Featuring the bioactive secondary metabolites of Bacillus in interspecies interactions

Bacillus velezensis is considered as a model species for plant-associated bacilli providing its host with benefits such as protection against phytopathogens. This remarkable attribute stems from the potential of this bacteria to secrete a wide range of bioactive secondary metabolites (BSMs) with specific and complementary bioactivities. Until now, research into those secondary metabolites has predominantly focused on the characterization of their biological activities (e.g. antimicrobials), mostly guided by practical concerns for the use of these rhizobacteria as biocontrol agents.
However, our understanding of the ecological roles played by Bacillus’ BSMs and the fate of these BSMs within the competitive rhizosphere niche remains limited. Likewise, although it has been reported that B. velezensis adapts its BSMs production upon microbial interaction, the mechanisms by which this bacterium orchestrates its secondary metabolome are mostly unknown. Therefore, in this work, we aim to understand how Bacillus velezensis uses its BSMs to engage in microbial interaction with Streptomyces and Pseudomonas as well as the reciprocal effect of the interaction on the BSMs of Bacillus. Additionally, we investigate the intracellular regulatory processes governing the BSMs synthesis thereby enabling B. velezensis to modulate its production according to environmental cues. We show that B. velezensis can mobilize a substantial part of its metabolome upon the perception of Pseudomonas, as a soil-dwelling competitor. This metabolite response reflects a multimodal defensive strategy involving the lipopeptide surfactin that promotes biofilm formation and motility and includes antimicrobials such as polyketides and the bacteriocin amylocyclicin. Furthermore, we identify pyochelin, a secondary siderophore of Pseudomonas, as info-chemical triggering this response via a mechanism independent of iron stress. We hypothesize that B. velezensis senses such chelator as chemical marker of the presence of competitors, illustrating a new facet of siderophore-mediated interactions beyond the concept of competition for iron and siderophore piracy. This phenomenon may thus represent a new component in the microbial conversations driving the behavior of members of the rhizosphere community. Likewise, we observed that the metabolomes of Bacillus and Streptomyces undergo unsuspected changes upon interaction. We identify multiple new compounds derived from the interaction. Among these, we observed a change in the blend of the macrolactin produced by Bacillus, the induction of the siderophore of bacillibactin
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and several new variants of this molecule and a sharp increase in the production of novel avilamycin variants by Streptomyces sp. MM99. Remarkably, we note that several strains of Streptomyces promptly degrade the cyclic lipopeptides (CLPs) of Bacillus.
We further investigated the enzymatic degradation of Bacillus and Pseudomonas CLPs by Streptomyces. We observe that Streptomyces venezuelae is able to degrade the three lipopeptides surfactin, iturin and fengycin upon interaction with B. velezensis in vitro and in planta according to specific mechanisms. S. venezuelae is also able to degrade the structurally diverse sessilin, tolaasin, orfamide, xantholisin and putisolvin-type lipopeptides produced by Pseudomonas, indicating that this trait is likely engaged in the interaction with various competitors. Additionally, the degradation of CLPs is associated with the release of free amino and fatty acids used by Streptomyces to sustain growth. We hypothesize that lipopeptide-producing rhizobacteria and their biocontrol potential are impacted by the degradation of their lipopeptides as observed with the surface colonization pattern of B. velezensis, avoiding the confrontation zone with Streptomyces and the loss of antifungal properties of degraded iturin.
Finally, we bring a first comprehensive picture of the role of global regulators in the regulation of BSMs synthesis in B. velezensis by combining reverse genetics with comparative metabolomics and functional assays. More specifically, we identify ComA and Spo0A as key regulators of BSMs synthesis. Moreover, we make a direct link between these regulation processes and biocontrol related functions. Lastly, we pinpoint unsuspected activities of BSMs and implications of global regulators leading to the discovery of the cryptic lanthipeptide amylolysin β, further illustrating the secondary metabolome plasticity and diversity in B. velezensis.