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Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

ABSTRACT: The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and ?-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.

SUBMITTER: Cesa-Luna C

PROVIDER: S-EPMC7561207 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

Cesa-Luna Catherine C Baez Antonino A Aguayo-Acosta Alberto A Llano-Villarreal Roberto Carlos RC Juárez-González Víctor Rivelino VR Gaytán Paul P Bustillos-Cristales María Del Rocío MDR Rivera-Urbalejo América A Muñoz-Rojas Jesús J Quintero-Hernández Verónica V

PloS one 20201015 10

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar ...[more]

PMID: 33057351

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Project description:UnlabelledInterspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria.ImportanceBiofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that inhibited biofilm gene expression in Bacillus subtilis. We identified Pseudomonas protegens as one such bacterium and found that the biofilm-inhibiting compound it produces was the antibiotic 2,4-diacetylphloroglucinol (DAPG). We showed that even at subinhibitory concentrations, DAPG inhibits biofilm formation and sporulation in B. subtilis. These findings have potential implications for understanding the interactions between these two microbes in the natural world and support the idea that many compounds considered antibiotics can impact bacterial development at subinhibitory concentrations.

Dataset Information

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

Publications

Growth inhibition of pathogenic microorganisms by Pseudomonas protegens EMM-1 and partial characterization of inhibitory substances.

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