Advertisement

Applied Microbiology and Biotechnology

, Volume 95, Issue 3, pp 777–788 | Cite as

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

  • Sayaka Ohno
  • Hironori Okano
  • Yasunori Tanji
  • Akiyoshi Ohashi
  • Kazuya Watanabe
  • Ken Takai
  • Hiroyuki ImachiEmail author
Methods and protocols

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.

Keywords

Phage Host range EdU Click chemistry Fluorescence in situ hybridization (FISH) 

Notes

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.

Supplementary material

253_2012_4174_MOESM1_ESM.pdf (4.9 mb)
ESM 1 (PDF 5036 kb)

References

  1. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925Google Scholar
  2. Anderson RE, Brazelton WJ, Baross JA (2011) Is the genetic landscape of the deep subsurface biosphere affected by viruses? Front Microbiol 2:Article 219Google Scholar
  3. Best MD (2009) Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules. Biochemistry 48:6571–6584CrossRefGoogle Scholar
  4. Bielke L, Higgins S, Donoghue A, Donoghue D, Hargis BM (2007) Salmonella host range of bacteriophages that infect multiple genera. Poult Sci 86:2536–2540CrossRefGoogle Scholar
  5. Breinbauer R, Köhn M (2003) Azide-alkyne coupling: a powerful reaction for bioconjugate chemistry. Chembiochem 4:1147–1149CrossRefGoogle Scholar
  6. Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2′-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2′-deoxyuridine antibodies. Biotechniques 44:927–929CrossRefGoogle Scholar
  7. Buckley PJ, Kosturko LD, Kozinski AW (1972) In vivo production of an RNA-DNA copolymer after infection of Escherichia coli by bacteriophage T4. Proc Natl Acad Sci USA 69:3165–3169CrossRefGoogle Scholar
  8. Carlson K (1973) Multiple initiation of bacteriophage T4 DNA replication: delaying effect of bromodeoxyuridine. J Virol 12:349–359Google Scholar
  9. Chen F, Lu J-R, Binder BJ, Liu Y-C, Hodson RE (2001) Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR Gold. Appl Environ Microbiol 67:539–545CrossRefGoogle Scholar
  10. Clark JR, March JB (2006) Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. Trends Biotechnol 24:212–218CrossRefGoogle Scholar
  11. Daims H, Brühl A, Amann R, Schleifer K-H, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444CrossRefGoogle Scholar
  12. Danovaro R, Corinaldesi C, Dell’Anno A, Fuhrman JA, Middelburg JJ, Noble RT, Suttle CA (2011) Marine viruses and global climate change. FEMS Microbiol Rev 35:993–1034CrossRefGoogle Scholar
  13. Diermeier-Daucher S, Clarke ST, Hill D, Vollmann-Zwerenz A, Bradford JA, Brockhoff G (2009) Cell type specific applicability of 5-ethynyl-2′-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry 75A:535–546CrossRefGoogle Scholar
  14. Edgar R, McKinstry M, Hwang J, Oppenheim AB, Fekete RA, Giulian G, Merril C, Nagashima K, Adhya S (2006) High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes. Proc Natl Acad Sci USA 103:4841–4845CrossRefGoogle Scholar
  15. Funatsu T, Taniyama T, Tajima T, Tadakuma H, Namiki H (2002) Rapid and sensitive detection method of a bacterium by using a GFP reporter phage. Microbiol Immunol 46:365–369Google Scholar
  16. Furukawa H, Kuroiwa T, Mizushima S (1983) DNA injection during bacteriophage T4 infection of Escherichia coli. J Bacteriol 154:938–945Google Scholar
  17. Goodridge L, Chen J, Griffiths M (1999a) Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7. Appl Environ Microbiol 65:1397–1404Google Scholar
  18. Goodridge L, Chen J, Griffiths M (1999b) The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk. Int J Food Microbiol 47:43–50CrossRefGoogle Scholar
  19. Hennes KP, Suttle CA (1995) Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy. Limnol Oceanogr 40:1050–1055CrossRefGoogle Scholar
  20. Hennes KP, Suttle CA, Chan AM (1995) Fluorescently labeled virus probes show that natural virus populations can control the structure of marine microbial communities. Appl Environ Microbiol 61:3623–3627Google Scholar
  21. Hua H, Kearsey SE (2011) Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2′-deoxyuridine. Nucleic Acids Res 39:e60CrossRefGoogle Scholar
  22. Hugenholtz P (2002) Exploring prokaryotic diversity in the genomic era. Genome Biol 3:reviews0003Google Scholar
  23. Hyman P, Abedon ST (2010) Chapter 7—bacteriophage host range and bacterial resistance. In: Laskin AI, Sariaslani S, Gadd GM (eds) Advaces in applied microbiology, vol 70. Academic Press, San Diego, CA, USA, pp 217–248Google Scholar
  24. Imachi H, Aoi K, Tasumi E, Saito Y, Yamanaka Y, Saito Y, Yamaguchi T, Tomaru H, Takeuchi R, Morono Y, Inagaki F, Takai K (2011) Cultivation of methanogenic community from subseafloor sediments using a continuous-flow bioreactor. ISME J 5:1913–1925CrossRefGoogle Scholar
  25. Jacobs WR, Barletta RG, Udani R, Chan J, Kalkut G, Sosne G, Kieser T, Sarkis GJ, Hatfull GF, Bloom BR (1993) Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 260:819–822CrossRefGoogle Scholar
  26. Jensen EC, Schrader HS, Rieland B, Thompson TL, Lee KW, Nickerson KW, Kokjohn TA (1998) Prevalence of broad-host-range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa. Appl Environ Microbiol 64:575–580Google Scholar
  27. Kenzaka T, Yamaguchi N, Prapagdee B, Mikami E, Nasu M (2001) Bacterial community composition and activity in urban rivers in Thailand and Malaysia. J Health Sci 47:353–361CrossRefGoogle Scholar
  28. Kenzaka T, Tani K, Nasu M (2010) High-frequency phage-mediated gene transfer in freshwater environments determined at single-cell level. ISME J 4:648–659CrossRefGoogle Scholar
  29. Kosaka T, Kato S, Shimoyama T, Ishii S, Abe T, Watanabe K (2008) The genome of Pelotomaculum thermopropionicum reveals niche-associated evolution in anaerobic microbiota. Genome Res 18:442–448CrossRefGoogle Scholar
  30. Kristensen DM, Mushegian AR, Dolja VV, Koonin EV (2010) New dimensions of the virus world discovered through metagenomics. Trends Microbiol 18:11–19CrossRefGoogle Scholar
  31. Kutateladze M, Adamia R (2010) Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol 28:591–595CrossRefGoogle Scholar
  32. Lee SH, Onuki M, Satoh H, Mino T (2006) Isolation, characterization of bacteriophages specific to Microlunatus phosphovorus and their application for rapid host detection. Lett Appl Microbiol 42:259–264CrossRefGoogle Scholar
  33. Lu TK, Koeris MS (2011) The next generation of bacteriophage therapy. Curr Opin Microbiol 14:524–531CrossRefGoogle Scholar
  34. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar BA, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüssmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer K-H (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371CrossRefGoogle Scholar
  35. Matsuzaki S, Tanaka S, Koga T, Kawata T (1992) A broad-host-range vibriophage, KVP40, isolated from sea water. Microbiol Immunol 36:93–97Google Scholar
  36. Miller RC, Taylor DM, MacKay K, Smith HW (1973) Replication of T4 DNA in Escherichia coli treated with toluene. J Virol 12:1195–1203Google Scholar
  37. Miyashita A, Mochimaru H, Kazama H, Ohashi A, Yamaguchi T, Nunoura T, Horikoshi K, Takai K, Imachi H (2009) Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs). FEMS Microbiol Lett 297:31–37CrossRefGoogle Scholar
  38. Mosier-Boss PA, Lieberman SH, Andrews JM, Rohwer FL, Wegley LE, Breitbart M (2003) Use of fluorescently labeled phage in the detection and identification of bacterial species. Appl Spectrosc 57:1138–1144CrossRefGoogle Scholar
  39. Nogami T (2002) Chapter 10 - Bacteriophage method. In: Ito T, Sato J (eds) Rapid detection and measurement techniques for food microbiology. Science Forum, Abiko, Chiba, Japan, pp 215–224 (in Japanese)Google Scholar
  40. Oda M, Morita M, Unno H, Tanji Y (2004) Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage. Appl Environ Microbiol 70:527–534CrossRefGoogle Scholar
  41. Rohwer F, Thurber RV (2009) Viruses manipulate the marine environment. Nature 459:207–212CrossRefGoogle Scholar
  42. Suttle CA (2007) Marine viruses—major players in the global ecosystem. Nat Rev Microbiol 5:801–812CrossRefGoogle Scholar
  43. Tadmor AD, Ottesen EA, Leadbetter JR, Phillips R (2011) Probing individual environmental bacteria for viruses by using microfluidic digital PCR. Science 333:58–62CrossRefGoogle Scholar
  44. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  45. Tanji Y, Furukawa C, Na S-H, Hijikata T, Miyanaga K, Unno H (2004) Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage. J Biotechnol 114:11–20CrossRefGoogle Scholar
  46. Tarahovsky YS, Ivanitsky GR, Khusainov AA (1994) Lysis of Escherichia coli cells induced by bacteriophage T4. FEMS Microbiol Lett 122:195–199CrossRefGoogle Scholar
  47. Thomas JA, Soddell JA, Kurtböke DÍ (2002) Fighting foam with phages? Water Sci Technol 46:511–518Google Scholar
  48. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Sayaka Ohno
    • 1
    • 2
  • Hironori Okano
    • 1
    • 3
  • Yasunori Tanji
    • 2
  • Akiyoshi Ohashi
    • 4
  • Kazuya Watanabe
    • 5
  • Ken Takai
    • 1
  • Hiroyuki Imachi
    • 1
    Email author
  1. 1.Subsurface Geobiology Advanced Research (SUGAR) Project, Extremobiosphere Research Program, Institute of BiogeosciencesJapan Agency for Marine-Earth Science and Technology (JAMSTEC)YokosukaJapan
  2. 2.Department of BioengineeringTokyo Institute of TechnologyYokohamaJapan
  3. 3.Department of Environmental Systems EngineeringNagaoka University of TechnologyNagaokaJapan
  4. 4.Department of Social and Environmental EngineeringHiroshima UniversityHigashi-HiroshimaJapan
  5. 5.School of Life SciencesTokyo University of Pharmacy and Life SciencesHachiojiJapan

Personalised recommendations