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.
journal_name
FEMS Microbiol Lettjournal_title
FEMS microbiology lettersauthors
Fossum S,De Pascale G,Weigel C,Messer W,Donadio S,Skarstad Kdoi
10.1111/j.1574-6968.2008.01103.xsubject
Has Abstractpub_date
2008-04-01 00:00:00pages
210-4issue
2eissn
0378-1097issn
1574-6968pii
FML1103journal_volume
281pub_type
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