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. 2017 Jul 5;8(4):e00723-17.
doi: 10.1128/mBio.00723-17.

Bacillus subtilis Protects Public Goods by Extending Kin Discrimination to Closely Related Species

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Bacillus subtilis Protects Public Goods by Extending Kin Discrimination to Closely Related Species

Nicholas A Lyons et al. mBio. .

Abstract

Kin discrimination systems are found in numerous communal contexts like multicellularity and are theorized to prevent exploitation of cooperative behaviors. The kin discrimination system in Bacillus subtilis differs from most other such systems because it excludes nonkin cells rather than including kin cells. Because nonkin are the target of the system, B. subtilis can potentially distinguish degrees of nonkin relatedness, not just kin versus nonkin. We examined this by testing a large strain collection of diverse Bacillus species against B. subtilis in different multicellular contexts. The effects of kin discrimination extend to nearby species, as the other subtilis clade species were treated with the same antagonism as nonkin. Species in the less-related pumilus clade started to display varied phenotypes but were mostly still discriminated against, while cereus clade members and beyond were no longer subject to kin discrimination. Seeking a reason why other species are perceived as antagonistic nonkin, we tested the ability of B. subtilis to steal communally produced surfactant from these species. We found that the species treated as nonkin were the only ones that made a surfactant that B. subtilis could utilize and that nonkin antagonism prevented such stealing when the two strains were mixed. The nonkin exclusion kin discrimination method thus allows effective protection of the cooperative behaviors prevalent in multicellularity while still permitting interactions with more distant species that are not a threat.IMPORTANCE Multicellular systems like bacterial biofilms and swarms rely on cooperative behaviors that could be undermined by exploitative invaders. Discriminating kin from nonkin is one way to help guard against such exploitation but has thus far been examined only intraspecifically, so the phylogenetic range of this important trait is unknown. We tested whether Bacillus subtilis treats other species as nonkin by testing a single strain against a diverse collection of Bacillus isolates. We found that the species in the same clade were treated as nonkin, which then lessened in more distant relatives. Further experiments showed that these nonkin species produced a cooperative good that could be stolen by B. subtilis and that treating each other as nonkin largely prevented this exploitation. These results impact our understanding of interspecies interactions, as bacterial populations can interact only after they have diverged enough to no longer be a threat to their cooperative existences.

Keywords: antagonism; cell-cell interaction; evolution; microbial ecology.

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Figures

FIG 1
FIG 1
Phylogenetic tree of species used in this study. Minimum-evolution tree based on full-length 16S rRNA gene sequences from the type strain of each species. Unless indicated, all are from the genus Bacillus. Three Lysinibacillus and five Paenibacillus species were used in experiments, but only the sequences from L. fusiformis and P. taichungensis were included in this tree. In parentheses are the numbers of strains of each species/genus used. Bootstrap values are based on 500 replicates.
FIG 2
FIG 2
Multicellular interaction assays used to assess kin versus nonkin. (A) Swarm interaction assay. Representative examples of B. subtilis NCIB 3610 swarming toward either itself (upper left) or a different species, resulting in either merging of the colonies (top two images, green borders) or formation of a boundary between them (bottom, red borders). Bar, 1 cm. (B) Biofilm meeting assay. Biofilms of B. subtilis that encountered or enveloped the indicated species were scored as nonantagonistic (top, green borders), while biofilms that stopped short of the other species or showed signs of impaired growth were counted as antagonistic interactions (bottom, red borders). Bar, 0.5 cm. (C) Halo formation assay. Colonies of the indicated species were spotted on biofilm-inducing medium after top-spreading (but not pregrowing) B. subtilis cells and then examined for inhibition of lawn growth (halos) around the colony. Bar, 0.5 cm.
FIG 3
FIG 3
Varied phenotypes between and within different assays. (A) Examples of strains that displayed different phenotypes in the three multicellular interaction assays. Red and green borders indicate interactions judged as antagonistic and not antagonistic, respectively, in the individual assays, leading to a varied score overall. B. subtilis is in the lower right corner in the swarm and biofilm images and is the lawn in the halo images. Bar, 0.5 cm. (B) Strains that displayed opposite phenotypes in the swarm assay on different types of medium (LB or B, both with 0.7% agar). This behavior was observed in two strains each of B. altitudinis and Paenibacillus taichungensis (four strains total). P. taichungensis formed a thin swarm on LB that merged with B. subtilis (thick white ring) but did not spread out on B medium and formed a wide zone of inhibition. The B. subtilis swarm was spotted in the lower right corner in each image. Bar, 1 cm.
FIG 4
FIG 4
Overall phenotypes from all three interaction assays, arranged by phylogeny. (A) The first three columns indicate the number of strains of each species with the given final phenotype, a sum of the phenotypes from the three assays. The tree is from Fig. 1, except that the P. mirabilis branch is abbreviated for brevity; horizontal lines in the table divide clades. Mann-Whitney two-tailed independent tests were performed comparing the set of B. subtilis phenotypes (35 antagonistic) to the set from each species; significant P values are in bold. The percent identities of 16S rRNA genes to B. subtilis are given in the last column, and the red gradient on the far right is the inferred phylogenetic range of kin discrimination behavior. (B) 16S rRNA gene identity to B. subtilis of strains in each interaction phenotype group. “Interspecies antagonism” refers to all the non-subtilis species that exhibited antagonistic interactions. Bars represent the averages ± standard errors of the means (SEM). Asterisks indicate a significant difference from both the no-antagonism and varied phenotypes (P < 0.0001), which are not different from each other (P = 0.2516).
FIG 5
FIG 5
Surfactant-stealing assays. (A) B. subtilis NCIB 3610 ΔsrfAA (constitutively expressing YFP) spotted on swarm-inducing medium 3 cm away from either itself or its wild-type (WT) parent (expressing red fluorescent protein). The enlarged image shows the spatial distribution of the two fluorescent strains (mutant in green, wild type in red; overlap shows up as yellow). (B) Examples of strains that did and did not elicit spreading of B. subtilis ΔsrfAA, implying production of a surfactant that the mutant could use. Strain identity was verified by fluorescence as in panel A. (C) Correlation between the percentage of each species that had a usable surfactant and the average phenotype of each species in both the swarm interaction assay (black squares) and the final overall phenotype from all three assays (gray triangles). Values can be found in Table 1 and Fig. S1A in the supplemental material; error bars represent the standard error of the mean for the phenotype score. (D) Micrographs of mixtures of B. subtilis ΔsrfAA (expressing YFP) with strains that elicited spreading as determined in panel B. Examples are shown from the three general phenotype categories as defined on the right: zero/little spreading (0-cm2 area shown), modest spread (5.7 cm2 shown), and good spread but spatial segregation (24 cm2 shown). The number of strains that exhibited each phenotype is indicated in parentheses on the right. All bars, 1 cm.
FIG 6
FIG 6
Analyses of surfactant-stealing results. (A to C) Graphs of 16S rRNA gene identity to B. subtilis (A), final interaction phenotypes (B), and swarm interaction phenotypes (C) of strains that did or did not have surfactant stolen in the surfactant-stealing assay. The varied phenotypes in the swarm interaction assay are the strains that gave different results on different media, as in Fig. 3B. (D) Total area occupied by B. subtilis ΔsrfAA when alone or when mixed with surfactant-producing self (wild-type B. subtilis NCIB 3610) or other strains with usable surfactants. The crowded values near the bottom are shown in an expanded scale on the right. (E) Area of B. subtilis ΔsrfAA in mixtures with other strains, arranged in increasing order to highlight the large number that showed very little spreading. For all graphs, long horizontal lines are the averages, error bars represent the standard errors of the means, and P values from Mann-Whitney tests comparing each set are given above the graphs. “No ant.” and “Antag.” are the no-antagonism and antagonism categories, respectively.
FIG 7
FIG 7
Model of the two types of kin discrimination systems. The depiction of which cells are targeted in each system is at the top. Large circles at the bottom represent phylogenetic distance: highest relatedness in the center, decreasing outward along the radius. Both systems restrict interactions to clonemates (“self”) and kin (green central circle). The kin association system is based on association with kin cells and therefore treats all other species as not kin regardless of their evolutionary distance or physiological similarity (white outer circle). The nonkin exclusion system used by B. subtilis is further able to distinguish between closely (red) and distantly (white) related species. Arrows pointing outward indicate selective pressure to diversify, which we hypothesize is stronger in the nonkin exclusion system.

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