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.

journal_name

FEMS Microbiol Lett

authors

Fossum S,De Pascale G,Weigel C,Messer W,Donadio S,Skarstad K

doi

10.1111/j.1574-6968.2008.01103.x

subject

Has Abstract

pub_date

2008-04-01 00:00:00

pages

210-4

issue

2

eissn

0378-1097

issn

1574-6968

pii

FML1103

journal_volume

281

pub_type

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