficient time resolution to detect these fluctuations. A major unanswered question concerns the possibility that the activity of Rok is somehow regulated, perhaps responding to the presence of an unknown corepressor. To extend our understanding of the role of Rok, we identified additional genes regulated by this protein and asked whether Rok was capable of acting positively or only as a repressor. By transcriptional profiling and the use of lacZ fusions, we have identified at least seven gene clusters that are negatively regulated by rok, including several involved in the production of bacteriocin...

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