A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125

  • Maria Giuliani
  • Ermenegilda Parrilli
  • Cinzia Pezzella
  • Valentina Rippa
  • Angela Duilio
  • Gennaro Marino
  • Maria Luisa TutinoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 824)


The sequencing and the annotation of the marine Antarctic Pseudoalteromonas haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of several genetic tools for studying it at the molecular level. To allow a deeper understanding of the PhTAC125 physiology a genetic system for the reverse genetics in this bacterium was developed. In the present work, we describe a practical technique for allelic exchange and/or gene inactivation by in-frame deletion and the use of a counterselectable marker in P. haloplanktis. The construction of suitable non-replicating plasmid and methods used to carry out a two-step integration–segregation strategy in this bacterium are reported in detail.

Furthermore two examples, in which the developed methodology was applied to find out gene function or to construct genetically engineered bacterial strains, were described.

Key words

Pseudoalteromonas haloplanktis TAC125 Allelic exchange Counterselectable marker Suicide vector 


  1. 1.
    Médigue, C., Krin, E., Pascal, G, et al. (2005). Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome Res. 15, 1325–35.PubMedCrossRefGoogle Scholar
  2. 2.
    Duilio, A., Tutino, M.L., Marino, G. (2004). Recombinant protein production in Antarctic Gram negative bacteria. Methods Mol. Biol., 267, 225237.Google Scholar
  3. 3.
    Parrilli, E., Duilio, A., Tutino, M.L. (2008). Heterologous protein expression in psychrophilic hosts. In Psychrophiles: from Biodiversity to Biotechnology. Edited by: RMea. Berlin Heidelberg: Springer-Verlag., 365–379.Google Scholar
  4. 4.
    Hensel, M., and Holden, D.W. (1996). Molecular genetic approaches for the study of virulence in both pathogenic bacteria and fungi. Microbiology 142, 1049–1058.PubMedCrossRefGoogle Scholar
  5. 5.
    Schwarzer, A. & PUhler, A. (1991). Manipulation of Corynebacterium glutamicum by gene disruption and replacement. BioTechnology 9, 84–87.Google Scholar
  6. 6.
    McFadden, J. (1996). Recombination in mycobacteria. Mol. Microbiol. 21, 205–211.PubMedCrossRefGoogle Scholar
  7. 7.
    Stibitz, S. (1994). Use of conditionally counterselectable suicide vectors for allelic exchange. Methods Enzymol. 235, 458–465.7Google Scholar
  8. 8.
    Parrilli, E., Cusano, A.M., Giuliani, M., (2006). Cell engineering of Pseudoalteromonas haloplanktis TAC125: construction of a mutant strain with reduced exo-proteolytic activity. Microb. Cell Fact. 5(1), P36Google Scholar
  9. 9.
    Parrilli, E., De Vizio, D., Cirulli, C., (2008). Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature. Microb. Cell Fact. 7, 2PubMedCrossRefGoogle Scholar
  10. 10.
    Kast, P. (1994). PKSS – a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 138, 109–114.PubMedCrossRefGoogle Scholar
  11. 11.
    Li, M.Z. and Elledge, S.J. (2005). MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules. Nat. Genet. 37, 311–319.PubMedCrossRefGoogle Scholar
  12. 12.
    Kristich, C.J., Chandler, J.R., and Dunny, G.M. (2007). Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis. Plasmid 57, 131–144.PubMedCrossRefGoogle Scholar
  13. 13.
    Tascon, R. I., Rodriguez-Ferri, E. F., Gutierrez-Martin, C. B., (1993). Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative. J. Bacteriol. 175, 5717–5722.PubMedGoogle Scholar
  14. 14.
    Tutino, M. L., Duilio, A., Moretti, M. A., (2000). A rolling-circle plasmid from Psychrobacter sp. TA144: evidence for a novel Rep subfamily. Biochem. Biophys. Res. Commun. 274, 488–495.PubMedCrossRefGoogle Scholar
  15. 15.
    Parrilli, E., Giuliani, M., Pezzella, C., (2010). PhPssA is required for alpha-amylase secretion in Antarctic Pseudoalteromonas haloplanktis. Microbiol., 156(1), 211–219.CrossRefGoogle Scholar
  16. 16.
    Wittenberg, J.B., Bolognesi, M., Wittenberg, B.A., (2002). Truncated haemoglobins: a new family of haemoglobins widely distributed in bacteria, unicellular eukaryotes and plants. J. Biol. Chem. 277, 871–874.PubMedCrossRefGoogle Scholar
  17. 17.
    Giordano, D., Parrilli, E., Dettaï, A. (2007). The truncated haemoglobins in the Antarctic psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125. Gene 398, 69–77.PubMedCrossRefGoogle Scholar
  18. 18.
    Parrilli, E., Giuliani, M., Giordano, D., (2010). The role of a 2-on-2 haemoglobin in oxidative and nitrosative stress resistance of Antarctic Pseudoalteromonas haloplanktis TAC125. Biochimie 92, 1003–1009.PubMedCrossRefGoogle Scholar
  19. 19.
    Cusano, A.M., Parrilli, E., Marino, G. (2006). A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125. Microb. Cell Fact. 5, 40.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Maria Giuliani
    • 1
  • Ermenegilda Parrilli
    • 1
  • Cinzia Pezzella
    • 1
  • Valentina Rippa
    • 1
  • Angela Duilio
    • 1
  • Gennaro Marino
    • 2
  • Maria Luisa Tutino
    • 1
    Email author
  1. 1.Department of Organic Chemistry and BiochemistryUniversità degli studi di Napoli Federico IINaplesItaly
  2. 2.Department of Organic Chemistry and BiochemistryUniversità degli studi di Napoli Federico II, Complesso Universitario M.S. AngeloNaplesItaly

Personalised recommendations