Genomic and chemical diversity ofBacillus subtilissecondary metabolites against plant pathogenic fungi

Abstract

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing.In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation.Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis.IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

Secondary metabolites of Bacillus subtilis impact the assembly of soil-derived semisynthetic bacterial communities

“…Furthermore, in vivo experiments could reveal the impact of NRPs on microbial communities in complex natural systems, similar to the study from Chowdhury et al from 2013 [59]. Noteworthy, our study focused only on NRPs, but additional SMs, such as bacteriocins, are predicted for B. subtilis P5_B1 as well [51]. Future investigations should investigate the impact of both bacteriocins and NRPs on microbial communities.…”

“…It is presumed that the antifungal plipastatin, expressed from the ppsA-E gene cluster, acts as an inhibitor of phospholipase A2, forming pores in the fungal membrane and causing morphological changes in the fungal membrane and cell wall [45,46]. This antifungal potential was demonstrated primarily against various filamentous fungi [47][48][49][50][51]. The broad-spectrum antibiotic bacillaene, synthesised by the pksB-S gene cluster, is mainly targeting bacterial protein synthesis [52].…”

“…Still, it was also shown that it could protect cells and spores from predation [ 53 ]. We recently demonstrated that the production of these NRPs varies among coisolated B. subtilis environmental strains due to missing core genes or potentially altered gene regulation, highlighting the existing natural diversity of SM production in this species [ 51 ].…”

“…With an NRP-mutant-based approach, we investigated the impact of NRPs on the establishment and composition of the bacterial communities. We previously demonstrated that the strain P5_B1 produces the NRPs surfactin and plipastatin and has further BGC predictions for the NRPs bacillaene and bacillibactin [ 51 ]. It was revealed by 16S rRNA amplicon sequencing that the established semisynthetic mock communities contained 13 genera with a relative abundance of >0.19% in at least one mock community.…”

Phylogenetic distribution of secondary metabolites in theBacillus subtilisspecies complex

“…Members of the B. subtilis group are producers of numerous well-known natural products, iturin, mycosubtilin, fengycin/plipastatin, or bacillaene. Only recent studies have emerged that investigated species-level distribution of the corresponding biosynthetic gene clusters (BGCs) in the B. subtilis group (3). Recent reviews provide an overview of various SMs produced by these Bacilli (1,4).…”