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. 2022 Mar;16(3):833-841.
doi: 10.1038/s41396-021-01124-4. Epub 2021 Oct 14.

Kin discrimination drives territorial exclusion during Bacillus subtilis swarming and restrains exploitation of surfactin

Barbara Kraigher  1 , Monika Butolen  2 , Polonca Stefanic  2 , Ines Mandic Mulec  3   4
Affiliations

Kin discrimination drives territorial exclusion during Bacillus subtilis swarming and restrains exploitation of surfactin

Barbara Kraigher et al. ISME J. 2022 Mar.

Abstract

Swarming is the collective movement of bacteria across a surface. It requires the production of surfactants (public goods) to overcome surface tension and provides an excellent model to investigate bacterial cooperation. Previously, we correlated swarm interaction phenotypes with kin discrimination between B. subtilis soil isolates, by showing that less related strains form boundaries between swarms and highly related strains merge. However, how kin discrimination affects cooperation and territoriality in swarming bacteria remains little explored. Here we show that the pattern of surface colonization by swarming mixtures is influenced by kin types. Closely related strain mixtures colonize the surface in a mixed swarm, while mixtures of less related strains show competitive exclusion as only one strain colonizes the surface. The outcome of nonkin swarm expansion depends on the initial ratio of the competing strains, indicating positive frequency-dependent competition. We find that addition of surfactin (a public good excreted from cells) can complement the swarming defect of nonkin mutants, whereas close encounters in nonkin mixtures lead to territorial exclusion, which limits the exploitation of surfactin by nonkin nonproducers. The work suggests that kin discrimination driven competitive territorial exclusion may be an important determinant for the success of cooperative surface colonization.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Swarm patterns of separately spotted and mixed kin and nonkin strains.
In encounter experiments (a, c), two kin or nonkin strains in the exponential growth phase were spotted on the swarming agar at a distance of 3–4 cm. In the mixture experiments (b, d, e) strains were mixed in liquid cultures (ratio 1:1) before spotting in the center of the swarming agar. The kin combination presented here is PS-218 mKate and PS-218 YFP (a, b), and the nonkin combination is PS-218 mKate and PS-216 YFP (c, d, e), which are representative of all the kin and nonkin strain combinations tested. Photographs of the 7-cm agar plates are shown at the top, and at the bottom, enlarged areas of the center of each plate are shown with merged and separate YFP and mKate fluorescent image captures taken by fluorescent stereomicroscope; magnification ×8, scale bar = 5 mm.
Fig. 2
Fig. 2. Influence of the initial ratio between strains on the colonization of swarming agar.
Mixtures of kin strains (strains PS-218 mKate and PS-218 YFP are shown here) or nonkin strains (strains PS-218 mKate and PS-216 YFP are shown here) in different ratios were spotted (2 µl) in the center of the swarming agar plates and visualized by flourescent stereomicroscope. In kin mixtures of all ratios (4:1, 1:1, and 1:4) both strains swarmed together over the entire agar surface, as indicated by the yellowish color (green–yellow or orange) in the merged image. In nonkin mixtures, in most cases only the more abundant strain in the inoculum mixture colonized the surface, indicated in the merged image by either red or green. Black panels under either mKate or YFP filters mean that the specific fluorescent proteins were not detected by fluorescent stereomicroscope, indicating a significant decrease in the abundance of the corresponding strain; magnification ×8, scale bar = 5 mm.
Fig. 3
Fig. 3. Swarming deficient mutants (srfA) can be complemented by surfactin from kin and nonkin strains.
a Surfactin from a kin strain restores swarming of the srfA mutant. PS-216 YFP srfA mutant was inoculated in the center of the swarming agar either alone or mixed with the PS-216 mKate (self-combination), with the PS-68 mKate (kin combination), or with 10 µl conditioned medium from the PS-216 mKate. When the conditioned medium was added, swarming of the mutant was restored but the pattern of swarming changed. Photographs of the 7-cm agar plates are shown at the top, and at the bottom, enlarged areas in the center of each plate are shown with merged YFP and mKate fluorescent images. Magnification ×8, scale bar = 5 mm. b, c Surfactin from the nonkin strain restores swarming of the srfA mutant. b The addition of 10 µl of conditioned medium from the nonkin wild-type strain (PS-218 YFP) promoted swarming of the PS-216 mKate srfA mutant. c Surfactin diffusing from the nonkin PS-218 mKate hag mutant was used by the PS-216 YFP srfA mutant spotted at 1-cm distance. d Swarming of the srfA mutant can be restored by a low proportion of wild-type kin cells. Strains PS-216 srfA and PS-216 were mixed in different proportions. Wild type restored swarming with only 0.2% in the initial mix.
Fig. 4
Fig. 4. Sharing of surfactin is affected by frequency-dependent competitive exclusion of nonkin.
a When the mutant PS-218 YFP srfA was mixed with the nonkin wild-type PS-216 mKate in different ratios (4:1, 1:1, and 1:4), the winning strain was the more abundant one. Swarming of the mutant was not rescued by the wild type at any ratio tested. Enlarged areas of the center of the plates are shown with merged and separated YFP and mKate fluorescent images; magnification ×8, scale bar = 5 mm. The experiment was repeated in three biological replicates (each with three technical replicates). Representative results of the experiments are shown. b Population size of the srfA mutant strain increases when it cooperates with the kin wild type, but not when mixed with the nonkin wild type. The PS-216 srfA YFP cells (2 µl) were mixed with 2 µl of sterile B medium or 2 μl of kin (PS-216 mKate) or nonkin strains (PS-218 mKate) and spotted in the center of the agar. After overnight incubation, the number of PS-216 srfA YFP cells in each colony was determined. The average number of three experiments (each represented by three replicate plates) with standard deviations are shown. A significant increase (P < 0.01) in the number of PS-216 YFP srfA cells was only detected when the mutant was mixed with kin wild-type cells. When PS-216 YFP srfA was mixed with the nonkin wild-type cells, one of two scenarios occurred: either the mutant conquered the plate and did not swarm, or the nonkin wild type conquered the plate and the mutant cell counts were less than 105 (*). Photographed 7-cm agar plates are shown at the bottom. * when the competing nonkin strain PS-218 mKate colonized the swarming plate, the number of PS-216 srfA YFP cells was lower than 105, which is less than 0,01% of the total population.
Fig. 5
Fig. 5. Flagella defective mutant hag can help srfA mutant to swarm.
a hag mutant can restore swarming of the kin srfA mutant. When two kin non-swarming mutants, PS-216 YFP hag and PS-216 mKate srfA, were mixed, the srfA mutant could swarm in all the ratios tested. b Swarming is restricted in nonkin hag and srfA mixtures. When nonkin mutants (PS-218 YFP srfA and PS-216 mKate hag) were mixed in different ratios, weak swarming occurred only if both strains survived (at a strain ratio of 1:1). Magnification ×8, scale bar = 5 mm.
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