Surfactin facilitates establishment of Bacillus subtilis in synthetic communities

Abstract

Soil bacteria are prolific producers of a myriad of biologically active secondary metabolites. These natural products play key roles in modern society, finding use as anti-cancer agents, as food additives, and as alternatives to chemical pesticides. As for their original role in interbacterial communication, secondary metabolites have been extensively studied under in vitro conditions, revealing many roles including antagonism, effects on motility, niche colonization, signaling, and cellular differentiation. Despite the growing body of knowledge on their mode of action, biosynthesis, and regulation, we still do not fully understand the role of secondary metabolites on the ecology of the producers and resident communities in situ. Here, we specifically examine the influence of Bacillus subtilis-produced cyclic lipopeptides during the assembly of a bacterial synthetic community, and simultaneously, explore the impact of cyclic lipopeptides on B. subtilis establishment success in a synthetic community propagated in an artificial soil microcosm. We found that surfactin production facilitates B. subtilis establishment success within multiple synthetic communities. Although neither a wild type nor a cyclic lipopeptide non-producer mutant had a major impact on the synthetic community composition over time, both the B. subtilis and the synthetic community metabolomes were altered during co-cultivation. Overall, our work demonstrates the importance of surfactin production in microbial communities, suggesting a broad spectrum of action of this natural product.

Keywords: Bacillus subtilissurfactin; chemical ecology; establishment; secondary metabolites; synthetic community.

Graphical Abstract

Figure 1

A schematic diagram illustrating the experimental design, and the research question of the main experiments conducted. B. subtilis P5_B1 and its NRP-deficient mutants were inoculated into a 4-member SynCom propagated in the hydrogel bead microcosms.

Figure 2

Surfactin production facilitates B. subtilis establishment in a SynCom but does not alter its composition in the soil-like environment over time. A set of gfp-labeled B. subtilis strains (Surfactin producers: WT and ppsC, non-producers: sfp and srfAC) were introduced into the SynCom, and their populations were followed over time to determine the role of LPs in SynCom assembly and their contribution to B. subtilis establishment in the simplified system. (A). SynCom assembly after the different B. subtilis variants were introduced. Members abundances are represented as the proportion occupied by each member relative to the total biomass in the system. (B) B. subtilis gfp-labeled population dynamics after introduction in the SynCom. Surfactin producers (WT and ppsC) population size remains stable over time whereas non-producers (sfp and srfAC) sharply decline by the end of the experiments suggesting that surfactin production is crucial for B. subtilis establishment in the SynCom. (C) B. subtilis WT and its derivates mutant growth dynamics when propagated individually in the hydrogel microcosms. (D) Complementation assay. A mixed population (1:1) of the WT and the srfAC was propagated in the hydrogel beads in the presence of the SynCom. The presence of the WT strain (surfactin producer) rescued the surfactin-deficient mutant. The letters represent significant differences among groups at day 14 (one-way ANOVA and Tukey honest tests). The experiment was conducted independently twice with n = 3 in both cases. The data were pooled and analyzed together.

Figure 3

Impact of individual SynCom members on B. subtilis growth. A set of B. subtilis strains were co-cultured with each SynCom member at different ratios. The GFP signal was used as a proxy for the respective B. subtilis strains’ growth in co-culture and the area under the curve of growth was used as culture productivity parameter. The impact of being co-culture with each SynCom member was estimated as % growth reduction = [(Growth^{Bsub Monoculture} - Growth^{Bsub Co-culture})/Growth^{Bsbu Monoculture})] × 100.

Figure 4

Changes in SynCom composition upon B. subtilis inoculation. Bray–Curtis distance NMDS ordination plot performed on the CFU data of the SynCom after B. subtilis introduction for comparing the effects of sampling time (colors) and the variant of B. subtilis (shapes) on SynCom composition. The multivariate analysis of SynCom composition was performed using a PERMANOVA on Bray–Curtis dissimilarity matrix obtained from the CFU counts dataset using the function adonis2 (R package vegan). The model was adjusted as: Y ~ time:variant. The P values and R² are reported as an inset within the figure.

Figure 5

The full SynCom and individual SynCom members induce surfactin production both in the bead system and in liquid culture. (A) Surfactin production in the bead system by P5_B1 increased when co-cultivated with the full SynCom compared to pure B. subtilis monoculture. The concentration was estimated from the last day of sampling in the B. subtilis, SynCom co-cultivation experiment. (B) Changes in surfactin production when P5_B1 was supplemented with cell-free supernatants obtained from individual SynCom members and the full SynCom in liquid cultures. In both experiments surfactin production was quantified by UHPLC and three replicates (n = 3) were performed per treatment.

Figure 6

Untargeted metabolic analysis revealed extensive metabolic changes in the co-cultivation experiments. (A) A heatmap visualization based on the significantly increased or decreased chemical features (m/z) in the co-cultivation experiments. A set of chemical features (m/z), related to OLS were strongly modulated in presence of surfactin producer (WT and ppsC) (Upper brackets). Similarly, m/z associated with surfactin isoforms were detected in the system (Bottom brackets). (B) Changes in the SynCom chemical features when supplemented with pure surfactin. The visualization shows the variation over the time (24, 48, and 72 h) of the main chemical features produced by the SynCom. Similarly, the same group, OLS-lipids, were produced less in presence of pure surfactin (upper brackets). The heatmaps were made on the feature abundances retrieved from the ESI-MS chromatogram.

Figure 7

B. subtilis P5_B1 establishment in different publicly available SynComs. The WT and its derivate mutant impaired in surfactin production (srfAC) were co-cultured with different SynCom at decreasing ratios (see Methods and SI for details). The gfp signal was used as proxy for Bacillus establishment in the co-cultures during 24 h incubation period. The area under the curve (AUC) ratio (B. subtilis growth in co-culture in each SynCom dilution to B. subtilis growth in monoculture) was used as the B. subtilis establishment parameter. “^*” indicates that the value of the AUC ratio was significant and its position denotated which strain had a higher value. “ns” indicates no significance (P < .05, Student’s t-test adjusted for multiple testing by the Benjamini–Hochberg method). The name inset the plot indicates the lab or the name of the SynCom, in parenthesis the number of members composing the community.