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TnAraOut, A transposon-based approach to identify and characterize essential bacterial genes

Nature Biotechnology volume 18, pages 740–745 (2000)Cite this article

Abstract

Identification of genes that encode essential products provides a promising approach to validation of new antibacterial drug targets. We have developed a mariner-based transposon, TnAraOut, that allows efficient identification and characterization of essential genes by transcriptionally fusing them to an outward-facing, arabinose-inducible promoter, PBAD, located at one end of the transposon. In the absence of arabinose, such TnAraOut fusion strains display pronounced growth defects. Of a total of 16 arabinose-dependent TnAraOut mutants characterized in Vibrio cholerae, four were found to carry insertions upstream of known essential genes (gyrB, proRS, ileRS, and aspRS) whereas the other strains carried insertions upstream of known and hypothetical genes not previously shown to encode essential gene products. One of the essential genes identified by this analysis appears to be unique to V. choleraeand thus may represent an example of a species-specific drug target.

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Figure 1: Isolation of arabinose-dependent strains.
Figure 2: Effect of arabinose concentration on the induction of β-galactosidase expression.
Figure 3: Arabinose-dependent growth phenotypes of TnAraOut insertions.

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Acknowledgements

We thank E.J. Rubin, J.H. Blum, and B.J. Akerley for useful discussions. This work was supported by grant AI-26289 from the National Institute of Allergy and Infectious Diseases (NIAID). Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org. Sequencing of Vibrio cholerae was accomplished also with support from NIAID.

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  1. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, 02115, MA

    Nicholas Judson, John J. Mekalanos & John J. Mekalanos

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Correspondence to Nicholas Judson.

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Judson, N., Mekalanos, J. TnAraOut, A transposon-based approach to identify and characterize essential bacterial genes. Nat Biotechnol 18, 740–745 (2000). https://doi.org/10.1038/77305

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