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

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.

References

  1. 1

    Clayton, R.A., White, O. & Fraser, C.M. Findings emerging from complete microbial genome sequences. Curr. Opin. Microbiol. 1, 562–566 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Tang, C.M., Hood, D.W. & Moxon, E.R. Microbial genome sequencing and pathogenesis. Curr. Opin. Microbiol. 1, 12–16 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Schmid, M.B., Kapur, N., Isaacson, D.R., Lindroos, P. & Sharpe, C. Genetic analysis of temperature-sensitive lethal mutants of Salmonella typhimurium. Genetics 123, 625–633 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Arigoni, F. et al. A genome-based approach for the identification of essential bacterial genes. Nat. Biotechnol. 16, 851–856 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Mushegian, A.R. & Koonin, E.V. A minimal gene set for cellular life derived by comparison of complete bacterial genomes [see comments]. Proc. Natl. Acad. Sci. USA 93, 10268–10273 (1996).

    CAS  Article  Google Scholar 

  6. 6

    Akerley, B.J. et al. Systematic identification of essential genes by in vitro mariner mutagenesis. Proc. Natl. Acad. Sci. USA 95, 8927–8932 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Hutchison, C.A. et al. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286, 2165–2169 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    CAS  Article  Google Scholar 

  9. 9

    Rubin, E.J. et al. In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad. Sci. USA 96, 1645–1650 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Gueiros-Filho, F.J. & Beverley, S.M. Trans-kingdom transposition of the Drosophila element mariner within the protozoan Leishmania (see comments). Science 276, 1716–1719 (1997). (Published erratum appears in Science 277, 753, 1997.)

    CAS  Article  Google Scholar 

  11. 11

    Harley, C.B. & Reynolds, R.P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 15, 2343–2361 (1987).

    CAS  Article  Google Scholar 

  12. 12

    Takiff, H.E., Baker, T., Copeland, T., Chen, S.M. & Court, D.L. Locating essential Escherichia coli genes by using mini-Tn10 transposons: the pdxJ operon. J. Bacteriol. 174, 1544–1553 (1992).

    CAS  Article  Google Scholar 

  13. 13

    Rappleye, C.A. & Roth, J.R. A Tn10 derivative (T-POP) for isolation of insertions with conditional (tetracycline-dependent) phenotypes. J. Bacteriol. 179, 5827–5834 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Chow, W.Y. & Berg, D.E. Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations. Proc. Natl. Acad. Sci.USA 85, 6468–6472 (1988).

    CAS  Article  Google Scholar 

  15. 15

    Fullner, K.J. & Mekalanos, J.J. Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect. Immun. 67, 1393–1404 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org.

  17. 17

    Heidelberg, J.F. et al. Whole genome sequencing of Vibrio cholerae, the etiologic agent of cholera. In Proceedings of the 35th US–Japan Cholera and Other Bacterial Enteric Infections Joint Panel Meeting 87 (Baltimore, MD; 1999).

    Google Scholar 

  18. 18

    Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

  19. 19

    Schimmel, P., 19. Tao, J. & Hill, J. Aminoacyl tRNA synthetases as targets for new anti-infectives. FASEB J. 12, 1599–1609 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Samuels, D.S., Marconi, R.T., Huang, W.M. & Garon, C.F. gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi. J. Bacteriol. 176, 3072–3075 (1994).

    CAS  Article  Google Scholar 

  21. 21

    Hirokawa, T., Boon-Chieng, S. & Mitaku, S. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378–379 (1998).

    CAS  Article  Google Scholar 

  22. 22

    Nakai, K. & Kanehisa, M. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11, 95–110 (1991).

    CAS  Article  Google Scholar 

  23. 23

    Trucksis, M., Michalski, J., Deng, Y.K. & Kaper, J.B. The Vibrio cholerae genome contains two unique circular chromosomes. Proc. Natl. Acad. Sci.USA 95, 14464–14469 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Curnow, A.W., Tumbula, D.L., Pelaschier, J.T., Min, B. & Soll, D. Glutamyl-tRNA(Gln) amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis. Proc. Natl. Acad. Sci.USA 95, 12838–12843 (1998).

    CAS  Article  Google Scholar 

  25. 25

    Ibba, M., Curnow, A.W. & Soll, D. Aminoacyl-tRNA synthesis: divergent routes to a common goal. Trends Biochem. Sci. 22, 39–42 (1997).

    CAS  Article  Google Scholar 

  26. 26

    Irani, M.H. & Maitra, P.K. Properties of Escherichia coli mutants deficient in enzymes of glycolysis. J. Bacteriol. 132, 398–410 (1977).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Chiang, S.L. & Mekalanos, J.J. Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization. Mol. Microbiol. 27, 797–805 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Schleif, R. Two positively regulated systems, ara and mal. In Escherichia coli and Salmonella, Vol. 1 (ed. Neidhardt, F.C.) 1300–1309 (ASM Press, Washington, DC; 1996).

    Google Scholar 

  29. 29

    Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

    Google Scholar 

  30. 30

    Metcalf, W.W. et al. Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. Plasmid 35, 1–13 (1996).

    CAS  Article  Google Scholar 

  31. 31

    Kolter, R., Inuzuka, M. & Helinski, D.R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15, 1199–1208 (1978).

    CAS  Article  Google Scholar 

  32. 32

    Guiney, D.G. & Helinski, D.R. The DNA–protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer. Mol. Gen. Genet. 176, 183–189 (1979).

    CAS  PubMed  Google Scholar 

  33. 33

    Mekalanos, J.J. Duplication and amplification of toxin genes in Vibrio cholerae. Cell 35, 253–263 (1983).

    CAS  Article  Google Scholar 

  34. 34

    Simon, R. et al. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1, 784–791 (1983).

    CAS  Article  Google Scholar 

  35. 35

    Miller, J.H. Experiments in molecular genetics. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1972).

    Google Scholar 

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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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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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