A Combinatorial Kin Discrimination System in Bacillus subtilis Nicholas A. Lyons, Barbara Kraigher, Polonca Stefanic, Ines Mandic-Mulec, Roberto Kolter  Current Biology  Volume 26, Issue 6, Pages 733-742 (March 2016) DOI: 10.1016/j.cub.2016.01.032 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Boundary Formation between Swarms of B. subtilis Strains Is Due to Cell Death (A) Swarms of strain NCIB 3610 form boundaries with swarms of the FENS 2-3-5 and CO39 strains on swarm-inducing B medium containing 0.7% agar. Scale bar, 1 cm. (B) Live/dead staining of the plates in (A), sampled from the meeting point of the same strain (first three bars) or the boundary region between two non-kin swarms (last two bars). Cells were incubated with impermeable propidium iodide (PI) to stain dead cells and a cell-permeable green dye to stain all cells; at least 200 total cells were counted using fluorescence microscopy; representative micrographs are shown. Percentages of PI-positive cells were averaged over three replicate samples. Data are represented as the mean ± SEM. Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Genetic Screen for Loss of Kin Recognition in NCIB 3610 (A) Example screen plate of Tn10 transposon insertion isolates grown on swarm-inducing medium, with negative and positive hits enlarged on the right. (B) The six transposon mutants that displayed a swarm boundary phenotype, swarming toward the wild-type NCIB 3610 parent strain. (C) Transposon mutants from (B) on swarming media containing supplemented phosphate (5 mM KH2PO4 instead of 0.6 mM). Scale bars, 1 cm. See also Figure S1. Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Candidate Gene Deletions Reinforce the Role of Antimicrobial Production and Defense in Kin Discrimination (A) Swarms of NCIB 3610 containing deletions of the indicated genes. See also Table S1. (B) The sigW promoter driving expression of yellow fluorescent protein (yfp) in swarms of wild-type and cannibalism mutants of NCIB 3610 and FENS 2-3-5. See also Figure S2. (C) Deletion of the antimicrobial response regulator sigW exacerbates the non-kin boundaries in some strain backgrounds. Scale bars, 1 cm (plates) and 0.5 cm (micrographs). Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Different Strains of B. subtilis Use Different Combinations of Kin Recognition Genes (A) The transposon insertions isolated from the screen in NCIB 3610 moved into the FENS 2-3-5 strain background, on swarm-inducing media. See also Figure S3. (B) Quadruple mutants of NCIB 3610 and FENS 2-3-5 swarms still form boundaries. (C) The extracellular matrix mutant (epsI) from the transposon screen performed in the PS-216 strain swarming toward the wild-type parent. (D) Deletions of the three main components of the extracellular matrix in the NCIB 3610 background have little effect on kin recognition with the parental strain. (E) Matrix mutants of the CO39 strain greatly exaggerate non-kin boundaries against the NCIB 3610 swarm. Scale bar, 1 cm. Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Gene Expression within Swarms Meeting Kin and Non-kin (A) NCIB 3610 swarms forming boundaries with FENS 2-3-5 and CO39 strains. Samples for gene expression analysis were taken from the boxed areas labeled a–h. Shown at the top are representative plates from three biological replicates. (B) Heatmap of all genes that displayed at least 2-fold difference in expression between samples. Each column is the ratio of the samples indicated in (A), ranging from higher in the denominator (blue) to higher in the numerator (orange), with white indicating a <2-fold change. See Data S1 for all expression values and the full list of genes tested. (C) The subset of genes with significant expression differences that are involved in antimicrobial production (red), cell-surface modification (cyan), or response to stresses (yellow). Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Whole-Genome Alignment of Strains 168 and RO-NN-1 The light-green graph depicts the percent identity between the two strains in a scanning window size of 4 kb. Genes of interest from many of the low-consensus regions are indicated above the graph: features present only in strain 168 are in orange text, genes only in RO-NN-1 are in blue, and genes found in both genomes are in black. “Toxin” is meant to be a general term for any genes with antimicrobial activity; Φ, phage. See also Figure S4. Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Conservation of Antimicrobial Genes across B. subtilis Strains (A) Twenty-three NCIB 3610 genes with antimicrobial activity were used to search all 52 fully sequenced B. subtilis genomes using BLASTn. Positive search results are represented by gray boxes; white boxes indicate the absence of an identified homolog. Strains are listed in order of decreasing relatedness to NCIB 3610 from left to right, with subspecies other than subtilis given in italics; bolded strains are those tested in (B). The total number of homologs of each gene is listed to the right of the table. Shown is an abbreviated collection of genomes searched; see Figure S5 for all 52 strains plus their phylogenetic relations. TA, toxin/antitoxin; CDI, contact-dependent inhibition; PF04740, polymorphic domain found in type II TA proteins [35]. (B) Swarms of various strains appearing in (A) were tested for kin discrimination with wild-type NCIB 3610 and NCIB 3610 ΔsunA, with relatedness decreasing counterclockwise. Scale bars, 1 cm. Current Biology 2016 26, 733-742DOI: (10.1016/j.cub.2016.01.032) Copyright © 2016 Elsevier Ltd Terms and Conditions