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

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 (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.

FIG. 1.

EL-FISH/NanoSIMS images of E. coli cells hybridized with probe EUB338-I. Cells were labeled with custom-synthesized fluorescently labeled tyramides containing either fluorine or bromine atoms. Secondary-electron image and corresponding image showing the relative abundance of ¹⁹F-fluorine derived from tyramide 517F (A) and tyramide 570F (B). (C) Secondary-electron image and corresponding image showing the relative abundance of ⁸¹Br derived from tyramide 544Br. (D) Secondary-electron image and ¹⁹F-fluorine distribution in negative control with no hybridization or tyramide labeling. The color scale bars indicate the relative halogen-to-carbon abundance and were adjusted to achieve optimal cell visualization while ensuring image comparability.

FIG. 2.

Fluorescence and NanoSIMS images of a microbial consortium consisting of filamentous cyanobacteria (Anabaena sp. strain SSM-00) and alphaproteobacteria (Rhizobium sp. strain WH2K) attached to heterocysts. Images taken after a 24-h incubation with ¹³C-bicarbonate and ¹⁵N-dinitrogen. (A) Fluorescence image of the microbial consortium after EL-FISH with probe ALF968. (B) NanoSIMS secondary-electron image corresponding to panels C to E. (C) Localization of fluorine relative to carbon after EL-FISH with ALF968. (D) Distribution of ¹⁵N-nitrogen enrichment. (E) Distribution of ¹³C-carbon enrichment. Color bars indicate relative fluorine abundance (C) and isotope enrichment (D and E) in the image. Het, heterocyst; Veg, vegetative cell; Epi, epibiont; unatt Epi, Epibiont cells not attached to heterocysts.

FIG. 3.

NanoSIMS images of microbial aggregates obtained from an oral biofilm from the human gingival sulcus after incubation with ¹³C-labeled amino acids and EL-FISH. (A) Secondary-electron image. (B) Distribution of ¹³C-carbon enrichment. (C) Relative abundance of ¹⁹F-fluorine to ¹²C-carbon after hybridization with probe CF319a. Arrows in panels B and C indicate probe-identified microorganisms that have incorporated the labeled substrate. Color bars indicate the relative isotopic/elemental enrichment/abundance in the image.