Lipopeptide Interplay Mediates Molecular Interactions between Soil Bacilli and Pseudomonads

Abstract

Some Bacillus species, such as B. velezensis, are important members of the plant-associated microbiome, conferring protection against phytopathogens. However, our knowledge about multitrophic interactions determining the ecological fitness of these biocontrol bacteria in the competitive rhizosphere niche is still limited. Here, we investigated molecular mechanisms underlying interactions between B. velezensis and Pseudomonas as a soil-dwelling competitor. Upon their contact-independent in vitro confrontation, a multifaceted macroscopic outcome was observed and characterized by Bacillus growth inhibition, white line formation in the interaction zone, and enhanced motility. We correlated these phenotypes with the production of bioactive secondary metabolites and identified specific lipopeptides as key compounds involved in the interference interaction and motile response. Bacillus mobilizes its lipopeptide surfactin not only to enhance motility but also to act as a chemical trap to reduce the toxicity of lipopeptides formed by Pseudomonas. We demonstrated the relevance of these unsuspected roles of lipopeptides in the context of competitive tomato root colonization by the two bacterial genera. IMPORTANCE Plant-associated Bacillus velezensis and Pseudomonas spp. represent excellent model species as strong producers of bioactive metabolites involved in phytopathogen inhibition and the elicitation of plant immunity. However, the ecological role of these metabolites during microbial interspecies interactions and the way their expression may be modulated under naturally competitive soil conditions has been poorly investigated. Through this work, we report various phenotypic outcomes from the interactions between B. velezensis and 10 Pseudomonas strains used as competitors and correlate them with the production of specific metabolites called lipopeptides from both species. More precisely, Bacillus overproduces surfactin to enhance motility, which also, by acting as a chemical trap, reduces the toxicity of other lipopeptides formed by Pseudomonas. Based on data from interspecies competition on plant roots, we assume this would allow Bacillus to gain fitness and persistence in its natural rhizosphere niche. The discovery of new ecological functions for Bacillus and Pseudomonas secondary metabolites is crucial to rationally design compatible consortia, more efficient than single-species inoculants, to promote plant health and growth by fighting economically important pathogens in sustainable agriculture.

Keywords: Bacillus velezensis; Pseudomonas; bioactive secondary metabolites; cyclic lipopeptides; microbial ecology; microbial interaction; molecular cross-talk; plant growth-promoting rhizobacteria.

FIG 1

Predicted and detected bioactive secondary metabolites (BSMs) produced by the Pseudomonas strains used in this study. Metabolites with biosynthetic gene clusters (BGCs) predicted by antiSMASH 6.0 (42) and detected ([M+H]⁺ or [M+2H]⁺, the latter is indicated with an asterisk) by UPLC-qTOF MS in crude cell-free supernatant after growth in CAA medium are represented by green squares, while undetected BSMs are represented by light red squares. Detected m/z and mass errors in parts per million (based on one measurement from one strain [orfamides, CMR12a; achromobactin, JV497; phenazines, CMR12a; 2,4-diacetylphloroglucinol, Pf-5]) corresponding to the main variant(s) (indicated with the letters) detected are presented. The data are representative of the two independent repetitions.

FIG 2

Phenotype and growth of B. velezensis GA1 following confrontation with different Pseudomonas strains. (A) B. velezensis GA1 (left colony) phenotypic response on solid CAA medium following short distance (1 mm) confrontation with different Pseudomonas strains (right colony). The control (CTRL) colony shows GA1 cultured alone. (B) GA1 growth curve in EM liquid medium supplemented with 4% (vol/vol) of different Pseudomonas cell-free supernatants. The control (CTRL) corresponds to unsupplemented GA1 culture (data represent means ± standard deviation [SD]; n = 9). (C) White line formation (indicated by arrow) between colonies of GA1 (left colony) and some Pseudomonas strains (right colony) on jellified CAA medium following longer distance (5 mm) confrontation. (D) GA1 (left colony) motile response on EM medium following confrontation with different Pseudomonas strains (right colony). The control (CTRL) colony shows GA1 cultured alone. Pictures in panels A, C, and D are representative of the response observed in three independent repetitions with three technical replicates (n = 9).

FIG 3

Surfactin attenuates sessilin-mediated toxicity via white line formation. (A) GA1 biomass level measured after 10 h of growth in EM liquid medium supplemented or not (CTRL) with 4% (vol/vol) of cell-free supernatants from CAA cultures of CMR12a wild type or its mutants repressed in the synthesis of orfamides and phenazines (ΔofaBC-phz), sessilins (ΔsesA), sessilins and orfamides (ΔsesA-ofaBC), sessilins and phenazines (ΔsesA-phz), or all compounds (ΔsesA-ofaBC-phz) (for metabolome, see Table S1 in the supplemental material). Data show mean ± SD calculated from two independent experiments each with three culture replicates (n = 6) and different letters indicating statistically significant differences between the treatments (ANOVA and Tukey’s HSD tests, α = 0.05). (B) Growth inhibition of GA1 wild type and its ΔsrfaA mutant repressed in surfactin synthesis after 10 h of culture and following delayed supplementation (added 6 h after incubation start) with cell-free supernatants from CMR12a wild type (alone or together with 10 µM pure surfactin as chemical complementation) and with cell-free supernatants from the sessilin mutant (ΔsesA). Unsupplemented cultures of GA1 were used as a control (CTRL). Experiments were replicated, and data were statistically processed as described in panel A (n = 6). (C) White line formation and/or Bacillus inhibition observed following confrontation of GA1 wild type or the surfactin mutant ΔsrfaA with (I) CMR12a or its ΔsesA derivative, (II) P. tolaasii CH36 or its tolaasin-defective mutant ΔtolA, and (III) other Pseudomonas CLP producers (for metabolome, see Table S1). Pictures are representative of three independent repeats. (D) 3D representation of UPLC-MS analysis of metabolites that are present in the white line zone between GA1 and CMR12a showing the specific accumulation of sessilin and surfactin molecular ions.

FIG 4

Distance- and surfactin-dependent enhanced motility of B. velezensis GA1 mediated by interaction with Pseudomonas. (A) GA1 motility phenotype on EM jellified medium when cultured alone (left) or in confrontation with CMR12a (1 mm) (right). (B) Motility pattern of GA1 or the ΔsrfaA surfactin-deficient mutant in confrontation with CMR12a (5 mm). (C) MALDI FT-ICR mass spectrometry imaging (MSI) heat maps showing spatial localization and relative abundance of ions corresponding to the C₁₄ surfactin homolog (most abundant) when GA1 is confronted with CMR12a at increasing distances. (D) Comparison of surfactin production (expressed in fold change) in GA1 culture supplemented with 4% (vol/vol) of CMR12a or JV497 supernatants. The unsupplemented culture was fixed at 1 (CTRL). Bars show mean ± SD (n = 9). Statistical comparisons between treatments were assessed with the Mann-Whitney test; ns, not significant; ****, P < 0.0001. (E) UPLC-MS extracted ion chromatogram (EIC) illustrating the relative abundance of surfactin produced in GA1 EM medium when cultured alone (CTRL in red) or supplemented with 4% (vol/vol) CMR12a (+CMR12a in green). The different peaks correspond to the structural variants differing in fatty acid chain length.

FIG 5

Competitive root colonization assays support the roles of BSMs in Bacillus-Pseudomonas interaction in planta. (A) GA1 population (CFU per gram of tomato root) recovered at 3 days (empty bars) and 6 days (hatched bars) after coinoculation (dpi) with JV497, CMR12a, and its sessilin-repressed mutant compared to GA1 inoculated alone. Box plots were generated based on data from a minimum of three biologically independent assays each involving 6 plants per treatment (n = 18). The whiskers extend to the minimum and maximum values, and the midline indicates the median. Statistical differences between data at the same dpi are compared to the control (CTRL) and are labeled in red, while differences between treatments at different dpi are indicated with horizontal lines and a black asterisk when significant. Statistical analyses were performed using a Mann-Whitney test; ns, no significant difference; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (B) Confocal laser scanning microscopy images of tomato root colonization by GA1 (elongation root zone) at 6 dpi after monoinoculation (CTRL) or coinoculation with JV497, CMR12a, or CMR12a ΔsesA. GA1 tagged with GFPmut3 is depicted in green, while mCherry- or eforRed-labeled cells in red correspond to CMR12a and JV497 wild types or CMR12a ΔsesA, respectively. (C) UPLC-MS EIC illustrating relative production in planta of sessilin (blue peak) and surfactin (green peak) after 6 dpi of GA1 alone (GA1) or coinoculated with wild-type Pseudomonas (GA1 + CMR12a) or its sessilin-impaired mutant (GA1 + ΔsesA). (D) Cell populations recovered at 3 dpi for GA1 wild type (GA1) or the surfactin-impaired mutant (ΔsrfaA) coinoculated with CMR12a wild type (CMR12a) or its sessilin-impaired mutant (ΔsesA). Box plots were generated based on data from four biologically independent assays each involving at least 4 plants per treatment (n = 16). The whiskers extend to the minimum and maximum values, and the midline indicates the median. Statistical differences between the treatments were calculated using a Mann-Whitney test; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.