title: Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors.
abstract: In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Russia.
authors.forename: Ilya A
authors.initials: IA
authors.lastname: Osterman
authors.affiliation: Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors.forename: Ekaterina S
authors.initials: ES
authors.lastname: Komarova
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
authors.forename: Dmitry I
authors.initials: DI
authors.lastname: Shiryaev
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
authors.forename: Ilya A
authors.initials: IA
authors.lastname: Korniltsev
authors.affiliation: Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors.forename: Irina M
authors.initials: IM
authors.lastname: Khven
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Lukyanov
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Tashlitsky
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Serebryakova
authors.affiliation: G. F. Gauze Institute for Search for New Antibiotics, Russian Academy of Medical Sciences, Moscow, Russia.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Efremenkova
authors.affiliation: Moscow Institute of Physics and Technology (State University), Moscow Region, Russia.
authors.forename: Yan A
authors.initials: YA
authors.lastname: Ivanenkov
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Bogdanov
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia petya@genebee.msu.ru.|Skolkovo Institute of Science and Technology, Skolkovo, Russia.
authors.forename: Petr V
authors.initials: PV
authors.lastname: Sergiev
authors.affiliation: Lomonosov Moscow State University, Department of Chemistry and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.|Skolkovo Institute of Science and Technology, Skolkovo, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Dontsova
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D005818:Genetic Engineering; D057166:High-Throughput Screening Assays; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000187:drug effects*; D012270:Ribosomes / Q000187:drug effects* / Q000235:genetics; D013014:SOS Response, Genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D000097573:Red Fluorescent Protein
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C053139:sulA protein, E coli