title: A robust screen for novel antibiotics: specific knockout of the initiator of bacterial DNA replication.
abstract: We have developed a novel type of a positive screen for the discovery of antibacterial compounds that target the Escherichia coli replication initiator protein DnaA. DnaA is an essential replication protein, conserved in (almost) all bacteria--including all human pathogens--and no existing antibiotics target the main components of the DNA replication machinery. This makes DnaA an attractive target and compounds discovered by this screen will constitute a new group of antibiotics. The conditional mutant, dnaA219, has a cold sensitive phenotype due to overreplication. In the screen, a DnaA inhibitor will reduce DnaA overactivity and thus restore growth at the nonpermissive temperature. This positive type of selection utilizes the rare phenomenon of lethal overactivity. In addition, the mutant strain has been made independent of DnaA activity by introduction of an alternative initiation pathway that allows growth under conditions of complete knockdown of DnaA. The resulting dnaA219rnhA strain is the basis of a robust, cell-based assay amenable to high-throughput screening. The screening assay has been validated against (1) a library of microbial fermentation extracts and (2) a known intracellular DnaA inhibitor.
authors.affiliation: Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Rikshospitalet, University of Oslo, Oslo, Norway.
authors.forename: Solveig
authors.initials: S
authors.lastname: Fossum
authors.forename: Gianfranco
authors.initials: G
authors.lastname: De Pascale
authors.forename: Christoph
authors.initials: C
authors.lastname: Weigel
authors.forename: Walter
authors.initials: W
authors.lastname: Messer
authors.forename: Stefano
authors.initials: S
authors.lastname: Donadio
authors.forename: Kirsten
authors.initials: K
authors.lastname: Skarstad
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D020868:Gene Silencing / Q000187:drug effects*; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria