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Applied and Environmental Microbiology, January 2004, p. 527-534, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.527-534.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

Masahito Oda, Masatomo Morita, Hajime Unno, and Yasunori Tanji*

Department of Bioengineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

Received 16 April 2003/ Accepted 22 September 2003

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.

* Corresponding author. Mailing address: Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Phone: 81-45-924-5763. Fax: 81-45-924-5818. E-mail: ytanji{at}bio.titech.ac.jp.

Applied and Environmental Microbiology, January 2004, p. 527-534, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.527-534.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Slootweg, E. J., Keller, H. J.H.G., Hink, M. A., Borst, J. W., Bakker, J., Schots, A. (2006). Fluorescent T7 display phages obtained by translational frameshift. Nucleic Acids Res 34: e137-e137 [Abstract] [Full Text]  


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