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An array of target-specific screening strains for antibacterial discovery

Abstract

As the global threat of drug- and antibiotic-resistant bacteria continues to rise, new strategies are required to advance the drug discovery process. This work describes the construction of an array of Escherichia coli strains for use in whole-cell screens to identify new antimicrobial compounds. We used the recombination systems from bacteriophages λ and P1 to engineer each strain in the array for low-level expression of a single, essential gene product, thus making each strain hypersusceptible to specific inhibitors of that gene target. Screening of nine strains from the array in parallel against a large chemical library permitted identification of new inhibitors of bacterial growth. As an example of the target specificity of the approach, compounds identified in the whole-cell screen for MurA inhibitors were also found to block the biochemical function of the target when tested in vitro.

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Figure 1: Construction of E. coli strains for parallel screening.
Figure 2: Kinetic growth analysis of engineered strains.
Figure 3: Cell-based parallel screening of engineered E. coli strains.

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Acknowledgements

We are grateful to Michael G. Kurilla for countless helpful insights, to Janet Kaczmarczyk and Jason Sparkowski for technical aid in the early phases of this work, to Don Court for strains, to Bob Weisberg for phage, and to Ron Hoess and Piet DiBoer for plasmids. This work was completed while the authors were employees of the DuPont Pharmaceuticals Company.

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Correspondence to David L. Pompliano.

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Competing interests

The work described in this manuscript was performed while the authors were all employees of the DuPont Pharmaceuticals Company. During the manuscript review period, DuPont Pharmaceuticals was purchased by, and became part of, Bristol-Myers Squibb. Many of the authors have since moved on to other opportunities. D.L.P. is currently employed by GlaxoSmithKline. J.A.M. is employed by Essential Therapeutics. A.A. is employed by Bristol-Myers Squibb. C.F.S. is a US Government employee. V.G.L. is employed by Centocor.

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DeVito, J., Mills, J., Liu, V. et al. An array of target-specific screening strains for antibacterial discovery. Nat Biotechnol 20, 478–483 (2002). https://doi.org/10.1038/nbt0502-478

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