BackgroundPhytopathogenic fungi affecting crop and post-harvested vegetables are a major threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management.ResultsThe genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42.ConclusionThe production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents.

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.

Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C FA chain.

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)).

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry dataset. The method enables an automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of family of homologous compounds from MSI images. We show that the KMD filtering, based on an in-house software, is suitable and robust for high resolution (full width at half-maximum, FWHM, at m/z 410 of 20 000) and very high-resolution (FWHM, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria co-cultures and brain tissue section.

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.

BackgroundLantibiotics 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/SignificanceApart 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.