Genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol of Escherichia coli

FEMS Microbiol Lett. 2009 Jul;296(1):67-71. doi: 10.1111/j.1574-6968.2009.01620.x. Epub 2009 May 7.

Abstract

Complications of chemotherapy, such as appearance of multidrug resistance, have persuaded researchers to consider phage therapy as a new method to combat bacterial infections. In vitro experiments were performed to assess the therapeutic value of genetically modified phages for controlling gastrointestinal Escherichia coli O157:H7 cells in Luria-Bertani (LB) media and contaminated cow milk. We constructed a modified nonreplicating M13-derived phage expressing a lethal catabolite gene activator protein (CAP) that is a Glu181Gln mutant of CAP. The modified phagemid was propagated in the lethal CAP-resistant strain XA3DII. Time-kill assay experiments showed a considerable reduction in the number of surviving bacteria in both LB media and contaminated cow milk. Our further study using other test strains demonstrated that the host range of lethal phage is limited to E. coli strains that produce pili. This study provides a possible strategy for the exploitation of genetically engineered nonlytic phages as bactericidal agents by minimizing the risk of release of progeny phages and endotoxins into the environment. The phage was engineered to remain lethal to its bacterial target, but incapable of replicating therein. Furthermore, the addition of an inducer to express the lethal protein is not required.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics
  • Animals
  • Bacteriophage M13 / genetics*
  • Bacteriophage M13 / growth & development
  • Cattle
  • Colony Count, Microbial
  • Culture Media
  • Cyclic AMP Receptor Protein / biosynthesis*
  • Cyclic AMP Receptor Protein / genetics
  • Escherichia coli O157 / growth & development*
  • Escherichia coli O157 / virology*
  • Microbial Viability
  • Milk / microbiology
  • Mutant Proteins / biosynthesis*
  • Mutant Proteins / genetics
  • Pest Control, Biological / methods*
  • Promoter Regions, Genetic

Substances

  • Culture Media
  • Cyclic AMP Receptor Protein
  • Mutant Proteins