title: Tools for characterizing bacterial protein synthesis inhibitors.
abstract: Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol.
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, Illinois, USA.
authors.forename: Cédric
authors.initials: C
authors.lastname: Orelle
authors.forename: Skylar
authors.initials: S
authors.lastname: Carlson
authors.forename: Bindiya
authors.initials: B
authors.lastname: Kaushal
authors.forename: Mashal M
authors.initials: MM
authors.lastname: Almutairi
authors.forename: Haipeng
authors.initials: H
authors.lastname: Liu
authors.forename: Anna
authors.initials: A
authors.lastname: Ochabowicz
authors.forename: Selwyn
authors.initials: S
authors.lastname: Quan
authors.forename: Van Cuong
authors.initials: VC
authors.lastname: Pham
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Squires
authors.forename: Brian T
authors.initials: BT
authors.lastname: Murphy
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mankin
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012270:Ribosomes / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D012407:rRNA Operon
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D011500:Protein Synthesis Inhibitors
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