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 .
各種検索
サポート
ヘルプ