Bacillus lipopeptides as key players in rhizosphere chemical ecology.

Microbial natural products are widely explored for their therapeutic potential. Understanding the underlying evolutionary and adaptive forces driving their production remains a fundamental question in biology. Amphiphilic cyclic lipopeptides (CLPs), a prominent category of bacterial specialized metabolites, show strong antimicrobial activity, particularly against phytopathogens. It is thus assumed that these compounds are deployed by soil- or rhizosphere-dwelling bacteria as microbial weapons in competitive natural environments. Here, we challenge this reductionist perspective and present evidence that Bacillus CLPs are prominent chemical mediators of ecological interactions. They help Bacillus to communicate, compete, defend against predators, or cooperate and establish mutualistic relationships with other (micro)organisms. Additional parallel examples are highlighted in other genera, such as Pseudomonas. This broader perspective underscores the need for further investigation into the role of CLPs in shaping the adaptive strategies of key rhizobacterial species.

Biosynthesis of the antioxidant γ-glutamyl-cysteine with engineered Yarrowia lipolytica.

The dipeptide γ-glutamylcysteine (γ-GC), the first intermediate of glutathione (GSH) synthesis, is considered as a promising drug to reduce or prevent plethora of age-related disorders such as Alzheimer and Parkinson diseases. The unusual γ-linkage between the two constitutive amino acids, namely cysteine and glutamate, renders its chemical synthesis particularly challenging. Herein, we report on the metabolic engineering of the non-conventional yeast Yarrowia lipolytica for efficient γ-GC synthesis. The yeast was first converted into a γ-GC producer by disruption of gene GSH2 encoding GSH synthase and by constitutive expression of GSH1 encoding glutamylcysteine ligase. Subsequently genes involved in cysteine and glutamate anabolism, namely MET4, CYSE, CYSF, and GDH1 were overexpressed with the aim to increase their intracellular availability. With such a strategy, a γ-GC titer of 464 nmol mg-1 protein (93 mg gDCW-1 ) was obtained within 24 h of cell growth.

Lipopeptides as rhizosphere public goods for microbial cooperation.

IMPORTANCE: Here, we provide new insights into the possible fate of cyclic lipopeptides as prominent specialized metabolites from beneficial bacilli and pseudomonads once released in the soil. Our data illustrate how the B. velezensis lipopeptidome may be enzymatically remodeled by Streptomyces as important members of the soil bacterial community. The enzymatic arsenal of S. venezuelae enables an unsuspected extensive degradation of these compounds, allowing the bacterium to feed on these exogenous products via a mechanism going beyond linearization, which was previously reported as a detoxification strategy. As soils are carbon-rich and nitrogen-poor environments, we propose a new role for cyclic lipopeptides in interspecies interactions, which is to fuel the nitrogen metabolism of a part of the rhizosphere microbial community. Streptomyces and other actinomycetes, producing numerous peptidases and displaying several traits of beneficial bacteria, should be at the front line to directly benefit from these metabolites as "public goods" for microbial cooperation.

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.

Bacillus lipopeptide-mediated biocontrol of peanut stem rot caused by Athelia rolfsii.

Introduction: Peanut (Arachis hypogaea L.) is a widespread oilseed crop of high agricultural importance in tropical and subtropical areas. It plays a major role in the food supply in the Democratic Republic of Congo (DRC). However, one major constraint in the production of this plant is the stem rot (white mold or southern blight) disease caused by Athelia rolfsii which is so far controlled mainly using chemicals. Considering the harmful effect of chemical pesticides, the implementation of eco-friendly alternatives such as biological control is required for disease management in a more sustainable agriculture in the DRC as in the other developing countries concerned. Bacillus velezensis is among the rhizobacteria best described for its plant protective effect notably due to the production of a wide range of bioactive secondary metabolites. In this work, we wanted to evaluate the potential of B. velezensis strain GA1 at reducing A. rolfsii infection and to unravel the molecular basis of the protective effect. Results and discussion: Upon growth under the nutritional conditions dictated by peanut root exudation, the bacterium efficiently produces the three types of lipopeptides surfactin, iturin and fengycin known for their antagonistic activities against a wide range of fungal phytopathogens. By testing a range of GA1 mutants specifically repressed in the production of those metabolites, we point out an important role for iturin and another unidentified compound in the antagonistic activity against the pathogen. Biocontrol experiments performed in greenhouse further revealed the efficacy of B. velezensis to reduce peanut disease caused by A. rolfsii both via direct antagonism against the fungus and by stimulating systemic resistance in the host plant. As treatment with pure surfactin yielded a similar level of protection, we postulate that this lipopeptide acts as main elicitor of peanut resistance against A. rolfsii infection.

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.

If All Else Fails: Impact of Silicon Accumulation in Maize Leaves on Volatile Emissions and Oviposition Site Selection of Spodoptera exigua Hübner.

Silicon (Si) fertilization alleviates biotic stresses in plants. Si enhances plant resistance against phytophagous insects through physical and biochemical mechanisms. In particular, Si modifies jasmonic acid levels and the emissions of herbivore-induced plant volatiles (HIPVs). Here, we investigated whether Si accumulation in the tissues of maize leaves modifies the emissions of constitutive and herbivore-induced plant volatiles, with cascade deterrent effects on oviposition site selection by Spodoptera exigua Hübner (Lepidoptera: Noctuidae). Maize plants were cultivated in a hydroponic system under three Si concentrations, resulting in three groups of plants expressing different Si concentrations in their tissues (0.31 ± 0.04, 4.69 ± 0.49, and 9.56 ± 0.30 g Si. Kg- 1 DW). We collected volatiles from undamaged and caterpillar-infested plants, and found that Si concentration in plant tissues had no significant impact. Jasmonic acid content was high in insect-infested plants, but was similar across all Si treatments. Oviposition site selection bioassays using fertilized S. exigua females showed that Si concentration in plant tissues did not affect the number of eggs laid on Si-treated plants. In conclusion, our study shows that the Si content in maize tissues does not impact the semiochemical interactions with S. exigua.

Relationship between lipopeptide biosurfactant and primary metabolite production by Bacillus strains in solid-state and submerged fermentation.

The relationship between lipopeptide and primary metabolite production by Bacillus spp. in solid-state fermentation (SSF) and submerged fermentation (SmF) was evaluated. Four wild-type strains and one mutant strain (unable to develop biofilm) were assessed in SSF and SmF, using a defined medium and polyurethane foam as inert support for SSF. Strain ATCC 21,332 in SSF presented the highest lipopeptide production. The wild-type strains revealed higher lipopeptide and biomass production and lower synthesis of primary metabolites in SSF than in SmF. However, the mutant strain showed a slightly higher production of primary metabolites in SSF than in SmF. Carbon balance analysis showed that the carbon flux was mainly directed to lipopeptides in SSF, whereas in SmF, it was directed to the production of primary metabolites and the carbon flux to lipopeptides is inversely related to primary metabolites in both types of cultures.

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.

In Yarrowia lipolytica erythritol catabolism ends with erythrose phosphate.

In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four-carbon sugar alcohol, from erythrose-P, an intermediate of the pentose phosphate pathway. Under non-stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose-P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose-1P isomerase that converts erythrulose-1P into erythrulose-4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.

Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions.

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized-cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g-1 and 0.16 mg g-1 respectively).

Stimulation of Fengycin-Type Antifungal Lipopeptides in Bacillus amyloliquefaciens in the Presence of the Maize Fungal Pathogen Rhizomucor variabilis.

Most isolates belonging to the Bacillus amyloliquefaciens subsp. plantarum clade retain the potential to produce a vast array of structurally diverse antimicrobial compounds that largely contribute to their efficacy as biocontrol agents against numerous plant fungal pathogens. In that context, the role of cyclic lipopeptides (CLPs) has been well-documented but still little is known about the impact of interactions with other soil-inhabiting microbes on the expression of these molecules. In this work, we wanted to investigate the antagonistic activity developed by this bacterium against Rhizomucor variabilis, a pathogen isolated from diseased maize cobs in Democratic Republic of Congo. Our data show that fengycins are the major compounds involved in the inhibitory activity but also that production of this type of CLP is significantly upregulated when co-cultured with the fungus compared to pure cultures. B. amyloliquefaciens is thus able to perceive fungal molecules that are emitted and, as a response, up-regulates the biosynthesis of some specific components of its antimicrobial arsenal.

Streptomyces lunaelactis sp. nov., a novel ferroverdin A-producing Streptomyces species isolated from a moonmilk speleothem.

A novel actinobacterium, designated MM109(T), was isolated from a moonmilk deposit collected from the cave 'Grotte des Collemboles' located in Comblain-au-Pont, Belgium. Based on a polyphasic taxonomic approach comprising chemotaxonomic, phylogenetic, morphological, and physiological characterization, the isolate has been affiliated to the genus Streptomyces. Multilocus sequence analysis based on the 16S rRNA gene and five other house-keeping genes (atpD, gyrB, rpoB, recA and trpB) showed that the MM109(T) isolate is sufficiently distinct from its closest relative, Streptomyces peucetius strain AS 4.1799(T), as to represent a novel species. The phylogenetic distinctiveness of the taxon represented by isolate MM109(T) was supported by the isolation and identification of additional twelve moonmilk-derived isolates, which according to multilocus sequence analysis were clustered along with MM109(T). Scanning electron microscopy observations revealed complex and diversified structures within a MM109(T) colony, made from branching vegetative mycelia. The spore chains of the MM109(T) isolate undergo complete septation at the late stages of the morphological differentiation process, leading to the formation of packs of smooth cylindrical-shaped spores. Isolate MM109(T) produces several intracellular and diffusible pigments, particularly an intracellular green-pigmented secondary metabolite, which was identified through UPLC-ESI-MS analysis as ferroverdin A, an iron-chelating molecule formerly extracted and characterized from Streptomyces sp. strain WK-5344. The isolate MM109(T) is thus considered to represent a novel species of Streptomyces, for which the name Streptomyces lunaelactis sp. nov. is proposed with the type strain MM109(T) (=DSM 42149(T) = BCCM/LMG 28326(T)).

Dual mode of action of amylolysin: a type-B lantibiotic produced by Bacillus amyloliquefaciens GA1.

The partial genome sequencing of Bacillus amyloliquefaciens GA1 led to the identification of the aml gene cluster involved in the synthesis of the novel lantibiotic named amylolysin. Pure amylolysin was shown to have an antibacterial activity toward Gram-positive bacteria including methicillin resistant Staphylococcus aureus. The lantibiotic was also found efficient to inhibit the growth of Listeria monocytogenes strains on poultry meat upon a long storage at 4°C. In silico analyses of the aml gene cluster revealed the presence of a characteristic motif involved in interaction with peptidoglycan precursor lipid II. In the present work, this interaction was further investigated using the LiaRS based reporter gene that is able to sense specifically antibiotics that interfere with lipid II cycle. Beside this, the pore-forming ability of amylolysin was evidenced by means of membrane depolarization measurements and cell leaking experiments.

Characterization of amylolysin, a novel lantibiotic from Bacillus amyloliquefaciens GA1.

BACKGROUND: Lantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity. METHODOLOGY/FINDINGS: Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact in vitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus. CONCLUSION/SIGNIFICANCE: Apart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens.

Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A.

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi.

Antimicrobial Activity of Bacillus amyloliquefaciens ANT1 Toward Pathogenic Bacteria and Mold: Effects on Biofilm Formation.

The intensive use and misuse of antibiotics over the last decades have generated a strong selective pressure for the emergence of multi-resistant strains and nosocomial infections. Biofilm has been demonstrated as a key parameter in spreading infections, especially in hospitals and healthcare units. Therefore, the development of novel anti-biofilm drugs is actually of the upmost importance. Here, the antimicrobial and antibiofilm activities toward pathogenic microorganisms of a set of non-ribosomal synthesized peptides and polyketides isolated from Bacillus amyloliquefaciens ANT1 culture supernatant are presented.

Antilisterial Activity on Poultry Meat of Amylolysin, a Bacteriocin from Bacillus amyloliquefaciens GA1.

This paper describes the production, the purification and the antilisterial activity of amylolysin, a novel bacteriocin from B. amyloliquefaciens GA1. The strain genome was first analysed using PCR techniques for the presence of gene clusters that direct the synthesis of characterised bacteriocins from B. amyloliquefaciens and the closely related B. subtilis. Our results suggest that amylolysin corresponds to a novel bacteriocin. The effect of amylolysin on the growth of different isolates of Listeria monocytogenes was evaluated in poultry meat during 21 days of storage at 4 °C. A potent antilisterial effect was observed for all the indicator strains tested, demonstrating that amylolysin is a novel bacteriocin that could be used as a food preservative.