Fig 1.
LXG toxins-antitoxin systems in B. subtilis.
(A) Multiple alignment of LXG toxins from B. subtilis strain 3610. Alignment was constructed using NCBI COBALT (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) with default settings. Red indicates highly conserved positions and blue indicates lower conservation. Red lines are gaps in alignment. The location of the LXG domain, the central region, and the toxin domain are shown below the alignment. (B) Intercellular competition mediated by LXG toxin-antitoxin systems. LXG toxins are neutralized by cognate antitoxins in toxin producers. The WXG100 protein promotes the T7SS-dependent delivery of LXG toxins from producers to recipients. If recipients do not express cognate antitoxins, delivered LXG toxins exert toxic effects. Cognate toxin domains and antitoxins are shown by the same color.
Table 1.
Classification and distribution of LXG toxin and antitoxin genes in B. subtilis strainsa).
Fig 2.
Expression of LXG toxin–antitoxin operons.
Strains harboring promoter-gfp reporters were grown to an OD600 of 0.7 to 0.8 in liquid LB (preculture, 0 h). The cultures were diluted to an OD600 of 0.5, and 2 μl of the dilutions were spotted on three solid media, MSgg, 2×SGG, and LB. After 12 h, 24 h, or 48 h of incubation at 30°C, expression of gfp reporters in colonies were measured by flow cytometry. Strain 3610 (with no gfp reporter) was used as a negative control. Two sets of data are shown for each strain. All histograms are shown on the same scale, and values for the x and y axes are shown only in the lower left histogram. The peak positions of fluorescence from strain 3610 are indicated as a background reference by dotted lines.
Fig 3.
In vivo activity of LXG toxins and antitoxins.
(A) Induction of LXG toxins prevents colony formation. Overnight cultures of Pspac-hy-LXG toxin gene strains (left panels) and Pspac-hy-LXG toxin-antitoxin operon strains (right panels) were serially diluted, and 2 μl of the dilutions was spotted onto LB solid media with or without 1 mM IPTG. The wild-type strain was used as a reference. The plates were then incubated at 37°C for 18 h. (B) Representative growth curves of wild-type (WT) and Pspac-hy-LXG toxin gene strains. Strains were grown at 37°C in LB with vigorous shaking, and optical density at OD600 was monitored over time. IPTG (final concentration 1 mM) was added at the indicated time point (0 h). (C) The DNA and RNA content of cells before and after induction of LXG toxins. Cells were collected at 0 h and 1 h shown in panel B, samples of total DNA and RNA were isolated, and the samples from 0.03 OD cells were then analyzed by agarose gel electrophoresis. The positions of chromosome DNA and rRNAs are indicated by arrows. Results of two independent experiments are shown.
Fig 4.
LXG toxins and antitoxins mediate intercellular competition.
(A) Time-course analysis of competition. The wild-type strain and LXG toxin–antitoxin deletion mutants carrying a constitutively expressed gfp reporter (GFP strains) were grown to an OD600 of 0.7–0.8. and precisely diluted cultures (OD600 of 0.5) of the wild-type and one of the mutants were mixed at a 1:1 ratio. Exact proportions of the GFP strains in the mixtures were determined by flow cytometry and used as time 0 samples. The mixtures were spotted onto solid MSgg medium, and the inoculated plates were then incubated at 30°C. The proportion of GFP-reporter strains within colonies at each time point was determined by flow cytometry using three independent colonies. Percentages are presented as mean ± standard deviation (n = 3). (B) Inducing antitoxins restored competitiveness to toxin–antitoxin deletion mutants. Antitoxin genes were ectopically induced from the IPTG-dependent spac-hy promoter at the amy locus of mutant chromosomes. The indicated strains were co-cultured on MSgg supplemented with 0.1 mM IPTG. Percentages are presented as mean ± standard deviation (n = 3).
Fig 5.
T7SS is required for LXG toxin delivery.
(A) Competition assays between LXG toxin–antitoxin deletion mutants and T7SS mutants. Strains were co-cultured for 48 h on MSgg. Competition assays using ΔyukE Pspac-hy-yukE and ΔyukC Pspac-hy-yukC mutants were done on MSgg supplemented with 0.1 mM IPTG. (B) Reducing the initial cell density induced spatially segregated growth. Cultures (OD600 of 0.5) of the wild-type with or without the gfp reporter were mixed at a 1:1 ratio. The mixtures were diluted 1 to 100-fold, and 2 μl of the diluted mixtures were spotted on MSgg solid medium. After 48 h, colonies were observed with a stereomicroscope. Top views are colony images, and bottom views are enlarged images of the colony center. Scale bars, 2 mm (top), and 0.2 mm (bottom). (C) Cluster formation prevented LXG toxin function. Competition assays were conducted using diluted inoculation mixtures. Percentages are presented as mean ± standard deviation (n = 3).
Fig 6.
LXG toxin–antitoxin systems specifically function in biofilms.
(A) Competition assays with the wild-type strain and LXG toxin–antitoxin deletion mutants on three different solid media. (B) Competition assays with the wild-type strain and LXG toxin–antitoxin deletion mutants in liquid shaking culture. Strains were co-cultured for 24 h in liquid MSgg medium with vigorous shaking. (C) Biofilm matrix polymers were required for LXG toxin function. Competition assays were conducted in the ΔepsA–O (top) or ΔtapA–tasA (bottom) mutant backgrounds. Percentages are presented as mean ± standard deviation (n = 3).
Fig 7.
Round-robin duels between six LXG toxin–antitoxin deletion mutants.
(A) The indicated LXG toxin–antitoxin deletion mutants, with or without the gfp reporter, were co-cultured at a 1:1 ratio on MSgg solid medium. After 48 h, the proportion of GFP-reporter strains within colonies was determined as mean ± standard deviation (n = 3). Fluorescent images of colonies are also shown. The experiments were performed at least three times, and representative examples are shown in the figures. The mutant strains are arranged from left (top) to right (bottom) in order of competitiveness. Scale bar, 2 mm. (B). Enlarged images of colony centers. The values of the GFP strain (%) are from panel A. Scale bar, 0.2 mm. (C) LXG toxins function synergistically. The ΔyxiB–E mutant with the gfp reporter was co-cultured with double LXG toxin–antitoxins mutants. The proportion of GFP-reporter strains within colonies was reported as mean ± standard deviation (n = 3). Fluorescent images of colonies are also shown. Scale bar, 2 mm.
Fig 8.
LXG toxins are effective against natural isolates of B. subtilis.
The GFP-labeled strain 3610 or the T7SS mutant ΔyukE–D was co-cultured with a B. subtilis natural isolate at a ratio of 1:1 on MSgg medium. After 48 h of cultivation, the morphology and GFP fluorescence of colonies were observed with a stereomicroscope. Single-strain cultures are shown as references. Selected results are shown; see S10 Fig for complete results. The experiments were repeated twice and confirmed the reproducibility. The classification of co-cultured colonies is indicated on the right. Scale bar, 2 mm.
Table 2.
Distribution of antibiotic and toxin biosynthesis genes in B. subtilis strainsa, b).
Fig 9.
WapA exhibits different properties from LXG toxins.
(A) Expression of the wapAI toxin-antitoxin operon. The PwapA-gfp strain was grown as described in Fig 1, and GFP fluorescence levels were measured by flow cytometry. Two sets of data are shown for each strain. (B) Competition assays with the wild-type strain and the ΔwapAI (gfp) mutant on MSgg, 2×SGG, and LB media in different genetic backgrounds. The proportion of GFP-reporter strains within colonies was reported as mean ± standard deviation (n = 3). (C) Herd protection did not protect against WapA. Competition assays were conducted on LB using diluted culture mixtures (n = 3). (D) Colony morphology in competition assays on LB. Colony morphology and GFP fluorescence were observed with a stereomicroscope 24 h after inoculation. (E) Competition assays with the wild-type strain and the ΔwapAI (gfp) mutant in LB under shaking conditions (n = 3).
Table 3.
B. subtilis strains used in this study.