An array of Escherichia coli clones over-expressing essential proteins: a new strategy of identifying cellular targets of potent antibacterial compounds.
With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.
Xu
H Howard
HH
Department of Biological Sciences, California State University, Los Angeles, 90032, USA. hxu3@calstatela.edu
Real
Lilian
L
Bailey
Melissa Wu
MW
D000900:Anti-Bacterial Agents / Q000008:administration & dosage*; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D016503:Drug Delivery Systems / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism
D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins