title: Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis.
abstract: Although most clinically used antibiotics are derived from natural products, identifying new antibacterial molecules from natural product extracts is difficult due to the complexity of these extracts and the limited tools to correlate biological activity with specific molecules. Here, we show that bacterial cytological profiling (BCP) provides a rapid method for mechanism of action determination on plates and in complex natural product extracts and for activity-guided purification. We prepared an extract from Bacillus subtilis 3610 that killed the Escherichia coli lptD mutant and used BCP to observe two types of bioactivities in the unfractionated extract: inhibition of translation and permeablization of the cytoplasmic membrane. We used BCP to guide purification of the molecules responsible for each activity, identifying the translation inhibitors bacillaene and bacillaene B (glycosylated bacillaene) and demonstrating that two molecules contribute to cell permeabilitization, the bacteriocin subtilosin and the cyclic peptide sporulation killing factor. Our results suggest that bacillaene mediates translational arrest, and show that bacillaene B has a minimum inhibitory concentration 10 × higher than unmodified bacillaene. Finally, we show that BCP can be used to screen strains on an agar plate without the need for extract preparation, greatly saving time and improving throughput. Thus, BCP simplifies the isolation of novel natural products, by identifying strains, crude extracts and fractions with interesting bioactivities even when multiple activities are present, allowing investigators to focus labor-intensive steps on those with desired activities.
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Poochit
authors.initials: P
authors.lastname: Nonejuie
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Rachelle M
authors.initials: RM
authors.lastname: Trial
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Newton
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Lamsa
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Varahenage
authors.initials: V
authors.lastname: Ranmali Perera
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Julieta
authors.initials: J
authors.lastname: Aguilar
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA, USA.
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Liu
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA, USA.|Center for Marine Biotechnology and Biomedicine, University of California, San Diego, CA, USA.
authors.forename: Pieter C
authors.initials: PC
authors.lastname: Dorrestein
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Joe
authors.initials: J
authors.lastname: Pogliano
authors.affiliation: Division of Biological Sciences, University of California, San Diego, CA, USA.
authors.forename: Kit
authors.initials: K
authors.lastname: Pogliano
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001430:Bacteriocins / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D010456:Peptides, Cyclic / Q000378:metabolism; D011090:Polyenes / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001430:Bacteriocins; D029968:Escherichia coli Proteins; C475958:LptD protein, E coli; D010456:Peptides, Cyclic; D011090:Polyenes; C096634:bacillaene; C549023:bacillaene B; C047828:sboA protein, Bacillus subtilis