Resolving the conflict between antibiotic production and rapid growth by recognition of peptidoglycan of susceptible competitors

Microbial communities employ a variety of complex strategies to compete successfully against competitors sharing their niche, with antibiotic production being a common strategy of aggression. Here, by systematic evaluation of four non-ribosomal peptides/polyketide (NRPs/PKS) antibiotics produced by Bacillus subtilis clade, we revealed that they acted synergistically to effectively eliminate phylogenetically distinct competitors. The production of these antibiotics came with a fitness cost manifested in growth inhibition, rendering their synthesis uneconomical when growing in proximity to a phylogenetically close species, carrying resistance against the same antibiotics. To resolve this conflict and ease the fitness cost, antibiotic production was only induced by the presence of a peptidoglycan cue from a sensitive competitor, a response mediated by the global regulator of cellular competence, ComA. These results experimentally demonstrate a general ecological concept – closely related communities are favoured during competition, due to compatibility in attack and defence mechanisms.

Arbitrary units (arb. units) for toxicity were determined as the ratio of WT B. subtilis growth alone/growth in competition against Bacilli: Biofilm cells were harvested 48 hours post inoculation and colony forming units (CFU) were calculated alone, and during co-inoculation. Graph represent mean ± SD from three independent experiments (n = 9). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc testing. P < 0.05 was considered statistically significant. No significant differences were observed between the toxicity Arbitrary units (arb. units) for toxicity were determined as the ratio of Bacilli growth alone/growth in competition against indicated mutants: Biofilm cells were harvested 48 hours post inoculation and colony forming units (CFU) were calculated alone, and during co-inoculation. Graphs represent mean ± SD from three independent experiments (n = 9). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc testing. P < 0.05 was considered statistically significant.
Significant differences between the toxicity towards WT B. subtilis and toxicity towards indicated Bacilli,     Figure 7. Analysis of the luciferase activity in a WT B. subtilis strain harboring P pksC -lux (bacillaene) reporter. Luminescence was monitored in B4 medium (No Treatment), and B4 medium supplemented with 15% v/v of the SF (<3kDa) of the conditioned medium from the indicated species. Graphs represent mean ± SD from three independent experiments (n = 9). Statistical analysis was performed using two-  Figure 10. Analysis of the luciferase activity in a WT B. subtilis strain harboring P srfAA -lux (surfactin) and P pksC -lux (bacillaene) reporter. Luminescence was monitored in B4 medium (No Treatment), and B4 medium supplemented with ESP (15% v/v) from the indicated species. Graphs represent mean ± SD from three independent experiments (n = 9). Statistical analysis was performed using two-way ANOVA followed by Dunnett's multiple comparison test. P < 0.05 was considered statistically significant. P values at different time points are shown in the  Figure 11. Analysis of the luciferase activity in a WT B. subtilis strain harboring P srfAA -lux (surfactin) and P pksC -lux (bacillaene) reporter. Luminescence was monitored in B4 medium (No Treatment), and B4 medium supplemented with DNA (≈100 ng/µl) from the indicated species. Graphs represent mean ± SD from three independent experiments (n = 9). Statistical analysis was performed using two-way ANOVA followed by Dunnett's multiple comparison test. P < 0.05 was considered statistically significant. P values at different time points are shown in the table. Source data are provided as a Source Data file.  Figure 13. Analysis of the luciferase activity in a WT B. subtilis strain harboring P srfAA -lux (surfactin) reporter. Luminescence was monitored in B4 medium (No Treatment), and B4 medium supplemented with PG (100 ng/µl) from the indicated species. Graphs represent mean ± SD from three independent experiments (n = 9).
Statistical analysis was performed using two-way ANOVA followed by Dunnett's multiple comparison test. P < 0.05 was considered statistically significant. P values at different time points are shown in the  and B4 medium supplemented with PG (100 ng/µl) from the indicated species. Supernatant was extracted from the samples at 16h and 24h using HCl treatment. Graphs represent mean ± SEM from four biological repeats (n = 4). Statistical analysis was performed using Brown-Forsthye and Welch's ANOVA with Dunnett's T3 multiple comparisons test. P < 0.05 was considered statistically significant.
Source data are provided as a Source Data file.   Quadrants with population (%) of the indicated reporters are mentioned.  Figure 1, 2 and S3. Toxicity values (shown in black) are means from three independent experiments (n = 9). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc testing. P < 0.05 was considered statistically significant. Significant differences between the toxicity towards indicated Bacilli when competed against WT B. subtilis and toxicity towards indicated Bacilli, when competed against the indicated NRP/PKS mutants are shown by their respective p values (in blue). Gene names and species name are in italics.

Strain construction
Deletions mutants were constructed using standard methods as described in 10,11 . For polymerase chain reactions, plasmids and primers used in this study are listed in Supplementary EPS was extracted 16 from the each indicated Bacilli biofilms grown at 48 hours at 30°C in B4 medium . Nine biofilm colonies from each Bacilli were scrapped and suspended in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), were mildly sonicated, and were then centrifuged at 8,000 × g to remove the cells. The supernatant was collected and mixed with five volumes of ice-cold isopropanol and incubated overnight at 4°C. Samples were then centrifuged at 10,000 × g for 10 min at 4°C. Pellets were suspended in a digestion mix of 0.1 M MgCl2, 0.1 mg/ml of DNase, and 0.1 mg/ml of RNase, and were incubated for 4 h at 37°C. Samples were extracted twice with phenol-chloroform. The aquatic fraction was dialyzed for 48 h with Slide-A-Lyzer dialysis cassettes by Thermo Fisher, 3,500 molecular weight cut-off, against distilled dH2O. Samples were lyophilized and lyophilized fraction was dissolved in 100 μl of dH2O.
The fractions were stored at −80 °C for further use.
Peptidoglycan (PG) was extracted from the indicated Bacilli in the corresponding figures legends 17,18 . Bacilli were grown in volume three hundred milliliter of B4 medium for 24 hours. Cells were collected by centrifugation (10,000 × g), washed with 0.8% NaCl, resuspended in hot 4% SDS, boiled for 30 min, and incubated at room temperature (RT) overnight. The suspension was then centrifuged (10,000 × g) to collect the pellet, and washed five times with dH2O to remove SDS. The pellet was then suspended in 25 mL 100 mM Tris-HCl, pH 7.5. with an addition of 25 μL RNAse solution (10 mg/mL), 25 μL of DNAse solution (10 mg/mL), and 250 μL of 1 M MgSO4 and incubated for 4h at 37 °C, with gentle shaking. This was followed by treatment with 25 μL of trypsin (10 mg/mL) and 250 μL of 1 M CaCl2 and incubated at 37 °C for 16 h, with gentle shaking. The insoluble material was then centrifuged at at 8,000 × g for 10 min at room temperature, washed once with water. The material was again resuspended in 4% SDS, boiled for 30 min, and incubated at room temperature (RT) overnight. The material was washed five times with dH2O to remove SDS. Pellet containing PG was dried, weighted and dissolved in dH2O. Material was further stored at −20 °C for further use . Protease Treated Conditioned Medium (CM)-Cells were grown to a mid-logarithmic phase of growth (OD=0. 6-0.8). Cells were diluted 1:100 in 300 ml of B4 medium and grown at 30°C for 24 h in a shaker incubator (Brunswick™ Innova® 42). Cells were removed by a centrifugation at (8,000 × g) and the growth media was filtered by 0.22μm filter (Corning).
For proteinase treatment, proteinase K (Sigma) was added to conditioned medium at a final concentration of 100 μg/ml and incubated for 2 hours at 37 °C, proteinase K was then removed with a 10-kDa cutoff spin filter Detecting the presence of PG using ELISA Detection of PG was carried out using Peptidoglycan ELISA kit (Abbexa®, Oxford, UK) according to the manufactures protocol. Briefly, B. thuringiensis biofilm colony was grown on B4 agar plates for 48 hours at 30 °C. 1.5 mm agar rings surrounding the colony were cut and scrapped out, and dissolved in of PBS (three agar rings were dissolved in 1 ml of PBS/ per observation) for 2 hours at room temperature by moderate shaking.
Positive controls were prepared at 100 ng/ml as described and provided by the manufactures. For negative control Standard Diluted buffers were used as provided by the manufacturer. The suspensions from PBS, positive and negative controls (50µl each) were then transferred to the 96-well ELISA plate, followed by instructions provided by the manufacturer. OD was measured at 450nm using a microplate reader (Synergy 2; BioTek, Winooski, VT, USA).