Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

Broad Host-Range Vector for Efficient Expression of Foreign Genes in Gram-Negative Bacteria

Bio/Technology volume 9, pages 477–479 (1991)Cite this article

Abstract

A broad host-range expression plasmid was constructed comprising the incQ rep-licon, the recA promoter from Escherichia coli and the g10-L. ribosome binding site (RBS) derived from bacteriophage T7. The structural genes for porcine soma-totropin (pst) and E. coli β-galactosidase (lacZ) were used to monitor gene expression in a diverse collection of Gram-negative bacterial hosts: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas testosteroni, Serratia marcescens and Erwinia herbicola. The E. coli recA promoter was functional in this wide range of hosts and was inducible by the addition of nalidixic acid. Moreover, the level of lacZ expression was often at least as high as that observed in E. coli. Previous studies had shown that the g10-L RBS was superior to a simple “consensus” RBS sequence for expression of foreign genes in E. coli. Here we demonstrate a 38 to 70 fold increase in expression in two Pseudomonas hosts using the g10-L RBS, indicating that the translational enhancer present in the g10-L RBS is also functional in other bacteria. The juxtaposition of these transcriptional and translational elements in a broad host-range vector provides a simple way to evaluate alternate hosts for recombinant protein production.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Jeenes, D.J., Soldati, L., Baur, H., Watson, J.M., Mercenier, A., Reimmann, C., Leisinger, T. and Haas, D. 1986. Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host. Mol. Gen. Genet. 203: 421–429.

    Article  CAS  Google Scholar 

  2. Mergeay, M., Boyen, A., Legrain, C. and Glansdorff, N. 1978. Expression of Escherichia coli K-12 arginine genes in Pseudomonas fluorescens. J. Bacteriol. 136: 1187–1188.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Martin, C., Cami, B., Borne, F., Jeenes, D.J., Haas, D. and Patte, J.C. 1986. Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli. Mol. Gen. Genet. 203: 430–434.

    Article  CAS  Google Scholar 

  4. Nagahari, K., Sano, Y. and Sakaguchi, K. 1977. Derepression of E. coli trp operon on interfamilial transfer. Nature 266: 745–746.

    Article  CAS  Google Scholar 

  5. Werneke, J.M., Sligar, S.G. and Schuler, M.A. 1985. Development of broad host-range vectors for expression of cloned genes in Pseudomonas. Gene 38: 73–84.

    Article  CAS  Google Scholar 

  6. Bagdasarian, M.M., Amann, E., Lurz, R., Ruckert, B. and Bagdasarian, M. 1983. Activity of the hybrid trp-lac (tac) promoter of E. coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 26: 273–282.

    Article  CAS  Google Scholar 

  7. Mermod, N., Ramos, J.L., Lehrbach, P.R. and Timmis, K.N. 1986. Vector for regulated expression of cloned genes in a wide range of Gram-negative bacteria. J. Bacteriol. 167: 447–454.

    Article  CAS  Google Scholar 

  8. Olins, P.O., Devine, C.S., Rangwala, S.H. and Kavka, K.S. 1988. The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Gene 73: 227–235.

    Article  CAS  Google Scholar 

  9. Olins, P.O. and Rangwala, S.H. 1989. A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J. Biol. Chem. 264: 16973–16976.

    CAS  PubMed  Google Scholar 

  10. Horii, T., Ogawa, T. and Ogawa, H. 1980. Organization of the recA gene of Escherichia coli. Proc. Natl. Acad. Sci. USA. 77: 313–317.

    Article  CAS  Google Scholar 

  11. Better, M. and Helinski, D.R. 1983. Isolation and characterization of the recA gene of Rhizobium meliloti. J. Bacteriol. 155: 311–316.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Keener, S.L., McNamee, K.P. and McEntee, K. 1984. Cloning and characterization of recA genes from Proteus vulgaris, Erwinia caratovora, Shigella flexneri, and Escherichia coli B/r. J. Bacteriol. 160: 153–160.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kokjohn, T.A. and Miller, R.V. 1985. Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO. J. Bacteriol. 163: 568–572.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Zink, R.T., Engwall, J.K., McEvoy, J.L. and Chatterjee, A.K. 1985. recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp. carotovora. J. Bacteriol. 164: 390–396.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Goldberg, I. and Mekelanos, J.J. 1986. Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant. J. Bacteriol. 165: 715–722.

    Article  CAS  Google Scholar 

  16. Hickman, M.J., Orser, C.S., Willis, D.K., Lindow, S.E. and Panopoulos, N.J. 1987. Molecular cloning and biological characterization of the recA gene from Pseudomonas syringae. J. Bacteriol. 169: 2906–2910.

    Article  CAS  Google Scholar 

  17. Benbrook, D.M. and Miller, R.V. 1986. Effects of norfloxacin on DNA metabolism in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 29: 1–6.

    Article  CAS  Google Scholar 

  18. Messing, J. 1979. A multipurpose cloning system based on single-stranded DNA bacteriophage M13. Recombinant DNA technical bulletin, NIH publication ♯79–99, Vol. 2, No. 2:43–48.

    Google Scholar 

  19. Murray, K., Dugglebey, C.J., Sala-Trepat, J.M. and Williams, P. 1972. The metabolism of benzoate and methylbenzoates via the meta-cleavage pathway by Pseudomonas arvilla mt-2. Eur. J. Biochem. 28: 301–310.

    Article  CAS  Google Scholar 

  20. Loper, J.E., Orser, C.S., Panopoulos, N.J. and Schroth, M.N. 1984. Genetic analysis of fluorescent pigment production in Pseudomonas syringae pv. syringae. J. Gen. Microbiol. 130: 1507–1515.

    CAS  Google Scholar 

  21. Drahos, D.J., Hemming, B.C. and McPherson, S. 1986. Tracking recombinant organisms in the environment: β-galactosidase as a selectable non-antibiotic marker for fluorescent pseudomonads. Bio/Technology 4: 439–444.

    CAS  Google Scholar 

  22. Hirota, Y. 1960. The effects of acridine dyes on mating type functions in Escherichia coli. Proc. Natl. Acad. Sci. USA. 46: 57–64.

    Article  CAS  Google Scholar 

  23. Barry, G. 1988. A broad host-range shuttle system for gene insertion into the chromosome of Gram-negative bacteria. Gene 71: 75–84.

    Article  CAS  Google Scholar 

  24. European Patent Application ♯193,515.

  25. Dente, L., Cesarini, G. and Cortese, R. 1983. pEMBL: a new family of single-stranded plasmids. Nucleic Acids Res. 11: 1645–1655.

    Article  CAS  Google Scholar 

  26. Maniatis, T., Fritsch, E. and Sambrook, J. 1982. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York.

    Google Scholar 

  27. Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.R. 1980. Broad host-range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA. 77: 7347–7351.

    Article  CAS  Google Scholar 

  28. Renart, J., Reiser, J. and Stark, G.R. 1979. Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with antisera: A method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA. 76: 3116–3120.

    Article  CAS  Google Scholar 

  29. Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  30. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Peter O. Olins: Corresponding author.

Authors and Affiliations

  1. Monsanto Corporate Research, 700 Chesterfield Village Parkway, St. Louis, MO, 63198

    Shaukat H. Rangwala, Roy L. Fuchs, David J. Drahos & Peter O. Olins

Authors
  1. Shaukat H. Rangwala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roy L. Fuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David J. Drahos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter O. Olins
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rangwala, S., Fuchs, R., Drahos, D. et al. Broad Host-Range Vector for Efficient Expression of Foreign Genes in Gram-Negative Bacteria. Nat Biotechnol 9, 477–479 (1991). https://doi.org/10.1038/nbt0591-477

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0591-477

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing