Gold-ISH: a nano-size gold particle-based phylogenetic identification compatible with NanoSIMS.
The linkage of microbial phylogenetic and metabolic analyses by combining ion imaging analysis with nano-scale secondary ion mass spectrometry (NanoSIMS) has become a powerful means of exploring the metabolic functions of environmental microorganisms. Phylogenetic identification using NanoSIMS typically involves probing by horseradish peroxidase-mediated deposition of halogenated fluorescent tyramides, which permits highly sensitive detection of specific microbial cells. However, the methods require permeabilization of target microbial cells and inactivation of endogenous peroxidase activity, and the use of halogens as the target atom is limited because of heavy background signals due to the presence of halogenated minerals in soil and sediment samples. Here, we present "Gold-ISH," a non-halogen phylogenetic probing method in which oligonucleotide probes are directly labeled with Undecagold, an ultra-small gold nanoparticle. Undecagold-labeled probes were generated using a thiol-maleimide chemical coupling reaction and they were purified by polyacrylamide gel electrophoresis. The method was optimized with a mixture of axenic (13)C-labeled Escherichia coli and Methanococcus maripaludis cells and applied to investigate sulfate-reducing bacteria in an anaerobic sludge sample. Clear gold-derived target signals were detected in microbial cells using NanoSIMS ion imaging. It was concluded that Gold-ISH can be a useful approach for metabolic studies of naturally occurring microbial ecosystems using NanoSIMS.
Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan. Electronic address: kengok@epl1.civil.tohoku.ac.jp.
Kengo
K
Kubota
Geomicrobiology Group, Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan; Geobio-Engineering and Technology Group, Submarine Resources Research Project, JAMSTEC, Natsushima-cho 2-15, Yokosuka 237-0061, Japan. Electronic address: morono@jamstec.go.jp.
Yuki
Y
Morono
Geochemical Research Group, Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan; Geobio-Engineering and Technology Group, Submarine Resources Research Project, JAMSTEC, Natsushima-cho 2-15, Yokosuka 237-0061, Japan.
Motoo
M
Ito
Department of Ocean Drilling Science Technical Support, Marine Works Japan LTD, Yokohama 236-0042, Japan.
Takeshi
T
Terada
Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
Shogo
S
Itezono
Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
Hideki
H
Harada
Geomicrobiology Group, Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan; Geobio-Engineering and Technology Group, Submarine Resources Research Project, JAMSTEC, Natsushima-cho 2-15, Yokosuka 237-0061, Japan.
Fumio
F
Inagaki
D004783:Environmental Microbiology; D006046:Gold; D008828:Microbiological Techniques / Q000379:methods*; D053758:Nanoparticles; D015345:Oligonucleotide Probes; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*
D015345:Oligonucleotide Probes; D006046:Gold
Gold-ISH; NanoSIMS ion microprobe; Undecagold