Skip to main content

Genetic Tools for the Manipulation of Moderately Halophilic Bacteria of the Family Halomonadaceae

  • Protocol
Recombinant Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 267))

Abstract

Moderately halophilic bacteria of the family Halomonadaceae (Halomonas, Chromohalobacter, and Zymobacter) have promising applications in biotechnology as a source of compatible solutes (stabilizers of biomolecules and cells), salt-tolerant enzymes, biosurfactants, and extracellular polysaccharides, among other products. In addition, they offer a number of advantages to be used as cell factories, alternative to conventional prokaryotic hosts like E. coli or Bacillus, for the production of recombinant proteins: (1) their high salt tolerance decreases to a minimum the necessity for aseptic conditions, resulting in cost-reducing conditions; (2) they are very easy to grow and maintain in the laboratory, and their nutritional requirements are simple; and (3) the majority can use a large range of compounds as a sole carbon and energy source. In this decade, the efforts of our group and others have made possible the genetic manipulation of this bacterial group. In this review, the most relevant tools are described, with emphasis given to cloning vectors, genetic exchange mechanisms, mutagenesis approaches, and reporter genes. Due to its relevance for genetic studies, complementary sections describing the influence of salinity on the susceptibility of moderately halophilic bacteria to antimicrobials, as well as the growth media most routinely used, culture conditions, and nucleic acid isolation procedures for these microorganisms, are included.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kushner, D. J. and Kamekura, M. (1988) Physiology of halophilic eubacteria, in Halophilic Bacteria (Rodríguez-Valera, F., ed.), CRC Press, Boca Raton, FL. pp. 109–140.

    Google Scholar 

  2. Ventosa, A., Nieto, J. J., and Oren, A. (1998) Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62, 504–544.

    PubMed  CAS  Google Scholar 

  3. Vreeland, R. H. (1992) The family Halomonadaceae, in The Prokaryotes, 2nd ed. (Balows, A., Trüper, H. G., Dworkin, M., Harder, W., and Schleifer, K. H., eds.), Springer-Verlag, New York, NY, pp. 3181–3188.

    Google Scholar 

  4. Arahal, D. R., Ludwig, W., Schleifer, K. H., and Ventosa, A. (2002) Phylogeny of the family Halomonadaceae based on 23S and 165 rDNA sequence analyses. Int. J. Syst. Evol. Microbiol. 52, 241–249.

    PubMed  CAS  Google Scholar 

  5. Coronado, M. J., Vargas, C., Mellado, E., Tegos, G., Drainas, C., Nieto, J. J., et al. (2000) The α-amylase gene of the moderate halophile Halomonas meridiana: cloning and molecular characterization. Microbiology 146, 861–868.

    PubMed  CAS  Google Scholar 

  6. Ventosa, A. and Nieto, J. J. (1995) Biotechnological applications and potentialities of halophilic microorganisms. World J. Microbiol. Biotechnol. 11, 85–94.

    Article  CAS  Google Scholar 

  7. Margesin, R. and Schinner, F. (2001) Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles 5, 73–83.

    Article  PubMed  CAS  Google Scholar 

  8. Galinski, E. A. (1995) Osmoadaptation in bacteria. Adv. Microb. Physiol. 37, 273–328.

    Article  CAS  Google Scholar 

  9. Da Costa, M. S., Santos, H., and Galinski, E. A. (1998) An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv. Biochem. Eng. Biotechnol. 61, 117–153.

    PubMed  Google Scholar 

  10. Bremer, E. and Kramer, R. (2000). Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes in bacteria, in Bacterial Stress Responses (Storz, G., and Hengge-Aronis, R., eds.), ASM Press, Washington, DC, pp. 77–97.

    Google Scholar 

  11. Sauer, T. and Galinski, E. A. (1998) Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol. Bioeng. 59, 128.

    Article  PubMed  CAS  Google Scholar 

  12. Knapp, S., Landstein, R., and Galinski, E. A. (1999) Extrinsic protein stabilization by the naturally occurring osmolytes b-hydroxyectoine and betaine. Extremophiles 3, 191–198.

    Article  PubMed  CAS  Google Scholar 

  13. Borges, N., Ramos, A., Raven, N. D., Sharp, R. J., and Santos, H. (2002) Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes. Extremophiles 6, 209–216.

    Article  PubMed  CAS  Google Scholar 

  14. Barth, S., Huhn, M., Matthey, B., Klimka, A., Galinski, E. A., and Engert, A. (2000) Compatible-solute supported periplasmic expression of functional recombinant proteins under stress conditions. Appl. Environ. Microbiol. 66, 1572–1579.

    Article  PubMed  CAS  Google Scholar 

  15. Nuccio, M. L., Rodhes, D., McNeil, S. D., and Hanson, A. D. (1999) Metabolic engineering of plants for osmotic stress resistance. Curr. Opin. Plant Biol. 2, 128–134.

    Article  PubMed  CAS  Google Scholar 

  16. Zhu, J. K. (2000) Genetic analysis of plant salt tolerance using Arabidopsis. Plant Physiol. 124, 941–948.

    Article  PubMed  CAS  Google Scholar 

  17. Holmberg, N. and Bülow, L. (1998) Improving stress tolerance in plants by gene transfer. Trends Plant Sci. 3, 61–66.

    Article  Google Scholar 

  18. Sakamoto, A. and Murata, N. (2000) Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance. J. Exp. Bot. 51, 81–88.

    Article  PubMed  CAS  Google Scholar 

  19. Chen, T. H. and Murata, N. (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Opin. Plant Biol. 5, 250–257.

    Article  PubMed  CAS  Google Scholar 

  20. Nakayama, H., Yoshida, K., Ono, H., Murooka, Y., and Shinmyo, A. (2000) Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiol. 122, 1239–1247.

    Article  PubMed  CAS  Google Scholar 

  21. Hezayen, F. F., Rehm, B. H. A., Eberhardt, R., and Steinbüchel, A. (2000) Polymer production by two newly isolated extremely halopilic archaea: application of a novel corrosion-resistant bioreactor. Appl. Microbiol. Biotechnol. 54, 319–325.

    Article  PubMed  CAS  Google Scholar 

  22. 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.

    PubMed  CAS  Google Scholar 

  23. Tegos, G., Vargas, C., Perysinakis, A., Koukkou, A. I., Nieto, J. J., Ventosa, A., 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.

    Article  PubMed  CAS  Google Scholar 

  24. Frillingos, S., Linden, A., Niehaus, F., Vargas, C., Nieto, J. J., Ventosa, A., et al. (2000) Cloning and expression of a-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata. J. Appl. Microbiol. 88, 495–503.

    Article  PubMed  CAS  Google Scholar 

  25. Nieto, J. J., Fernández-Castillo, R., Márquez, M. C., Ventosa A., and Ruiz-Berraquero, F. (1989) A survey of metal tolerance in moderately halophilic eubacteria. Appl. Environ. Microbiol. 55, 2385–2390.

    PubMed  CAS  Google Scholar 

  26. Rodríguez-Valera, F., Ruiz-Berraquero, F., and Ramos-Cormenzana, A. (1981) Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. Microb. Ecol. 7, 235–243.

    Article  Google Scholar 

  27. Vreeland, R. H., Anderson, R., and Murray, R. G. E. (1984) Cell wall and phospholipid composition and their contribution to the salt tolerance of Halomonas elongata. J. Bacteriol. 160, 879–883.

    PubMed  CAS  Google Scholar 

  28. James, S. R., Dobson, S. J., Franzmann, P. D., and McMeekin, T. A. (1990) Halomonas meridiana, a new species of extremely halotolerant bacteria isolated from Antarctic saline lakes. Syst. Appl. Microbiol. 13, 270–278.

    Google Scholar 

  29. Severin, J., Wohlfarth, A., and Galinski, E. A. (1992) The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J. Gen. Microbiol. 138, 1629–1638.

    CAS  Google Scholar 

  30. Cohen, G. N. and Rickenberg, R. H. (1956). Concentration specifique reversible des amino-acides chez E. coli. Ann. Ins. Pasteur. Paris 91, 693–720.

    CAS  Google Scholar 

  31. Kamekura, M., Wallace, R., Hipkiss, A. R., and Kushner, D. J. (1985) Growth of Vibrio costicola and other moderate halophiles in a chemically defined minimal medium. Can. J. Microbiol. 31, 870–872.

    Article  PubMed  CAS  Google Scholar 

  32. Cummings, E. P. and Gilmour, D. J. (1995) The effect of NaCl on the growth of a Halomonas species: accumulation and utilization of compatible solutes. Microbiology 141, 1413–1418.

    Article  CAS  Google Scholar 

  33. Nieto, J. J., Fernández-Castillo, R., García, M. T., Mellado, E., and Ventosa, A. (1993) Survey of antimicrobial susceptibility of moderately halophilic eubacteria and extremely halophilic aerobic archaeobacteria: utilization of antimicrobial resistance as a genetic marker. Syst. Appl. Microbiol. 16, 352–360.

    CAS  Google Scholar 

  34. Kunte, H. J. and Galinski, E. A. (1995) Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion on transposon Tn5 and Tn1732 in Halomonas elongata. FEMS Microbiol. Let. 128, 293–299.

    Article  CAS  Google Scholar 

  35. 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.

    Article  PubMed  CAS  Google Scholar 

  36. Borghese, R., Zagnoli, A., and Zannoni, D. (2001) Plasmid transfer and susceptibility to antibiotics in the halophilic phototrophs Rhodovibrio salinarum and Rhodothalassium salexigens. FEMS Microbiol. Lett. 197, 117–121.

    Article  PubMed  CAS  Google Scholar 

  37. Morelle, G. (1989) A plasmid extraction procedure on a miniprep scale. BRL Focus 11, 7–8.

    Google Scholar 

  38. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  39. Eckhardt, T. (1978) A rapid method for the identification of plasmid DNA in bacteria. Plasmid 1, 584–588.

    Article  PubMed  CAS  Google Scholar 

  40. Plazinski, J., Cen, Y. H., and Rolfe, B. G. (1985) General method for the identification of plasmid species in fast-growing soil microorganisms. Appl. Environ. Microbiol. 48, 1001–1003.

    Google Scholar 

  41. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al. (1989). Current protocols in molecular biology. Greene Publishing Associates, John Wiley & Sons, New York.

    Google Scholar 

  42. Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208–218.

    Article  CAS  Google Scholar 

  43. Fernández-Castillo, R., Vargas, C., Nieto, J. J, Ventosa, A., and Ruiz-Berraquero, F. (1992) Characterization of a plasmid from moderately halophilic eubacteria. J. Gen. Microbiol. 138, 1133–1137.

    PubMed  Google Scholar 

  44. Vargas, C., Férnandez-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.

    Article  PubMed  CAS  Google Scholar 

  45. 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, 851–861.

    Article  PubMed  CAS  Google Scholar 

  46. 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.

    PubMed  CAS  Google Scholar 

  47. Mellado, E., Asturias, M. A., Nieto, J. J., Timmis, K. N., and Ventosa, A. (1995) Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui. J. Bacteriol. 177, 3433–3445.

    Google Scholar 

  48. Llamas, I., del Moral, A., Béjar, V., Girón, M. D., Salto, R., and Quesada, E. (1997) Plasmids from Halomonas eurihalina, a microorganism which produces an exopolysaccharide of biotechnological interest. FEMS Microbiol. Lett. 156, 251–257.

    Article  CAS  Google Scholar 

  49. Mellado, E., García, M. T., Nieto, J. J., Kaplan, S., and Ventosa, A. (1997) Analysis of the genome of Vibrio costicola: pulsed-field gel electrophoresis analysis of genome size and plasmid content. Syst. Appl. Microbiol. 20, 20–26.

    Google Scholar 

  50. Mellado, E., Nieto, J. J., and Ventosa, A. (1995) Construction of novel shuttle vectors for use between moderately halophilic bacteria and Escherichia coli. Plasmid 34, 157–164.

    Article  PubMed  CAS  Google Scholar 

  51. Vargas, C., Coronado, M. J., Ventosa, A., and Nieto, J. J. (1997) Host range, stability and compatibility of broad-host-range plasmids and a shuttle vector in moderately halophilic bacteria. Evidence of intragenic and intergenic conjugation in moderate halophiles. Syst. Appl. Microbiol. 20, 173–181.

    Google Scholar 

  52. Knauf, V. C. and Nester, E. W. (1982) Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid 8, 45–54.

    Article  PubMed  CAS  Google Scholar 

  53. Spaink, H. P., Okker, R. J. H., Wiffelman, C. A., Pees, E., and Lutenberg, B. J. J. (1987). Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRLJI. Plant Mol. Biol. 9, 27–39.

    Article  CAS  Google Scholar 

  54. Bagdasariam, M., Lurz, R., Rückert, B., Franklin, F. C. H., Bagdasariam, M. M., Frey, J., et al. (1981) Specific purpose plasmid cloning vectors, II. Broad-host-range, high-copy-number RSF 1010-derived vectors and host-vector system for gene cloning in Pseudomonas. Gene 16, 237–247.

    Article  Google Scholar 

  55. Labes, M., Pühler, A., and Simon, R. (1990). A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for Gram-negative bacteria. Gene 89, 37–46.

    Article  PubMed  CAS  Google Scholar 

  56. Leemans, J., Langenakens, J., de Greve, H., Deblaere, R., van Monatgu, M., and Schell, J. (1982). Broad-host-range cloning vectors derived from the W-plasmid Sa. Gene 19, 361–364.

    Article  PubMed  CAS  Google Scholar 

  57. Konarska-Kozlowska, M., Thatte, V., and Iyer, V. N. (1983). Inverted repeats in the DNA of plasmid pCU1. J. Bacteriol. 153, 1502–1512.

    PubMed  CAS  Google Scholar 

  58. Cánovas, D., Vargas, C., Ventosa, A., and Nieto, J. J. (1997) Salt-sensitive and auxotrophic mutants of Halomonas elongata and H. meridiana by use of hydroxylamine mutagenesis. Curr. Microbiol. 34, 85–90.

    Article  Google Scholar 

  59. Ubben, D. and Schmitt, R. (1986) Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis. Gene 41, 145–152.

    Article  PubMed  CAS  Google Scholar 

  60. Schmitt, R., Bernhard, E., and Mattes, R. (1979) Characterization of Tn1721, a new transposon containing a tetracycline resistance gene capable of amplification. Mol. Gen. Genet. 172, 53–65.

    Article  PubMed  CAS  Google Scholar 

  61. Allmeier, H., Cresnar, B., Greck, M., and Schmitt, R. (1992) Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein. Gene 111, 11–20.

    Article  PubMed  CAS  Google Scholar 

  62. Göller, K., Ofer, A., and Galinski, E. A. (1998) Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis. FEMS Microbiol. Let. 161, 293–300.

    Article  Google Scholar 

  63. Cánovas, D., Vargas, C., Iglesias-Guerra, F., Csonka, L. N., Rhodes, D., Ventosa, A., et al. (1997) Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes. J. Biol. Chem. 272, 25794–25801.

    Article  PubMed  Google Scholar 

  64. Grammann, K., Volke, A., and Kunte, H. J. (2002) New type of osmoregulated solute transporter identified in halophilic members of the Bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581§. J. Bacteriol. 184, 3078–3085.

    Article  PubMed  CAS  Google Scholar 

  65. Ubben, D. and Schmitt, R. (1987) A transposable promoter and transposable promoter probes derived from Tn1721. Gene 53, 127–134.

    Article  PubMed  CAS  Google Scholar 

  66. De Lorenzo, V., Herrero, M., Jakubzik, U., and Timmis, K. (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative bacteria. J. Bacteriol. 172, 6568–6572.

    PubMed  Google Scholar 

  67. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.

    Article  PubMed  CAS  Google Scholar 

  68. Schäfer, A., Tauch, A., Jäger, W, Kalinowski, J., Thierbach, G., and Pühler, A. (1994) Small mobilizable multipurpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69–73.

    Article  PubMed  Google Scholar 

  69. Prentki, P. and Krisch, H. M. (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–313.

    Article  PubMed  CAS  Google Scholar 

  70. Quandt, J. and Hynes, M. (1993) Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127, 15–21.

    Article  PubMed  CAS  Google Scholar 

  71. Kessler, B., de Lorenzo, V., and Timmis, N. K. (1992) A general system to integrate lacZ fusions into the chromosome of Gram negative bacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol. Gen. Genet. 233, 293–301.

    Article  PubMed  CAS  Google Scholar 

  72. Douka, E., Christogianni, A., Koukkou, A. I., Afendra, A., and Drainas, C. (2001) Use of a green fluorescent protein as a reporter in Zymomonas mobilis and Halomonas elongata. FEMS Microbiol. Lett. 201, 221–227.

    Article  PubMed  CAS  Google Scholar 

  73. Lindgren, P. B., Frederick, R., Govindarajan, A. G., Panopoulos, N. J., Staskawicz, B. J., and Lindow, S. E. (1989) An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. Phaseolicola. EMBO J. 8, 2990–3001.

    Google Scholar 

  74. Tegos, C., Vargas, C., Vartholomatos, G., Perysinakis, A., Nieto, J. J., Ventosa, A., et al. (1997) Identification of a promoter region on the Halomonas elongata plasmid pHE1 employing the inaZ reporter gene of Pseudomonas syringae. FEMS Microbiol. Lett. 154, 45–51.

    Article  PubMed  CAS  Google Scholar 

  75. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.

    Article  PubMed  CAS  Google Scholar 

  76. Cormark, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Vargas, C., Nieto, J.J. (2004). Genetic Tools for the Manipulation of Moderately Halophilic Bacteria of the Family Halomonadaceae . In: Balbás, P., Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 267. Humana Press. https://doi.org/10.1385/1-59259-774-2:183

Download citation

  • DOI: https://doi.org/10.1385/1-59259-774-2:183

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-262-9

  • Online ISBN: 978-1-59259-774-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics