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.