title: Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli.
abstract: While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain--using chromosomal gene knock-in techniques--that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.
authors.affiliation: Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany rainer.nikolay@uni-konstanz.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Nikolay
authors.affiliation: Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Schloemer
authors.affiliation: Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: Screening Center Konstanz, University of Konstanz, Konstanz 78457, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Mueller
authors.affiliation: Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Heubach
authors.affiliation: Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany rainer.nikolay@uni-konstanz.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Deuerling
mesh_terms: D060846:Cell Engineering; D002701:Chloramphenicol / Q000494:pharmacology; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D055786:Gene Knockout Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry / Q000378:metabolism*; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry; D000097573:Red Fluorescent Protein
chemical_list: D002701:Chloramphenicol; D004917:Erythromycin; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein; C037084:ribosomal protein S17