Gene Transfer and Expression of Recombinant Proteins in Moderately Halophilic Bacteria

  • Amalia S. Afendra
  • Carmen Vargas
  • Joaquín J. Nieto
  • Constantin Drainas
Part of the Methods in Molecular Biology book series (MIMB, volume 267)


Moderately halophilic bacteria (MHB) of the genera Halomonas and Chromohalobacter have been used as hosts for the expression of heterologous proteins of biotechnological interest, thus expanding their potential to be used as cell factories for various applications. This chapter deals with the methodology for the construction of recombinant plasmids, their transfer to a number of MHB, and the assaying of the corresponding heterologous proteins activity. The transferred genes include (1) inaZ, encoding the ice nucleation protein of the plant pathogen Pseudomonas syringae, (2) gfp, encoding a green fluorescent protein from the marine bioluminescent jellyfish Aequorea victoria, and (3) the α -amylase gene from the hyperthermophilic archeon Pyrococcus woesei. Vector pHS15, which was designed for expression of heterologous proteins in both E. coli and MHB, was used for the subcloning and transfer of the above genes. The recombinant constructs were introduced to MHB by assisted conjugal transfer from E. coli donors. The expression and function of the recombinant proteins in the MHB transconjugants is described.

Key Words

Halophilic bacteria Halomonadaceae Chromohalobacter GFP α-amylase inaZ 


  1. 1.
    Ventosa, A., Nieto, J. J., and Oren, A. (1998) Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62, 504–544.PubMedGoogle Scholar
  2. 2.
    Vargas, C., Fernández-Castillo, R., Cánovas, D., Ventosa, A., and Nieto, J. J. (1995) Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction. Mol. Gen. Genet. 246, 411–418.PubMedCrossRefGoogle Scholar
  3. 3.
    Baertlein, D. A., Lindow, S. E., Panopoulos, N. J., Lee, S. P., Mindrinos, M. N., and Chen, T. H. H. (1992) Expression of a bacterial ice nucleation gene in plants. Plant Physiol. 100, 1730–1736.PubMedCrossRefGoogle Scholar
  4. 4.
    Drainas, C., Vartholomatos, G., and Panopoulos, N. J. (1992) The ice nucleation gene from Pseudomonas syringae as a sensitive gene reporter for promoter analysis in Zymomonas mobilis. Appl. Environm. Microbiol. 61, 273–277.Google Scholar
  5. 5.
    Reynen, M., Reipen, I., Sahm, H., and Sprenger, G. A. (1990) Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis. Mol. Gen. Genet. 223, 335–341.PubMedCrossRefGoogle Scholar
  6. 6.
    Miller, W. G. and Lindow, S. E. (1997) An improved GFP cloning cassette designed for prokaryotic transcriptional fusions. Gene 191, 149–153.PubMedCrossRefGoogle Scholar
  7. 7.
    Frillingos, S., Linden, A., Niehaus, F., Vargas, C., Nieto, J. J., Ventosa, A., et al. (2000) Cloning and expression of α-amylase from the hyperthermophilic archeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata. J. Appl. Microbiol. 88, 495–503.PubMedCrossRefGoogle Scholar
  8. 8.
    Ventosa, A., Nieto, J. J., and Oren, A. (1998) Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62, 504–544.PubMedGoogle Scholar
  9. 9.
    Vargas, C., Tegos, G., Drainas, C., Ventosa, A., and Nieto, J. J. (1999) Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata. Mol. Gen. Genet. 261, 851–856.PubMedCrossRefGoogle Scholar
  10. 10.
    Vargas, C., Tegos, G., Vartholomatos, G., Drainas, C., Ventosa, A., and Nieto, J. J. (1999) Genetic organization of the mobilization region of the plasmid pHE1 from Halomonas elongata. Syst. Appl. Microbiol. 22, 520–529.PubMedGoogle Scholar
  11. 11.
    Wolber, P. K. (1992) Bacterial ice nucleation. Adv. Microb. Physiol. 31, 203–237.Google Scholar
  12. 12.
    Margaritis, A. and Singh-Bassi, A. (1991) Principles and biotechnological applications of bacterial ice nucleation. Crit. Rev. Biotechnol. 11, 277–295.PubMedCrossRefGoogle Scholar
  13. 13.
    Panopoulos, N. (1995) Ice nucleation genes as reporters, in Biological Ice Nucleation and its Applications (Lee, R. E. Jr., Warren, G. J. and Gusta, L. V., eds.) APS Press, St. Paul, MN, pp. 271–281.Google Scholar
  14. 14.
    Arvanitis, N., Vargas, C., Tegos, G., Perysinakis, A., Nieto, J. J., Ventosa, A., et al. (1995) Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae. Appl. Environ. Microbiol. 61, 3821–3825.PubMedGoogle Scholar
  15. 15.
    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescence protein as a marker for gene expression. Science 263, 802–805.PubMedCrossRefGoogle Scholar
  16. 16.
    Cormark, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.CrossRefGoogle Scholar
  17. 17.
    Douka, E., Christoyianni, A., Koukkou, A.-I., Afendra, A.-S., and Drainas, C. (2001) Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata. FEMS Microbiol. Lett. 201, 221–227.PubMedCrossRefGoogle Scholar
  18. 18.
    Jørgensen, S., Vorgias, C. E., and Antranikian, G. (1997) Cloning, sequencing, characterization, and expression of an extracellular α-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J. Biol. Chem. 268, 16335–16342.CrossRefGoogle Scholar
  19. 19.
    Dong, G., Vieille, C., Savchenko, A., and Zeikus, J. G. (1997) Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl. Environm. Microbiol. 63, 3569–3576.Google Scholar
  20. 20.
    Janecek, S. (1994) Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases. Eur. J. Biochem. 224, 519–524.PubMedCrossRefGoogle Scholar
  21. 21.
    Janecek, S. (1998) Sequence of archeal Methanococcus jannaschii alpha-amylase contains features of families 13 and 57 of glycosyl hydrolases: a trace of their common ancestor? Folia Microbiol. (Praha) 43, 123–128.CrossRefGoogle Scholar
  22. 22.
    Vali, G. (1971) Quantitative evaluation of experimental results on the heterologous freezing nucleation of supercooled liquids. J. Atmos. Sci. 28, 402–409.CrossRefGoogle Scholar
  23. 23.
    Tegos, G., Vargas, C., Perysinakis, A., Koukkou, A. I., Christogianni, A., Nieto J. J., et al. (2000) Release of cell-free ice nuclei from Halomonas elongata expressing the ice nucleation gene inaZ of Pseudomonas syringae. J. Appl. Microbiol. 89, 785–792.PubMedCrossRefGoogle Scholar
  24. 24.
    de Palencia, P. F., Nieto, C., Acebo, P., Espinosa, M., and Lopez, P. (2000) Expression of green fluorescent protein in Lactococcus lactis. FEMS Microbiol. Lett. 183, 229–234.Google Scholar
  25. 25.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  26. 26.
    Lissemore, J. L., Jankowski, J. T., Thomas, C. B., Mascotti, D. P., and de Haseth, P. L. (2000) Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli. BioTechniques 28, 82–89.PubMedGoogle Scholar
  27. 27.
    Laderman, K., Davis, B. R., Krutzsch, H. C., Lewis, M. S., Griko, Y. V., Privalov, P. L., et al. (1993) The purification and characterization of an extremely thermostable α-amylase from the hyperthermophilic archaeobacterium Pyrococcus furiosus. J. Biol. Chem. 268, 24394–24401.PubMedGoogle Scholar
  28. 28.
    Koch, R., Spreinat, A., Lemke, K., and Antranikian, G. (1991) Purification and properties of a hyperthermoactive α-amylase from the archaeobacterium Pyrococcus woesei. Arch. Microbiol. 155, 572–578.CrossRefGoogle Scholar
  29. 29.
    Coronado, M. J., Vargas, C., Kunte, H. J., Galinski, E., Ventosa, A., and Nieto, J. J. (1995) Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies. Curr. Microbiol. 31, 365–371.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Amalia S. Afendra
    • 1
  • Carmen Vargas
    • 2
  • Joaquín J. Nieto
    • 2
  • Constantin Drainas
    • 1
  1. 1.Department of ChemistryUniversity of IoanninaIoanninaGreece
  2. 2.Department of Microbiology and Parasitology, Faculty of PharmacyUniversity of SevilleSevillaEspaña

Personalised recommendations