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Bacteriophages pp 147-162 | Cite as

Screening for Growth-Inhibitory ORFans in Pseudomonas aeruginosa-Infecting Bacteriophages

  • Hanne HendrixEmail author
  • Ines Staes
  • Abram Aertsen
  • Jeroen WagemansEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1898)

Abstract

Like all viruses, bacteriophages heavily depend on their host’s physiology for reproduction. Therefore, phages have evolved numerous proteins that influence the host metabolism to facilitate the infection process. Some of these proteins strongly perturb the host cell, ultimately leading to cell death. These growth-inhibitory phage proteins presumably target key metabolic processes, which may provide a basis for innovative phage-derived antibacterials. Unfortunately, most of these proteins are the so-called ORFans, since they have no known function or sequence homology to any other gene. We here describe a screening method for the identification of growth-inhibitory ORFans of bacteriophages infecting gram-negative bacteria (e.g., Pseudomonas aeruginosa), using the pUC18-mini-Tn7T-Lac vector system, which allows for stable single-copy integration of the phage ORFans in the Pseudomonas genome under the control of an IPTG-inducible promoter. Furthermore, we describe a method to examine the effect of the phage proteins in different hosts, using different vector copy numbers. Finally, we explain how to investigate the effect of ORFan expression on the host morphology using time-lapse microscopy.

Key words

Bacteriophage Pseudomonas aeruginosa Gateway cloning Chromosomal integration Spot test Bio-screen Time-lapse microscopy 

References

  1. 1.
    Roucourt B, Lavigne R (2009) The role of interactions between phage and bacterial proteins within the infected cell: a diverse and puzzling interactome. Environ Microbiol 11:2789–2805CrossRefGoogle Scholar
  2. 2.
    Choi KH, Gaynor JB, White KG et al (2005) A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2:443–448CrossRefGoogle Scholar
  3. 3.
    Amann E, Brosius J, Ptashne M (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25:167–178CrossRefGoogle Scholar
  4. 4.
    Liu J, Dehbi M, Moeck G et al (2004) Antimicrobial drug discovery through bacteriophage genomics. Nat Biotechnol 22:185–191CrossRefGoogle Scholar
  5. 5.
    Stover CK, Pham XQ, Erwin AL et al (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964CrossRefGoogle Scholar
  6. 6.
    Qiu D, Damron FH, Mima T et al (2008) PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in pseudomonas and Burkholderia spp. and other bacteria. Appl Environ Microbiol 74:7422–7426CrossRefGoogle Scholar
  7. 7.
    Nash HA (1981) Integration and excision of bacteriophage lambda: the mechanism of conservation site specific recombination. Annu Rev Genet 15:143–167Google Scholar
  8. 8.
    Choi KH, Kumar A, Schweizer HP (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397CrossRefGoogle Scholar
  9. 9.
    Sambrook J, Russell DW (2001) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.YGoogle Scholar
  10. 10.
    Sriramulu DD, Lunsdorf H, Lam JS et al (2005) Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung. J Med Microbiol 54:667–676CrossRefGoogle Scholar
  11. 11.
    de Jong IG, Beilharz K, Kuipers OP, Veening J-W (2011) Live cell imaging of bacillus subtilis and streptococcus pneumoniae using automated time-lapse microscopy. J Vis Exp (53):3145Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Gene TechnologyKU LeuvenLeuvenBelgium
  2. 2.Laboratory of Food MicrobiologyKU LeuvenLeuvenBelgium

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