Journal
SYSTEMATIC AND APPLIED MICROBIOLOGY
Volume 37, Issue 4, Pages 261-266Publisher
ELSEVIER GMBH, URBAN & FISCHER VERLAG
DOI: 10.1016/j.syapm.2014.02.003
Keywords
Gold-ISH; NanoSIMS ion microprobe; Undecagold
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Funding
- Japan Society for the Promotion of Science (JSPS)
- Strengthening Leading Edge Research and Development
- JSPS Funding Program for Next Generation World-Leading Researchers (NEXT Program)
- JSPS [24687004, 25630221]
- Japan Science and Technology Agency (JST)/Japan International Cooperation Agency (JICA) Science and Technology Research Partnership for Sustainable Development (SATREPS)
- Grants-in-Aid for Scientific Research [25630221, 26251041, 24687004] Funding Source: KAKEN
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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 C-13-labeled Escherichia colt 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. (C) 2014 Elsevier GmbH. All rights reserved.
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