title: A fluorescence-based bioassay for antibacterials and its application in screening natural product extracts.
abstract: The reliable assessment of the biological activity of a minor component embedded in a complex matrix of several hundred compounds is a difficult but common task in the search for natural product-based antibiotics, for example, by bioassay-guided fractionation. To quantify the antibiotic properties, it is necessary to assess the cell viability. Direct measurements use CFU counts, OD measurements or detection via fluorescent or reducible dyes. However, natural extracts often already possess intrinsic dye, fluorescent, reducing or protein denaturing properties, or they contain insoluble compounds or general protein-binding (tanning) polyphenols as disturbing features, while at the same time very little of the selective antibiotic sought after is present. A promising alternative is provided by intrinsically produced bright fluorescent proteins. In this paper, a rapid, robust and concentration-dependent assay for screening antibiotics with genetically modified mutants of Bacillus subtilis 168 (PabrB-iyfp) is presented. The Gram-positive bacteria exhibit a native fluorescence during their exponential growth phase due to the expression of improved yellow fluorescent protein. To demonstrate the applicability in the field of natural product research, several compounds and extracts were screened for antibacterial activity, with an emphasis on those from the fungal genus Hygrophorus (waxy caps).
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Michels
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Heinke
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
authors.forename: Pia
authors.initials: P
authors.lastname: Schöne
authors.affiliation: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Arnold
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
authors.forename: Ludger A
authors.initials: LA
authors.lastname: Wessjohann
mesh_terms: D000363:Agaricales / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D001688:Biological Products / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D005453:Fluorescence; D008164:Luminescent Proteins / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001688:Biological Products; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria