Pectic homogalacturonan sensed by Bacillus acts as host associated cue to promote establishment and persistence in the rhizosphere.

Bacillus velezensis isolates are among the most promising plant-associated beneficial bacteria used as biocontrol agents. However, various aspects of the chemical communication between the plant and these beneficials, determining root colonization ability, remain poorly described. Here we investigated the molecular basis of such interkingdom interaction occurring upon contact between Bacillus velezensis and its host via the sensing of pectin backbone homogalacturonan (HG). We showed that B. velezensis stimulates key developmental traits via a dynamic process involving two conserved pectinolytic enzymes. This response integrates transcriptional changes leading to the switch from planktonic to sessile cells, a strong increase in biofilm formation, and an accelerated sporulation dynamics while conserving the potential to efficiently produce specialized secondary metabolites. As a whole, we anticipate that this response of Bacillus to cell wall-derived host cues contributes to its establishment and persistence in the competitive rhizosphere niche and ipso facto to its activity as biocontrol agent.

Plant-associated Bacillus mobilizes its secondary metabolites upon perception of the siderophore pyochelin produced by a Pseudomonas competitor.

Bacillus velezensis is considered as model species for plant-associated bacilli providing benefits to its host such as protection against phytopathogens. This is mainly due to the potential to secrete a wide range of secondary metabolites with specific and complementary bioactivities. This metabolite arsenal has been quite well defined genetically and chemically but much remains to be explored regarding how it is expressed under natural conditions and notably how it can be modulated upon interspecies interactions in the competitive rhizosphere niche. Here, 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 as it includes polyketides and the bacteriocin amylocyclicin, with broad antibiotic activity, as well as surfactin lipopeptides, contributing to biofilm formation and enhanced motility. Furthermore, we identified the secondary Pseudomonas siderophore pyochelin as an info-chemical, which triggers this response via a mechanism independent of iron stress. We hypothesize that B. velezensis relies on such chelator sensing to accurately identify 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 of the microbial conversations driving the behavior of members of the rhizosphere community.

Lipopeptide Interplay Mediates Molecular Interactions between Soil Bacilli and Pseudomonads.

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.

Surfactin Stimulated by Pectin Molecular Patterns and Root Exudates Acts as a Key Driver of the Bacillus-Plant Mutualistic Interaction.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction.

Biosurfactants in Plant Protection Against Diseases: Rhamnolipids and Lipopeptides Case Study.

Biosurfactants are amphiphilic surface-active molecules that are produced by a variety of microorganisms including fungi and bacteria. Pseudomonas, Burkholderia, and Bacillus species are known to secrete rhamnolipids and lipopeptides that are used in a wide range of industrial applications. Recently, these compounds have been studied in a context of plant-microbe interactions. This mini-review describes the direct antimicrobial activities of these compounds against plant pathogens. We also provide the current knowledge on how rhamnolipids and lipopeptides stimulate the plant immune system leading to plant resistance to phytopathogens. Given their low toxicity, high biodegradability and ecological acceptance, we discuss the possible role of these biosurfactants as alternative strategies to reduce or even replace pesticide use in agriculture.

Genome plasticity is governed by double strand break DNA repair in Streptomyces.

The linear chromosome of the bacterium Streptomyces exhibits a remarkable genetic organization with grossly a central conserved region flanked by variable chromosomal arms. The terminal diversity co-locates with an intense DNA plasticity including the occurrence of large deletions associated to circularization and chromosomal arm exchange. These observations prompted us to assess the role of double strand break (DSB) repair in chromosome plasticity following. For that purpose, DSBs were induced along the chromosome using the meganuclease I-SceI. DSB repair in the central region of the chromosome was mutagenic at the healing site but kept intact the whole genome structure. In contrast, DSB repair in the chromosomal arms was mostly associated to the loss of the targeted chromosomal arm and extensive deletions beyond the cleavage sites. While homologous recombination occurring between copies of DNA sequences accounted for the most part of the chromosome rescue events, Non Homologous End Joining was involved in mutagenic repair as well as in huge genome rearrangements (i.e. circularization). Further, NHEJ repair was concomitant with the integration of genetic material at the healing site. We postulate that DSB repair drives genome plasticity and evolution in Streptomyces and that NHEJ may foster horizontal transfer in the environment.

Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens.

Most bacterial organisms rely on homologous recombination to repair DNA double-strand breaks and for the post-replicative repair of DNA single-strand gaps. Homologous recombination can be divided into three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a recA-dependent synaptic step allowing the invasion of an intact copy and the formation of Holliday junctions, and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step. In Escherichia coli, branch migration and resolution are performed by the RuvABC complex, but could also rely on the RecG helicase in a redundant manner. In this study, we show that recG and ruvABC are well-conserved among Streptomyces. ΔruvABC, ΔrecG and ΔruvABC ΔrecG mutant strains were constructed. ΔruvABC ΔrecG is only slightly affected by exposure to DNA damage (UV). We also show that conjugational recombination decreases in the absence of RuvABC and RecG, but that intra-chromosomal recombination is not affected. These data suggest that RuvABC and RecG are indeed involved in homologous recombination in Streptomyces ambofaciens and that alternative factors are able to take over Holliday junction in Streptomyces.

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens.

Non-homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the "core" NHEJ gene set constituted of conserved loci and the "variable" NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC23877, not only the deletion of "core" genes but also that of "variable" genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.