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Applied and Environmental Microbiology, May 2008, p. 3143-3150, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.00191-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS ^,

Departments of Chemical Engineering and of Civil and Environmental Engineering, Stanford University, Stanford, California 94305,¹ Departments of Microbiology and Immunology and of Medicine, Stanford University, Stanford, California 94305,² Glenn T. Seaborg Institute, Chemistry, Materials, Earth and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94551,³ Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019,⁴ VA Palo Alto Health Care System, Palo Alto, California 94304⁵

Received 21 January 2008/ Accepted 12 March 2008

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with ¹³C-carbon and ¹⁵N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

Published ahead of print on 21 March 2008.

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