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A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

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Abstract

The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2′-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.

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Acknowledgments

We thank Masaru Kawato and Yuto Yashiro for help with flow cytometry techniques; Norika Meguro, Hiromi Kazama, and Masayuki Ehara for the help with 16S rRNA gene analysis; Drs. Kengo Kubota, Myong-Ok Park, Takako Nogami, Hiroshi Tsukamoto, Kazuhiko Miyanaga, Hideki Kobayashi, Tadashi Maruyama, Sanae Sakai, Takuro Nunoura, Aidan J. Synnott, Roland Hatzenpichler, and Jennifer Glass for the helpful discussions and useful comments. We greatly appreciate Professor Hideki Harada for his continuous encouragement. We also thank Yuji Suzuki at the Yokohama city office for his assistance in sampling the Kanazawa-ku municipal sewage treatment plant. This study was partly supported by grants from the Japan Society for the Promotion of Science, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Subsurface Geobiology Advanced Research (SUGAR) Project, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa, 237-0061, Japan

    Sayaka Ohno, Hironori Okano, Ken Takai & Hiroyuki Imachi

  2. Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Kanagawa, 226-8501, Japan

    Sayaka Ohno & Yasunori Tanji

  3. Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, 940-2188, Japan

    Hironori Okano

  4. Department of Social and Environmental Engineering, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8527, Japan

    Akiyoshi Ohashi

  5. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan

    Kazuya Watanabe

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  1. Sayaka Ohno
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  2. Hironori Okano
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Correspondence to Hiroyuki Imachi.

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Ohno, S., Okano, H., Tanji, Y. et al. A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU). Appl Microbiol Biotechnol 95, 777–788 (2012). https://doi.org/10.1007/s00253-012-4174-1

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