PMID- 22660768
OWN - NLM
STAT- MEDLINE
DCOM- 20121107
LR  - 20171116
IS  - 1432-0614 (Electronic)
IS  - 0175-7598 (Linking)
VI  - 95
IP  - 3
DP  - 2012 Aug
TI  - A method for evaluating the host range of bacteriophages using phages 
      fluorescently labeled with 5-ethynyl-2'-deoxyuridine (EdU).
PG  - 777-88
LID - 10.1007/s00253-012-4174-1 [doi]
AB  - 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.
FAU - Ohno, Sayaka
AU  - Ohno S
AD  - Subsurface Geobiology Advanced Research-SUGAR Project, Institute of 
      Biogeosciences, Japan Agency for Marine-Earth Science and Technology-JAMSTEC, 
      2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan.
FAU - Okano, Hironori
AU  - Okano H
FAU - Tanji, Yasunori
AU  - Tanji Y
FAU - Ohashi, Akiyoshi
AU  - Ohashi A
FAU - Watanabe, Kazuya
AU  - Watanabe K
FAU - Takai, Ken
AU  - Takai K
FAU - Imachi, Hiroyuki
AU  - Imachi H
LA  - eng
SI  - GENBANK/AB697981
SI  - GENBANK/AB697982
SI  - GENBANK/AB697983
SI  - GENBANK/AB697984
SI  - GENBANK/AB697985
SI  - GENBANK/AB697986
SI  - GENBANK/AB697987
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120603
PL  - Germany
TA  - Appl Microbiol Biotechnol
JT  - Applied microbiology and biotechnology
JID - 8406612
RN  - 0 (DNA, Bacterial)
RN  - 0 (DNA, Ribosomal)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - G373S00W2J (5-ethynyl-2'-deoxyuridine)
RN  - W78I7AY22C (Deoxyuridine)
SB  - IM
MH  - Bacteria/*classification/genetics
MH  - Bacteriophages/growth & development/*physiology
MH  - DNA, Bacterial/chemistry/genetics
MH  - DNA, Ribosomal/chemistry/genetics
MH  - Deoxyuridine/*analogs & derivatives/metabolism
MH  - *Host Specificity
MH  - Microbiological Techniques/*methods
MH  - Molecular Sequence Data
MH  - RNA, Ribosomal, 16S/genetics
MH  - Sequence Analysis, DNA
MH  - Staining and Labeling/*methods
EDAT- 2012/06/05 06:00
MHDA- 2012/11/08 06:00
CRDT- 2012/06/05 06:00
PHST- 2012/03/23 00:00 [received]
PHST- 2012/05/10 00:00 [accepted]
PHST- 2012/05/07 00:00 [revised]
PHST- 2012/06/05 06:00 [entrez]
PHST- 2012/06/05 06:00 [pubmed]
PHST- 2012/11/08 06:00 [medline]
AID - 10.1007/s00253-012-4174-1 [doi]
PST - ppublish
SO  - Appl Microbiol Biotechnol. 2012 Aug;95(3):777-88. doi: 10.1007/s00253-012-4174-1. 
      Epub 2012 Jun 3.