Genomic and Chemical Diversity of Bacillus subtilis Secondary 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 nonribosomal peptides 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 secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated 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 spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide 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 IMPORTANCE Secondary 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 nonribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate the 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 the production of certain secondary metabolites.

Keywords: Bacillus subtilis; antiSMASH; biosynthetic gene clusters; chemodiversity; fungal inhibition; secondary metabolites.

FIG 1

(A) Antagonism assays between the plant pathogenic fungi Fusarium oxysporum, Fusarium graminearum, and Botrytis cinerea and the B. subtilis soil isolates (left) as well as their NRP-deficient sfp mutants (right). (B) Antagonism assays between the plant pathogenic fungi and B. subtilis soil isolates (upper left) as well as their single nonribosomal peptide srfAC (upper right, no surfactin), ΔppsC (lower right, no plipastatin), and ΔpksL (lower left, no bacillaene) mutants. A 5-μl quantity of bacterial overnight culture and fungal spore suspension was spotted onto the edges (bacteria) and in the center (fungi) of potato dextrose agar (PDA) plates. Strains were cocultivated at 21 to 23°C for 6 days. (C) Extracted ion chromatograms (m/z 1,000 to 1,600) display various levels of production of surfactin and plipastatin among B. subtilis soil isolates. The standard mixtures of plipastatin and surfactin are shown at the bottom. Multiple peaks in the LC-MS traces among the isolates and standards show different surfactin and plipastatin analogs with different fatty acid substitutions. The presence of surfactin and plipastatin in the isolates’ extracts was confirmed by retention time comparisons with the standards and by tandem mass spectrometry (MS/MS) fragmentation studies.

FIG 2

(A) Overview of qualitative evaluation of antagonisms assays assigned to inhibition, minor inhibition, and no inhibition. Strains P5_B2 and P8_B2 were not naturally competent, and no NRP mutants could be created. (B) Overview of NRP production of wild-type soil isolates based on the detection of surfactin and plipastatin in the extracts by ESI-MS. The production of the compounds was classified as production (detected), reduced production (detected but at a lower level), and no production (undetected).

FIG 3

Overview of predicted biosynthetic gene clusters (BGCs) by antiSMASH of 13 B. subtilis and 1 B. licheniformis (right) soil isolate. The color code visualizes the similarity of BGCs to a reference BGC, whereby the gray color (0%) indicates their absence. The cladogram is based on a core gene alignment by the pan-genome pipeline Roary.

FIG 4

Comparison of core genes of the biosynthetic gene clusters surfactin (A) and plipastatin (B) from coisolated B. subtilis strains, which were classified into producer (+) and nonproducer/production-hampered (−) strains based on the targeted LC-MS analysis. (C) Comparison of core genes of the biosynthetic gene clusters of bacillaene from two coisolated B. subtilis strains.

FIG 5

(A) Overview of intraspecies inhibition of coisolated B. subtilis strains. Focal strains were tested for their capability to inhibit each coisolated target strain. The inhibition potential was evaluated by examining the zone surrounding the focal strain colony and classified into inhibition (occurrence of a cell-free zone and growth reduction), minor inhibition (only growth reduction), and no inhibition (neither cell-free zone nor growth reduction). The target strains were embedded in 1% LB agar, and the focal strains (8 μl) were spotted on top. Plates were incubated at 37°C for 24 h. (B) Overview of predicted and known accessory BGCs by antiSMASH.