MIBiG 3.0 : a community-driven effort to annotate experimentally validated biosynthetic gene clusters

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/

Antifungal and plant-defence stimulating activities of a strain of Streptomyces sp. used as a biocontrol agent

La rhizosphère est abondamment colonisée par un ensemble de micro-organismes qui ont un impact sur la nutrition et la résistance des plantes aux stress biotiques et abiotiques. Les streptomycètes sont une composante majeure du microbiote rhizosphérique. Ce genre de bactérie à Gram positif filamenteuse est connu pour produire un large spectre de métabolites spécialisés aux activités biologiques diverses, notamment antimicrobiennes. Cependant, leur rôle dans la rhizosphère reste encore largement méconnu. L’objectif de ce travail a été d’étudier le mode d’action de la souche Streptomyces sp. AgN23 isolée de la rhizosphère d’un cep de vigne puis sélectionnée lors d’un criblage pour sa capacité à induire fortement les défenses de la plante. Cette activité élicitrice a été analysée par une approche transcriptomique sur la plante modèle Arabidopsis thaliana montrant que la souche induit plusieurs voies de défense participant à la protection contre les micro-organismes pathogènes. Outre l’activité immunostimulante, une activité antifongique d’AgN23 a aussi été observée. Afin d’identifier les métabolites actifs, le génome de la souche a été séquencé et annoté conduisant à la description de clusters de gènes codants pour les enzymes impliquées dans leur biosynthèse (Biosyntethic Gene Cluster – BGC). Une analyse métabolomique a permis d’identifier plusieurs métabolites antifongiques dont les BGC sont effectivement retrouvés dans le génome de la souche. Afin d’identifier les métabolites majeurs responsables de l’activité antifongique et d’aborder la question de la régulation de leur biosynthèse, des mutants d’AgN23 produits par mutagenèse UV ont été criblés pour détecter des variants. Des mutants sur- ou sous-producteurs d’activité antifongique ont été séquencés, ce qui a permis de repérer des éléments régulateurs candidats du métabolisme spécialisé de la souche. L’analyse métabolomique d’un mutant sur-producteur a montré une biosynthèse accrue d’un des composés candidats identifiés. En parallèle, une approche de type OSMAC (One Strain Many Compound) a montré que la production des métabolites antifongiques est fortement régulée par une source de carbone alternative au glucose qui modifie la nature des métabolites spécialisés produits par la souche et augmente le pouvoir antifongique de son surnageant. De façon concordante, l’analyse du transcriptome d’AgN23 cultivé dans la source de carbone candidate a révélé une forte reprogrammation du métabolisme central de la souche qui est susceptible d’expliquer la modification de son métabolome. L’ensemble de ces résultats a consolidé notre compréhension des mécanismes d’actions de Streptomyces sp. AgN23 envers les plantes et leurs parasites fongiques et ouvre des pistes d’optimisation pour son intégration dans de nouvelles stratégies de protection des cultures.The rhizosphere is abundantly colonized by microorganisms that influence plant nutrition and resistance to biotic and abiotic stresses. Streptomyces spp. are a major component of the rhizosphere microbiome. These Gram positive filamentous bacteria are well known for producing a broad range of specialized metabolites, notably antimicrobial compounds. However, their role in rhizosphere remains largely unknown. The aim of this project was to study the mechanisms of action of Streptomyces sp. AgN23. This strain was isolated from grapevine rhizosphere and selected for strongly inducing plant defense responses. A transcriptomic approach using Arabidopsis thaliana showed that the strain induces many defense pathways taking part in the observed protection against phytopathogenic microorganisms. Beside the immunostimulant activity, the strain also showed a direct antifungal activity. In order to identify active metabolites, Streptomyces sp. AgN23 genome was sequenced and the biosynthetic gene clusters (BGC) annotated. A global metabolomic analysis identified candidate compounds responsible for the antifungal activity and, for most of them, corresponding to BGC found in the strain genome. In order to investigate the role of identified metabolites and the regulation of their biosynthesis, mutants were produced by UV mutagenesis and screened for hypo- or hyper producers of antifungal compounds. The most interesting mutants were sequenced and putative regulatory elements implicated in the control of their biosynthesis were identified. A Streptomyces sp. AgN23 mutant overproducer of antifungal activity showed a higher rate of biosynthesis of one of the candidate compounds in comparison with the wild strain. Complementarily, an OSMAC-type approach (One Strain Many Compound) was designed and lead to identification of a carbon source alternative to glucose which resulted in strong changes regarding the identity of produced metabolites and resulted in stronger antifungal activity. Transcriptomic analysis showed that this alternative carbon source deeply reprogrammed the central metabolism of AgN23, probably causing the shift observed in specialised metabolites biosynthesis. Together, these results increased our understanding of a Streptomyces sp. mode of action to plants and against fungal phytopathogens and pave the way to the use of these microorganisms strain as microbial amendment in agriculture

Colonisation of Arabidopsis leaves by a Streptomyces sp. results in a protection against fungal diseasesrelying on plant immunity

International audienc

Study of a Streptomyces sp. derived signals and their plant perception mechanisms

International audienc

Identification of a Streptomyces necrotic elicitor involved in antifungal activity, plant defense stimulation and bacteria fitness in the rhizosphere

International audienc

Genome mining of Streptomyces violaceusniger AgN23 highlights its high potential as plant stimulant and biological control agent

International audienc

Phyllosphere colonisation by a soil Streptomyces sp. promotes plant defense responses against fungal infection

International audienc

Molecular basis of plant defenses stimulation by the soil borne Streptomyces strain AgN23: characterization of a novel necrotic elicitor

International audienc

Molecular basis of plant defenses stimulation by the soil borne Streptomyces strain AgN23: characterization of a novel necrotic elicitor

International audienc

Phyllosphere Colonization by a Soil Streptomyces sp. Promotes Plant Defense Responses Against Fungal Infection

International audienceStreptomycetes are soil-dwelling, filamentous actinobacteria and represent a prominent bacterial clade inside the plant root microbiota. The ability of streptomycetes to produce a broad spectrum of antifungal metabolites suggests that these bacteria could be used to manage plant diseases. Here, we describe the identification of a soil Streptomyces strain named AgN23 which strongly activates a large array of defense responses when applied on Arabidopsis thaliana leaves. AgN23 increased the biosynthesis of salicylic acid, leading to the development of salicylic acid induction deficient 2 (SID2)-dependent necrotic lesions. Size exclusion fractionation of plant elicitors secreted by AgN23 showed that these signals are tethered into high molecular weight complexes. AgN23 mycelium was able to colonize the leaf surface, leading to plant resistance against Alternaria brassicicola infection in wild-type Arabidopsis plants. AgN23-induced resistance was found partially compromised in salicylate, jasmonate, and ethylene mutants. Our data show that Streptomyces soil bacteria can develop at the surface of plant leaves to induce defense responses and protection against foliar fungal pathogens, extending their potential use to manage plant diseases