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. 2018 Sep 4:1:133.
doi: 10.1038/s42003-018-0136-1. eCollection 2018.

A single mutation in rapP induces cheating to prevent cheating in Bacillus subtilis by minimizing public good production

Nicholas A Lyons  1 , Roberto Kolter  2
Affiliations

A single mutation in rapP induces cheating to prevent cheating in Bacillus subtilis by minimizing public good production

Nicholas A Lyons et al. Commun Biol. .

Abstract

Cooperation is beneficial to group behaviors like multicellularity, but is vulnerable to exploitation by cheaters. Here we analyze mechanisms that protect against exploitation of extracellular surfactin in swarms of Bacillus subtilis. Unexpectedly, the reference strain NCIB 3610 displays inherent resistance to surfactin-non-producing cheaters, while a different wild isolate is susceptible. We trace this interstrain difference down to a single amino acid change in the plasmid-borne regulator RapP, which is necessary and sufficient for cheater mitigation. This allele, prevalent in many Bacillus species, optimizes transcription of the surfactin operon to the minimum needed for full cooperation. When combined with a strain lacking rapP, NCIB 3610 acts as a cheater itself-except it does not harm the population at high proportions since it still produces enough surfactin. This strategy of minimal production is thus a doubly advantageous mechanism to limit exploitation of public goods, and is readily evolved from existing regulatory networks.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Effect of a non-producer mutant in different B. subtilis strain backgrounds. a Representative examples of swarm plates of wild type, surfactin mutant (∆srfAA), and varying ratios of wild type + ∆srfAA mixtures in two closely related strains of B. subtilis, NCIB 3610 and PS-216. Plates are 8 cm in diameter. b Total cellular yield, as determined by OD600 readings of entire swarms, normalized to the value of wild-type alone (0% mutant). Averages of biological replicates (0% and 100% n = 2, others n = 4) ± standard error of the mean (SEM). The values statistically different from wild-type alone are 67%, 90%, and 100% for NCIB 3610 (P = 0.0024, <0.0001, <0.0001) and all for PS-216 (P = 0.0197, <0.0001, <0.0001, 0.0011, <0.0001). c Mean absolute values of wild-type swarm yields of both strains, in OD600 units. n = 4, error bars = SEM, P = 0.0200. d Relative fitness of the ∆srfAA mutant in swarms with wild type as determined by flow cytometry; each point is the mean ± SEM (wild-type alone n = 4, others n = 2), some error bars are not visible because they are smaller than the symbol. The wild-type alone values represent the fitness of mixtures of wild-type strains expressing different fluorescent proteins in varying ratios. All PS-216 values were significantly different from wild-type alone (P < 0.0001) except 90% mutant (P = 0.142), no NCIB 3610 ratios had significant fitness differences
Fig. 2
Fig. 2
Cheater resistance is lost by removing endogenous plasmid pBS32 or the rapP gene on it. a ∆srfAA cheating assays as in Fig. 1, with strains of NCIB 3610 either missing the plasmid (∆pBS32) or with the rapP gene interrupted by a transposon insertion (∆rapP). Total cell yield of each swarm normalized to the value of wild-type alone (0% mutant); averages of four (0% and 100%) or two replicates (all others). Relative fitness of the ∆srfAA mutant; each point is the mean ± SEM of independent biological replicates (0% n = 4, others n = 2). All yields were significantly different from wild-type alone (P < 0.0001) except ∆rapP 10% and 90% (P = 0.0388 and 0.0950); for fitness measurements, 10% and 33% mutant were statistically significant in both ∆pBS32 (P < 0.0001 and 0.0009) and ∆rapP (P = 0.0019 and 0.0477). b Cheating assays in which rapPphrP or rapP(T236N)phrP has been inserted into the chromosome of PS-216 or NCIB 3610 ∆pBS32. Statistically significant swarm yields included PS-216 + rapP 90% and 100% (P = 0.0003 and <0.0001), ∆pBS32 + rapP 67%, 90%, and 100% (P = 0.0102, <0.0001, <0.0001), and all ∆pBS32 + rapP(T236N) values (10% P = 0.0006, others P < 0.0001). Neither rapP addback had significant fitness values, but all ∆pBS32 + rapP(T236N) were significant (P < 0.0001 for all but 90% P = 0.0004)
Fig. 3
Fig. 3
Effect of RapP on gene expression. a Heat map of genes with at least two-fold change in expression between swarming strains. Each column is the log2 ratio of wild-type NCIB 3610, PS-216, or NCIB 3610 ∆pBS32. Only the expression differences that were statistically significant (P < 0.05 by two-tailed t-test) are shown; see Supplementary Data 1 for full results of all genes examined. The extreme differences displayed by sunA and rapP, which are in the SPβ prophage and plasmid pBS32, are due to the absence of the gene in one of the strains. b Percentage of swarming cells transcribing yfp from the indicated promoters in each strain, as determined by flow cytometry. Controls without yfp (no fluor) were used to set thresholds for YFP-positive events for each strain. NCIB 3610 is statistically different from both PS-216 and NCIB 3610 ∆pBS32 reporters for srfAA (P = 2.44 × 10−6 and 3.41 × 10−6) and sspB (P = 1.03 × 10−3 and 1.72 × 10−3). All are averages of three biological replicates; error bars represent SEM. c Transcriptional reporter expression in strains with rapPphrP inserted into their chromosomes. NCIB 3610 ∆pBS32+rapP(T236N) is significantly different from PS216+rapP and NCIB 3610 ∆pBS32+rapP in srfAA (P = 4.93 × 10−4 and 7.95 × 10−7) and sspB (P = 1.04 × 10−5 and 1.78 × 10−6) reporters. All are averages of at least three biological replicates, error bars represent SEM
Fig. 4
Fig. 4
Unicellular growth eliminates differences in public good production and cheating. a Percentage of cells expressing the PsrfAA-yfp transcriptional reporter during logarithmic growth in liquid LB medium. Averages of two (no fluor) or three (PsrfAA-yfp) biological replicates with SEM. wild type and ∆pBS32 reporters are not statistically different (P = 0.641). b Relative fitness of ∆srfAA in LB co-cultures with wild type in log phase; each point is the mean ± SEM of independent biological replicates (wild-type alone n = 4, others n = 2). No co-cultures were significantly different from wild-type alone cultures
Fig. 5
Fig. 5
RapP turns host cells into cheaters. a Cheating assays in NCIB 3610, except inoculating ∆pBS32 swarms with increasing proportion of wild-type cells instead of ∆srfAA. OD600 readings of total swarm yield, normalized to the value of 0% wild type. Relative fitness of the wild-type strain when mixed with ∆pBS32 with or without rapPphrP added in to the chromosome. The only swarm yields significantly different from 0% wild type were from wild type+∆pBS32 (all P < 0.0001). The wild type + ∆pBS32 fitness measurements of 10% and 33% were statistically significant (P < 0.0001 and 0.0008), as was the wild type + addback 10% (gray triangle) from both ∆pBS32-alone (P < 0.0001) and from wild type + ∆pBS32 10% (black circle, P = 0.0042). b Cheating assays with PS-216 or NCIB 3610 ∆pBS32 combined with increasing proportion of cells containing rapPphrP inserted into the chromosome (+rapP strain). All swarm yields were statistically significant except ∆pBS32 33% (P = 0.507), as were fitness values of PS-216 10% (P = 0.0080) and ∆pBS32 10% and 33% (P < 0.0001 and 0.0009). All points in figure are averages of at least two biological replicates, error bars represent SEM.
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