Biostimulation of Bacteria in Liquid Culture for Identification of New Antimicrobial Compounds

Abstract

We hypothesized that environmental microbiomes contain a wide range of bacteria that produce yet uncharacterized antimicrobial compounds (AMCs) that can potentially be used to control pathogens. Over 600 bacterial strains were isolated from soil and food compost samples, and 68 biocontrol bacteria with antimicrobial activity were chosen for further studies based on inhibition assays against a wide range of food and plant pathogens. For further characterization of the bioactive compounds, a new method was established that used living pathogens in a liquid culture to stimulate bacteria to produce high amounts of AMCs in bacterial supernatants. A peptide gel electrophoresis microbial inhibition assay was used to concurrently achieve size separation of the antimicrobial peptides. Fifteen potential bioactive peptides were then further characterized by tandem MS, revealing cold-shock proteins and 50S ribosomal proteins. To identify non-peptidic AMCs, bacterial supernatants were analyzed by HPLC followed by GC/MS. Among the 14 identified bioactive compounds, 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2-acetyl-3-methyl-octahydropyrrolo[1,2-a]piperazine-1,4-dione were identified as new AMCs. Our work suggests that antimicrobial compound production in microbes is enhanced when faced with a threat from other microorganisms, and that this approach can rapidly lead to the development of new antimicrobials with the potential for upscaling.

Keywords: antibiotic; antimicrobial compound; antimicrobial peptide; food pathogen; plant pathogen.

Figure 1

AMC antimicrobial discovery platform. Steps include (1) the isolation of microbes from environmental samples on different media (Supplementary File S1, Table S3), (2) purifying and (3) screening by plate growth inhibition assays using plant and food pathogens (hatched background), (4, 5) the AMC induction method by co-cultivation with living Gram-positive and -negative pathogens at 28 °C, (6) centrifugation and concentration of cell-free supernatants by freeze-drying, (7) testing of the concentrated supernatants with or without proteinase K to distinguish between AMPs and AMMs, followed by (8) peptide gel microbial growth inhibition assays to (9) extract bioactive peptides and MS/MS of trypsin-digested peptides or coculture supernatant fractionation by HPLC (high-performance liquid chromatography) followed by MS/MS (tandem mass spectrometry) or GC/MS (gas chromatography–mass spectrometry) analysis.

Figure 2

Antimicrobial peptide analysis. Top: Electrophoresis of bacterial supernatants on Tris–Tricine gels. (A,B) One half of the gel containing bioactive supernatants of the isolates 32YE and 9th, respectively, was challenged with C. michiganensis as a “gel plate assay” and the other half of the gel, with identical samples, was stained with Coomassie Brilliant Blue. (C,D) Gel plate assay of the isolates 33YE or 32LM, respectively, tested against L. monocytogenes and its half-stained gel with identical samples to visualize and recover the protein band(s) for further characterization by MS/MS. (E) MS/MS data from all peptides and protein fragments’ excised bands from all isolates (Supplementary File S6, Table S1). CS: crude supernatant; OP: organic phase; AP: aqueous phase; L: protein standard.

Figure 2

Antimicrobial peptide analysis. Top: Electrophoresis of bacterial supernatants on Tris–Tricine gels. (A,B) One half of the gel containing bioactive supernatants of the isolates 32YE and 9th, respectively, was challenged with C. michiganensis as a “gel plate assay” and the other half of the gel, with identical samples, was stained with Coomassie Brilliant Blue. (C,D) Gel plate assay of the isolates 33YE or 32LM, respectively, tested against L. monocytogenes and its half-stained gel with identical samples to visualize and recover the protein band(s) for further characterization by MS/MS. (E) MS/MS data from all peptides and protein fragments’ excised bands from all isolates (Supplementary File S6, Table S1). CS: crude supernatant; OP: organic phase; AP: aqueous phase; L: protein standard.

Figure 3

Potential synthetic pathway for AMMs that presented protein fragments that may have originated from metabolic pathway enzymes. The figure shows the proposed AMM pathways that biosynthesize both metabolites (based on GC/MS data) and protein fragments of metabolic enzymes (based on MS/MS data). Pathways include the tricarboxylic acid cycle (TCA), as well as the butanediol, glycerol and acetic acid pathways.