Linking microbial phylogeny to metabolic activity at the single cell level using enhanced element labeling - catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS. Export

@article{citeulike:2587542, abstract = {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 (CARD-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 alpha-proteobacterium. 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 (13)C-carbon and (15)N-nitrogen-labled substrates, 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.}, address = {Departments of Chemical Engineering, and Civil \& Environmental Engineering, Stanford University, Stanford, CA 94305, USA; Departments of Microbiology \& Immunology, and of Medicine, Stanford University, Stanford, CA 94305, USA; Glenn T. Seaborg Institute, Chemistry, Materials, Earth and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA; Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA; VA Palo Alto Health Care System, Palo Alto, CA, 94304, USA.}, author = {Behrens, Sebastian and L\"{o}sekann, Tina and Pett-Ridge, Jennifer and Weber, Peter K K. and Ng, Wing-On O. and Stevenson, Bradley S S. and Hutcheon, Ian D D. and Relman, David A A. and Spormann, Alfred M M.}, citeulike-article-id = {2587542}, citeulike-linkout-0 = {http://dx.doi.org/10.1128/AEM.00191-08}, citeulike-linkout-1 = {http://aem.asm.org/cgi/content/abstract/74/10/3143}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/18359832}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=18359832}, day = {21}, doi = {10.1128/AEM.00191-08}, issn = {1098-5336}, journal = {Appl Environ Microbiol}, keywords = {fish, methods, sims, stable\_isotopes}, month = {March}, posted-at = {2008-05-14 11:57:42}, priority = {2}, title = {Linking microbial phylogeny to metabolic activity at the single cell level using enhanced element labeling - catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS.}, url = {http://dx.doi.org/10.1128/AEM.00191-08}, year = {2008} }

TY - JOUR ID - citeulike:2587542 L3 - citeulike-article-id:2587542 N2 - 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 (CARD-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 alpha-proteobacterium. 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 (13)C-carbon and (15)N-nitrogen-labled substrates, 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. JF - Appl Environ Microbiol SN - 1098-5336 AD - Departments of Chemical Engineering, and Civil & Environmental Engineering, Stanford University, Stanford, CA 94305, USA; Departments of Microbiology & Immunology, and of Medicine, Stanford University, Stanford, CA 94305, USA; Glenn T. Seaborg Institute, Chemistry, Materials, Earth and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA; Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA; VA Palo Alto Health Care System, Palo Alto, CA, 94304, USA. TI - Linking microbial phylogeny to metabolic activity at the single cell level using enhanced element labeling - catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS. KW - fish KW - methods KW - sims KW - stable_isotopes AU - Behrens, Sebastian AU - Lösekann, Tina AU - Pett-Ridge, Jennifer AU - Weber, Peter K AU - Ng, Wing-On AU - Stevenson, Bradley S AU - Hutcheon, Ian D AU - Relman, David A AU - Spormann, Alfred M PY - 2008/03/21/ UR - http://dx.doi.org/10.1128/AEM.00191-08 ER -