title: Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens.
abstract: Interspecies 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.
  
  
  OBJECTIVE
  Biofilms 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.
authors.affiliation: University of North Carolina, Department of Biology, Chapel Hill, North Carolina, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Powers
authors.affiliation: John Jay College, Department of Sciences, The City University of New York, New York, New York, USA.
authors.forename: Edgardo
authors.initials: E
authors.lastname: Sanabria-Valentín
authors.affiliation: University of North Carolina, Division of Chemical Biology and Medicinal Chemistry, School of Pharmacy, Chapel Hill, North Carolina, USA.
authors.forename: Albert A
authors.initials: AA
authors.lastname: Bowers
authors.affiliation: University of North Carolina, Department of Biology, Chapel Hill, North Carolina, USA eshank@unc.edu.|University of North Carolina, Department of Microbiology and Immunology, Chapel Hill, North Carolina, USA.|University of North Carolina, Curriculum in Genetics and Molecular Biology, Chapel Hill, North Carolina, USA.
authors.forename: Elizabeth A
authors.identifier: http://orcid.org/0000-0002-4804-1966
authors.initials: EA
authors.lastname: Shank
mesh_terms: D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development; D018920:Coculture Techniques; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008826:Microbial Sensitivity Tests; D015394:Molecular Structure; D010696:Phloroglucinol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011549:Pseudomonas / Q000502:physiology*; D012988:Soil Microbiology; D013171:Spores, Bacterial / Q000187:drug effects
chemical_list: C059817:2,4-diacetylphloroglucinol; D010696:Phloroglucinol
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