PMID- 18359832 OWN - NLM STAT- MEDLINE DCOM- 20080527 LR - 20211020 IS - 1098-5336 (Electronic) IS - 0099-2240 (Print) IS - 0099-2240 (Linking) VI - 74 IP - 10 DP - 2008 May TI - 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. PG - 3143-50 LID - 10.1128/AEM.00191-08 [doi] AB - 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 (13)C-carbon and (15)N-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. FAU - Behrens, Sebastian AU - Behrens S AD - Department of Chemical Engineering and of Civil, Stanford University, Stanford, California 94305-5429, USA. FAU - Losekann, Tina AU - Losekann T FAU - Pett-Ridge, Jennifer AU - Pett-Ridge J FAU - Weber, Peter K AU - Weber PK FAU - Ng, Wing-On AU - Ng WO FAU - Stevenson, Bradley S AU - Stevenson BS FAU - Hutcheon, Ian D AU - Hutcheon ID FAU - Relman, David A AU - Relman DA FAU - Spormann, Alfred M AU - Spormann AM LA - eng GR - DP1 OD000964/OD/NIH HHS/United States GR - DP1OD000964/OD/NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20080321 PL - United States TA - Appl Environ Microbiol JT - Applied and environmental microbiology JID - 7605801 RN - 0 (Carbon Isotopes) RN - 0 (Nitrogen Isotopes) RN - 0 (RNA, Ribosomal, 16S) RN - 284SYP0193 (Fluorine) RN - SBV4XY874G (Bromine) SB - IM MH - Adult MH - Bacteria/classification/genetics/*metabolism MH - Biofilms MH - Bromine/metabolism MH - Carbon Isotopes/metabolism MH - Fluorine/metabolism MH - Humans MH - In Situ Hybridization/*methods MH - Isotope Labeling/*methods MH - Male MH - Microscopy, Fluorescence MH - Mouth/microbiology MH - Nitrogen Isotopes/metabolism MH - RNA, Ribosomal, 16S/genetics MH - Staining and Labeling/*methods PMC - PMC2394947 EDAT- 2008/03/25 09:00 MHDA- 2008/05/28 09:00 PMCR- 2008/11/01 CRDT- 2008/03/25 09:00 PHST- 2008/03/25 09:00 [pubmed] PHST- 2008/05/28 09:00 [medline] PHST- 2008/03/25 09:00 [entrez] PHST- 2008/11/01 00:00 [pmc-release] AID - AEM.00191-08 [pii] AID - 0191-08 [pii] AID - 10.1128/AEM.00191-08 [doi] PST - ppublish SO - Appl Environ Microbiol. 2008 May;74(10):3143-50. doi: 10.1128/AEM.00191-08. Epub 2008 Mar 21.