Open AccessA robust screen for novel antibiotics: specific knockout of the initiator of bacterial DNA replication
Author(s) -
Fossum Solveig,
De Pascale Gianfranco,
Weigel Christoph,
Messer Walter,
Donadio Stefano,
Skarstad Kirsten
Publication year - 2008
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2008.01103.x
Subject(s) - dnaa , dna replication , mutant , biology , dnab helicase , microbiology and biotechnology , escherichia coli , antibiotics , dna , phenotype , genetics , computational biology , origin of replication , gene , rna , helicase
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.