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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2008, p. 31433150 Vol. 74, No. 100099-2240/08/$08.000 doi:10.1128/AEM.00191-08Copyright 2008, American Society for Microbiology. All Rights Reserved.

Linking Microbial Phylogeny to Metabolic Activity at the Single-CellLevel by Using Enhanced Element Labeling-Catalyzed Reporter

Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMSSebastian Behrens,1 Tina Losekann,2 Jennifer Pett-Ridge,3 Peter K. Weber,3 Wing-On Ng,1

Bradley S. Stevenson,4 Ian D. Hutcheon,3 David A. Relman,2,5 and Alfred M. Spormann1*

Departments of Chemical Engineering and of Civil and Environmental Engineering, Stanford University, Stanford, California 943051;Departments of Microbiology and Immunology and of Medicine, Stanford University, Stanford, California 943052;

Glenn T. Seaborg Institute, Chemistry, Materials, Earth and Life Sciences Directorate, Lawrence Livermore National Laboratory,Livermore, California 945513 ; Department of Botany and Microbiology, University of Oklahoma, Norman,

Oklahoma 730194; and 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 communi-ties, we developed a method which combines rRNA-based in situ hybridization with stable isotope imagingbased 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 individualcells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified thecurrent catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, weobtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundanceof fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold andallowed 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 wasthen applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacte-rium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggre-gates 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 wereidentified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studiesof the ecophysiology of known and uncultured microorganisms in complex environments and communities.

Linking phylogenetic information to function of microorgan-isms in complex environmental communities is a key challengein microbial ecology. Isotope-labeling experiments provide auseful means to investigate the ecophysiology of microbialpopulations and cells in the environment (29, 47). The incor-poration of stable or radioisotopes into populations of micro-bial communities has been shown to reveal information abouttheir metabolic activities (40). Subsequent analysis of biomar-kers from cells which assimilated isotope-labeled compoundsmade it possible to link metabolic activities to the phylogeneticidentity of the respective microorganisms. Established biomar-kers in stable isotope probing experiments include phospho-

lipid fatty acids (6, 22), DNA (41), and RNA (23).The combination of fluorescence in situ hybridization

(FISH) and microautoradiography (MAR) enables the directmicroscopic observation of radioisotope incorporation into in-dividual cells within complex microbial communities (18, 30,34). However, the application of MAR-FISH is limited to

radio-isotopes with a suitable half-life and may be unsuitablefor many environmental settings. Also, the biologically relevantelements nitrogen and oxygen cannot be tracked by MAR-FISH. Recently, MAR-FISH and stable isotope probing (28,31, 49) protocols have been refined, and new, related tech-niques such as isotope arrays (2), Raman microscopy (13), andhigh-resolution nanometer-scale secondary-ion mass spec-trometry (NanoSIMS) (11) have been developed, thereby ex-panding the applications of isotope labeling experiments.These techniques have recently been reviewed by Neufeld etal. with respect to spatial resolution, sensitivity, and applicationto complex microbial communities and yet-to-be cultivated

microorganisms (29).By using secondary-ion mass spectrometry (SIMS), the iso-

topic/elemental composition of microorganisms can be deter-mined for natural abundance levels (32) or for levels afterisotopic enrichment (9). In studies of anaerobic methane-oxi-dizing archaea, SIMS was coupled with FISH to determineisotopic composition and microbial identity (32, 33, 46). Thisapproach requires the separate determination of microbialidentity by epifluorescence microscopy. However, the low spa-tial resolution of the isotopic analysis (5 m) limits its ap-plication. Recent improvements in SIMS design have in-creased sensitivity and spatial resolution (50 nm) (14) so thatit is now possible to quantify the isotopic composition of single

* Corresponding author. Mailing address: Department of Civil andEnvironmental Engineering, James H. Clark Center, 318 CampusDrive, E250, Stanford University, Stanford, CA 94305-5429. Phone:(650) 723-3668. Fax: (650) 724-4927. E-mail: [email protected].

Supplemental material for this article may be found at http://aem.asm.org/.

Published ahead of print on 21 March 2008.

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microbial cells in environmental samples (7, 17). Li et al. com-bined NanoSIMS and FISH using iodine- and fluorine-labeledoligonucleotide probes and stable isotopes of carbon and ni-trogen (19). The approach allowed the simultaneous analysisof microbial identity and stable isotope-labeled cells throughthe parallel detection of probe-conferred iodine labeling andisotopic cell composition in NanoSIMS measurements. How-ever, the environmental applicability of this approach is limitedbecause elemental labeling by standard FISH techniques isdirectly dependent on cellular ribosome content. This restrictsthe application to environments of high microbial activity. Li etal. (19) also reported that signal-to-background ratios for flu-orine after hybridization with a 19F-labeled oligonucleotideprobe were low and limited the application of this element forcell labeling.

Here, we report a new combination of FISH and NanoSIMSthat expands the applicability of the approach for environmen-tal studies. We used in situ hybridization with horseradishperoxidase (HRP)-conjugated oligonucleotide probes andtyramide signal amplification for elemental labeling of probe-

targeted cells to achieve microbial identification by Nano-SIMS. Labeled tyramide substrates were custom-synthesizedusing fluorescent dyes that contained halogen atoms. The useof tyramides labeled with both a fluorophore and with halogenatoms enabled simultaneous fluorescence and element label-ing. We named this new method EL-FISH, for element label-ing-FISH. Using EL-FISH, we increased the sensitivity of celldetection and extended the range of elements for cell labelingto bromine and fluorine. We demonstrate the methodologicalimprovement of EL-FISH/NanoSIMS with pure cultures ofEscherichia coli and Vibrio cholerae. We used the new protocolto study the metabolic interactions of a dual-species microbialconsortium consisting of a heterotrophic alphaproteobacte-

rium and a filamentous cyanobacterium. To show that EL-FISH/NanoSIMS can be applied to study single cells in com-plex microbial communities, we also applied the new methodto microbial aggregates obtained from the human gingivalsulcus.

MATERIALS AND METHODS

Culture preparation. E. coli strain K12 was grown until mid-log phase at 37Cin Luria-Bertani (LB) broth. Cells were washed twice in 1 phosphate-bufferedsaline (PBS) and fixed with 1% paraformaldehyde at room temperature for 30

min. After fixation, cells were washed twice in 1 PBS and stored in 50%ethanol1 PBS (vol/vol) at 20C until use. V. cholerae strain 92A1552 (50) was

grown at 30C in LB broth to an optical density at 600 nm of 3.0. For isotopiclabeling, cells were harvested by centrifugation at 6,000 g for 2 min, washedtwice in 1 PBS, and then resuspended in M9 mineral medium (26), which was

amended with 500 M 13C-labeled complex algal amino acid mixture (98 atom%13C; Sigma-Aldrich, MO). Cells were grown at 30C and fixed as described above

after a 6-h incubation. Pseudomonas aeruginosa strain PAO1 and Bacillus subtilisstrain JH642 were grown at 30C in LB broth to an optical density at 600 nm of1.0. Cells were fixed as described above.

A coculture ofAnabaena sp. strain SSM-00 and Rhizobium sp. strain WH2Kwas grown without agitation at 22C in 0.5 SO medium (45) under 21 to 30mol of photons/m2/s of cool, white fluorescent light. Prior to isotopic labeling,

cells were harvested by centrifugation at 6,000 g for 2 min, washed twice in0.5 SO medium without sodium bicarbonate, and then resuspendend in the

same medium. Fifty microliters of 500 mM NaH13CO3 (99 atom%13C) was

added to a clear sample vial before the addition of 1 ml of the cell suspension.Afterwards, the vial was sealed with a Teflon/silicone septum. One milliliter of15N2 (98 atom%

15N) was added to each vial with a syringe. The vials wereincubated upside down under a cool, white fluorescent light for 24 h at room

temperature. Following incubation, cells were fixed as described above.

Microbial aggregates from oral biofilms were sampled from the gingival sulcus

of a healthy individual (male, 32 years of age) after 2 days without tooth brush-

ing. These oral specimens were dispersed and suspended in dental transportmedium (Anaerobe Systems, CA). 13C-labeled complex algal amino acid mixture

(98 atom% 13C; Sigma-Aldrich, MO) was added to a final concentration of 500M. Samples were incubated for 3 h at 37C under anoxic conditions and fixedas described above.

FISH with HRP-conjugated oligonucleotides and tyramide signal amplifica-

tion. Catalyzed reporter deposition (CARD)-FISH was performed as previouslydescribed by Pernthaler et al. (38). Samples were spotted on silicon wafers (Ted

Pella, CA), air dried, and processed according to the protocol of Pernthaler et al.

(38). Hybridized samples were imaged with an epifluorescence microscope(Zeiss Axiophot; Germany) to map wafers and localize hybridized cells for

subsequent NanoSIMS measurements. Element labeling was achieved by usingcustom-labeled tyramides. Tyramides were labeled as described in Pernthaler et

al. (39). The following fluorescent dyes containing bromine and fluorine atoms

were used: 5-carboxy-2,4,5,7-tetrabromosulfonefluorescein, succinimidyl ester(544Br; emission wavelength/element label); Oregon Green 488-X, succinimidyl

ester (517F); and 6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxphenyl)-4-bora-

3a,4a-diaza-s-indacene-2-propionyl)amino) hexanoic acid, succinimidyl ester(BODIPY TMR-X; 570F). All dyes were obtained from Molecular Probes, Inc.

(Eugene, OR). HRP-conjugated oligonucleotide probes used in this study were

as follows: EUB338-I (5-GCTGCCTCCCGTAGGAGT-3) (3), a domain-spe-cific probe targeting most Bacteria; NON338 (5-ACTCCTACGGGAGGCAG

C-3) (48), a background control probe; ALF968 (5 -GGTAAGGTTCTGCGCGTT-3) (27), a probe specific to Alphaproteobacteria, except Rickettsiales; andCF319a (5-TGGTCCGTGTCTCAGTAC-3) (24), a probe targeting the Cyto-

phaga-Flavobacterium cluster of the Bacteroidetes, including some Sphingobac-

teria (20).Hybridization with a covalently halogen-Cy3-labeled probe was performed as

described previously (43). The EUB338-I oligonucleotide containing three

5-fluoro-2deoxyuridine (5Fl-dU) nucleotides was obtained from Operon Bio-technologies, Inc. (Table 1; footnote b gives the label positions).

NanoSIMS. Analyses were performed using the Lawrence Livermore NationalLaboratory NanoSIMS 50 instrument (Gennevilliers, France). Samples were

prepared by spotting cells onto a 7- by 7-mm silica wafer. Images were generatedwith a 2.6 pA Cs primary beam, focused to a nominal spot size of150 nm, and

stepped over the sample in a 256- by 256-pixel raster to generate secondary ions.Dwell time was 1 ms/pixel, and raster size ranged between 5 by 5 and 30 by 30

m. Samples were presputtered at high beam currents (1 nA) to a depth of100 nm before measurements to achieve sputtering equilibrium. Secondaryions were detected in the simultaneous collection mode by pulse counting to

generate 20 to 60 serial quantitative secondary-ion images (i.e., layers); thenumber of layers was adjusted according to the scanning frame to ensure com-

parable coverage between analyses with different raster sizes.For analyses where cells were targeted with fluorine (F)-labeled tyramides,

electron multiplier detectors were positioned to collect 12C, 13C, 19F,12C14N, and 12C15N ions. For bromine (Br)-targeted cells, electron multipliersettings were 12C12C, 12C13C, 12C14N, and 81Br. Nitrogen in the sample

was detected as the CN cluster ion. Samples were also imaged simultaneouslyby secondary electrons. The secondary mass spectrometer was tuned for 6,800

mass resolving power to resolve isobaric interferences. The depth of analysisduring a measurement ranged from 200 to 900 nm. For pure cultures, 5 to 20 cells

were analyzed individually, and measurements were repeated on selected cells toensure measurement accuracy. For the samples with mixed cell types, analyses

were conducted in 5 to 10 different locales.

Data were processed as quantitative isotopic ratio images using custom soft-

ware and were corrected for effects of quasi-simultaneous arrival, detector deadtime, and image shift from layer to layer. Each cell was defined as a region ofinterest by encircling pixels with 12C14N counts of 30% of the maximum

counts in the image. The isotopic composition and/or relative abundance ofhalogen ions present in each region of interest was calculated by averaging over

all replicate layers where both C and N isotopes were at sputtering equilibrium.Halogen ion (19F and 81Br) counts are normalized to 12C.

A B. subtilis spore preparation was used as a reference standard for the C and

N isotopic measurements (13C/12C 0.0110; 15N/14N 0.00370). Isotopic en-richment of standards was independently determined at the University of Utah.

Measurement precision, internal, was 0.4 to 1.4% (2) for individual13C/12C and

15N/15N measurements, and replicate analyses of the standard yielded an ana-

lytical precision, std, of 2.1% (2) for an individual measurement. Isotopeenrichment data are presented as atom percent excess (APE); calculations of

APE and associated precision for replicate analyses follow the example in Popa

et al. (40). APE is calculated based on the initial isotopic ratios of the organism

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at time zero (Ri) and the isotopic ratio of the sampled organism (Rf): APE

[Rf/(Rf 1) Ri/(Ri 1)] 100%.

RESULTS

CARD-FISH is an rRNA-based technique for fluorescentlystaining microorganisms that has broad applicability in micro-bial ecology. We demonstrate that CARD-FISH can be used tolabel microbial cells with halogen atoms, such as bromine andfluorine, in order to identify single cells phylogenetically in asensitive manner by NanoSIMS analysis.

Element labeling of microorganisms with CARD-FISH. Inorder to identify microorganisms by NanoSIMS, we increasedthe intracellular abundance of fluorine and bromine, whichhave a relatively low natural abundance in biological samples(relative fluorine and bromine abundances in tissue are 0.05and 7 ppm, respectively [10]). We chose the halogens fluorineand bromine, but the application of other elements/isotopes isalso feasible (e.g., the relative iodine abundance in tissue is0.05 to 0.7 ppm) (10, 19). We used rRNA-targeted FISH withHRP-conjugated oligonucleotide probes to introduce the ele-ment label selectively into probe-targeted cell populations. El-ement labeling was achieved by the use of tyramides that con-tained halogen atoms. Halogen-labeled tyramides were customsynthesized using fluorescent dyes, such as Oregon Green(Molecular Probes, Inc.), which contains two fluorine atoms.The fluorescent tyramides were deposited inside cells by thecatalytic activity of the probe-bound HRP. In this way cells thathybridized with the selected probe were labeled with bothstable halogen isotopes and fluorescent dye molecules, allow-ing identification of these cells in mixed communities by bothNanoSIMS and epifluorescence microscopy.

We tested the CARD-FISH/NanoSIMS protocol by per-forming hybridizations with E. coli and V. cholerae cells. Todemonstrate the applicability of different elemental labels, wehybridized E. coli cells with a general probe for the domainBacteria (EUB338-I) and performed signal amplification with

different tyramides. Three fluorescent dyes with different op-tical properties and halogen content were chosen for tyramidelabeling. Figure 1 and Table 1 summarize the results of thelabeling of E. coli with fluorine- and bromine-containing tyra-mides. All tyramides were applied under standard conditionsas described by Pernthaler et al. (39). Cellular abundance ofthe respective halogen was expressed relative to 12C-carbon.We found that hybridized cells in comparison with nonhybrid-ized background control cells had higher relative ratios ofhalogen to carbon (Fig. 1). The signal amplification with tyra-mide 517F was most efficient. The probe-conferred fluorinesignal was 180-fold above the natural background of fluorine tocarbon (Table 1). Use of tyramide 570F resulted in an eight-

fold increase over the fluorine-to-carbon background ratio. A20-fold increase in relative bromine abundance was achieved inE. coli cells labeled with tyramide 544Br (Table 1).

In contrast, EL-FISH with rRNA-targeted oligonucleotideprobes carrying a single fluorescent dye molecule at the 5 endand a number of halogen-containing nucleotides, such as 5Fl-dU,increased the cellular fluorine-to-carbon ratios only twofold.Hence, the signal-to-noise ratio of the EL-FISH approach overthe standard FISH technique is increased 10- to 100-fold, depend-ing on the fluorescent tyramide and element label used (Table 1).

To confirm that the increased relative elemental halogenabundance was due solely to probe hybridization, we hybrid-ized a background control probe (NON338) to 13C-enriched V.

TABLE 1. Relative fluorine and bromine abundance ofE. coli and V. cholerae cells after FISH

Method, probe, and/ordye (n)a

No. ofsinglecells

analyzed

Relative halogen abundance of a set of single cellsfSignal/background

ratio

19F/12C 19F/14N 81Br/12C 81Br/14NHalogen/

CHalogen/

N

Standard FISH withhalogen-containing

probe EUB338-Iwith Cy3 and5Fl-dUb

16 4.5 103 1.3 104 5.0 103 1.2 104 2 6

EL-FISH with differenttyramides onE. colic

517F 7 4.1 101 1.4 102 2.3 101 4.2 103 181 265570F 6 1.8 102 4.6 104 1.1 102 2.1 104 8 13544Br 11 1.8 102 1.4 104 6.5 103 8.7 105 22 21Background controld 2.3 103 7.0 105 8.8 104 4.2 105 8.1 104 2.1 105 3.1 104 1.3 105 1 1

EL-FISH on Vibrio sp.e

EUB338-I 22 8.4 101 1.8 102 2.2 101 6.0 103 42 37NON338 9 2.0 102 9.0 104 6.0 103 2.0 103 1 1

a Standard FISH, fluorine-labeling of E. coli cells with directly fluorine-labeled oligonucleotide probes; EL-FISH on E. coli, comparison of different halogen-containing tyramides after EL-FISH on E. coli cells with probe EUB338-I; EL-FISH on Vibrio sp., comparison of positive (probe EUB338-I) and negative (probe

NON338) control hybridizations on V. cholerae cells.b 5Fl-dU nucleotides were substituted for deoxythymidine nucleotides. The EUB338-I probe sequence was Cy3GC5Fl-dUGCC5Fl-dUCCCG5Fl-dUAGGAGT. The probe was Cy3 labeled for hybridization control by epifluorescence microscopy.

cAll hybridizations performed with HRP-conjugated EUB338-I probe. See Materials and Methods section for tyramide abbreviations. Tyramide signal amplificationwas for 10 min at 46C with a 1:500 dilution of tyramide stock.

d Negative control. Analysis was performed on fixed cells without hybridization and tyramide signal amplification.e Hybridizations performed with HRP-conjugated EUB338-I or NON338 probe. Tyramide signal amplification using tyramide Oregon Green 488-X (517 F). Mass

images provided in supplemental data.fNumbers given in the table are average values with standard errors for the number of single cells analyzed.

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cholerae cells. After hybridization with the background controlprobe and incubation with the tyramide 517F, V. cholerae cells

did not show elevated fluorine abundance (Table 1; see alsoFig. S1 in the supplemental material). Hybridization with aBacteria-specific probe (EUB338), on the other hand, resultedin a 42-fold increase of intracellular fluorine abundance (rel-ative to 12C-carbon) compared with NON338 background con-trol levels (see Fig. S1 in the supplemental material). We alsotested whether our technique allowed for specific discrimina-tion of microbes with mismatches at the probe target site. Wehybridized cultures of the gram-positive B. subtilis (1.3 weightedmismatches) and the gammaproteobacterium P. aeruginosa (1.5weighted mismatches) with the oligonucleotide probe CF319a(21). Nonspecific FISH staining or fluorine enrichment ofthese strains by probe CF319a was not observed (see Fig. S2 inthe supplemental material).

Dual-species microbial consortium (Anabaena and epibiont).

Having shown that EL-FISH can be used to increase relativecellular halogen abundance in probe-targeted cells for cellidentification by NanoSIMS, we combined the phylogeneticisotope labeling with visualization of metabolic activity. Wechose a dual-species microbial consortium consisting of a fila-mentous cyanobacterium and a heterotrophic alphaproteobac-terium (45) to examine whether the combination of EL-FISH/NanoSIMS can be used both to identify microbial partnersphylogenetically and to monitor the intra- and intercellularfates of carbon and nitrogen. The distinct morphology of thetwo microbial species in this sample served as a visual controlof label distribution.

Associations of heterotrophic bacteria and photosyntheticcyanobacteria occur in a variety of aquatic habitats (36). These

interactions have been described as mutualistic, with fitnessbenefits for both associated species (35). The heterotrophicbacterium (epibiont) attaches to the cyanobacterial heterocyst,which the supplies the epibiont with organic carbon and nitro-gen compounds (35). The respiratory activity of the epibiont isbelieved to decrease local oxygen concentrations and produceCO2, thereby stimulating photosynthetic growth of the cya-nobacterium (42).

We used pure cultures and cocultures of Anabaena sp. strainSSM-00 and Rhizobium sp. strain WH2K. To investigate whether the physical interaction between the Anabaena andthe heterotrophic epibiont involves the transfer of metabo-lite(s), we incubated the pure cultures and the coculture with13C-bicarbonate (25 mM; 99 atom% 13C) and 15N-dinitrogen(70% 15N2 of total N2; 98 atom%

15N). Anabaena sp. strainSSM-00 fixed carbon and nitrogen in the absence of the Rhi-zobium species, while the Rhizobium sp. strain WH2K did notshow any 13C-bicarbonate and 15N-dinitrogen incorporation inpure culture (data not shown). PCRs using epibiont DNA anddegenerate primers targeting the nifH gene, which encodes anitrogenase subunit, did not result in a product (45).

When the Rhizobium species was grown in coculture, isoto-pically labeled carbon and nitrogen were detected, suggestingthat carbon and nitrogen compounds fixed by the cyanobacte-rium were assimilated by the epibiont (Fig. 2D and E). In Fig.2D newly fixed 15N-nitrogen is apparent in all three cell types,the heterocyst, vegetative cells, and epibionts. As previously

FIG. 1. EL-FISH/NanoSIMS images ofE. coli cells hybridized with probe EUB338-I. Cells were labeled with custom-synthesized fluorescentlylabeled tyramides containing either fluorine or bromine atoms. Secondary-electron image and corresponding image showing the relative abundanceof 19F-fluorine derived from tyramide 517F (A) and tyramide 570F (B). (C) Secondary-electron image and corresponding image showing therelative abundance of81Br derived from tyramide 544Br. (D) Secondary-electron image and 19F-fluorine distribution in negative control with nohybridization or tyramide labeling. The color scale bars indicate the relative halogen-to-carbon abundance and were adjusted to achieve optimalcell visualization while ensuring image comparability.


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shown, the export of newly fixed nitrogen from the heterocystto vegetative cells occurs rapidly (12, 25, 40), resulting in ahigher relative enrichment of newly fixed 15N-nitrogen in thevegetative cells than in the heterocyst and epibionts (Fig. 2D).Carbon dioxide fixation through oxygenic photosynthesis en-riched the vegetative cells in 13C-carbon (Fig. 2E). Enrichmentof13C-carbon was also visible in epibiont cells attached to theheterocyst (Fig. 2E). 13C-carbon enrichment in the heterocystswas relatively low, consistent with heterocysts being nongrow-ing and devoid of photosystem II and of autotrophy (Fig. 2E).

We used EL-FISH to label one partner of the dual speciesconsortium with fluorine to enable visualization by NanoSIMS(Fig. 2A). The power of elemental and isotopic imaging byNanoSIMS was revealed when images corresponding to differ-ent isotope masses were compared. Figure 2C shows the se-lective labeling of the epibiont cell with fluorine after in situhybridization of the consortium with an alphaproteobacteriaprobe (ALF968). Anabaena cells, which were not targeted bythe alphaproteobacteria probe, contained only backgroundlevels of fluorine. Rhizobium cells not attached to a heterocystdid not show 13C or 15N enrichment (Fig. 2D and E). However,these cells were visible in secondary electron images and de-tected with EL-FISH because of their increased fluorine abun-dance (Fig. 2B and C). These findings show that EL-FISH

enables cell detection and phylogenetic identification indepen-dent of the cells metabolic state.

Multispecies microbial aggregates obtained from oral bio-

films. To demonstrate the applicability of our method for theidentification of metabolically active microbial cells in complexmicrobial communities, we examined aggregates obtainedfrom an oral biofilm from the human gingival sulcus. Oralbiofilms encompass more than 750 bacterial species, of whichapproximately 40% can be cultivated while the remaining spe-cies have only been identified by sequencing of their 16S rRNAgenes (1). Microorganisms belonging to the Cytophaga-Fla-vobacterium cluster of the Bacteroidetes are present in oralbiofilms (8). The predominant genera Capnocytophaga, Pre-votella, and Porphyromonas have been implicated in the patho-genesis of periodontal disease (37, 44).

Microbial aggregates from oral biofilms were incubated in amineral medium with 500 M 13C-labeled amino acids for 3 h,fixed, and hybridized with a group-specific Cytophaga-Flavo-bacterium probe (CF319a) (24). Following CARD-FISH, car-bon and fluorine enrichment was visualized by NanoSIMS(Fig. 3). The probe-targeted cell population was identified byelevated intracellular fluorine levels. By comparing the 13CAPE image (Fig. 3B) with the fluorine image (Fig. 3C), Cyto-phaga-Flavobacterium species were identified as metabolizing

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 and15N-dinitrogen. (A) Fluorescence image of the microbial consortium after EL-FISH with probe ALF968. (B) NanoSIMS secondary-electron imagecorresponding to panels C to E. (C) Localization of fluorine relative to carbon after EL-FISH with ALF968. (D) Distribution of15N-nitrogenenrichment. (E) Distribution of13C-carbon enrichment. Color bars indicate relative fluorine abundance (C) and isotope enrichment (D and E) inthe image. Het, heterocyst; Veg, vegetative cell; Epi, epibiont; unatt Epi, Epibiont cells not attached to heterocysts.


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the 13C-labeled amino acid because of their enrichment in both13C and 19F (mean of 19F/12C ratio for 13C-enriched cells,0.0891 0.0059; mean of background except 13C-enrichedcells, 0.0698 0.0007) (Fig. 3, arrows). Figure 3 also showsthat microorganisms other than the probe-targeted Cytophaga-Flavobacterium species metabolized 13C-labeled amino acids.Notably, not all cells with elevated fluorine were 13C enriched.The mostly slender, long filamentous cells were Cytophaga-Flavobacterium species that did not metabolize 13C-labeledamino acids. Cells that were both enriched in 13C-carbon andexhibited a relative increase in fluorine abundance mostly hada rod shape or coccoid morphology.

DISCUSSION

We developed a FISH protocol (EL-FISH) to label probe-targeted cell populations in a specific manner with the halo-gens bromine and fluorine to enable rRNA-based cell identi-fication by NanoSIMS. In combination with stable isotopetracer experiments, we obtained functional and phylogeneticinformation simultaneously from individual cells in microbialcommunities in a single NanoSIMS analysis.

Tyramides containing a fluorophore and halogen atomsproved to be very useful during sample preparation becausethey enabled direct correlation of fluorescence and halogensignals. First, samples were imaged by epifluorescence or con-focal laser scanning microscopy for specific probe hybridiza-tion signal. Fluorescence images obtained from microbial pop-ulations and single cells helped guide subsequent NanoSIMSanalyses to spots of interest on the silicon wafer. By analyzingfluorescence first, optimization of in situ hybridization wasachieved simultaneously with sample preparation for Nano-SIMS and obviated the need for additional experimental ef-forts.

EL-FISH offers advantages in the sensitivity of detectionover standard FISH techniques. In many environmental sam-ples, cell detection by rRNA-directed in situ hybridization ishampered by the low ribosome content of target microorgan-isms. When directly labeled oligonucleotide probes are used,fluorescence signal intensity and element-labeling efficiencydepend on the cellular ribosome content. Using EL-FISH,

deposition of fluorescent dye and element label is mediated by

the catalytic activity of the probe-conjugated HRP. The enzy-matic signal amplification reaction allows the adjustment ofprobe-conferred signal intensities by variations in tyramideconcentration and incubation times. Thus, cells from oligotro-phic environments, which have low ribosome contents, caneffectively be enriched with halogens to allow identification byNanoSIMS. We showed that elements of relatively low abun-dance in biological tissue, such as bromine and fluorine (10),can be effectively used for EL-FISH cell labeling. The EL-FISH halogen labeling takes advantage of the very high sensi-tivity of NanoSIMS for fluorine and bromine.

The rRNA-based element labeling of individual microbialcells for cell identification by NanoSIMS does not require the

modification of existing CARD-FISH protocols. The only al-teration is the use of halogen-containing tyramides. Manyhalogen-containing fluorescent dyes are commercially avail-able for custom tyramide conjugate synthesis and can be usedfor NanoSIMS analysis. In contrast, oligonucleotide probes,which are covalently modified through the addition of halogenatoms, might exhibit an altered melting behavior, dependingon the number and physiochemical properties of the addedelement. While hybridizations with these probes might be lesschallenging, their altered elemental composition will requireadditional controls and optimization of the hybridization con-ditions and is associated with lower detection sensitivity.

We conducted in situ hybridizations with pure cultures ofE.

coli to characterize fluorine and bromine labeling of cells byEL-FISH (Table 1). The relative halogen abundance obtainedafter EL-FISH was high compared to the natural backgroundof the respective halogen in nonhybridized cells (Fig. 1). Wealso compared the elemental labeling efficiency of EL-FISHwith standard FISH and showed that standard FISH resultedin lower cellular halogen abundance than EL-FISH whenoligonucleotide probes with three 5Fl-dU nucleotides wereused (Table 1).

Successful binding of the HRP-oligonucleotide conjugate toits probe sequence-defined ribosomal target site is a prerequi-site for enzyme-mediated tyramide deposition. Control hybrid-ization experiments with 13C-enriched V. cholerae cells showed

FIG. 3. NanoSIMS images of microbial aggregates obtained from an oral biofilm from the human gingival sulcus after incubation with13C-labeled amino acids and EL-FISH. (A) Secondary-electron image. (B) Distribution of 13C-carbon enrichment. (C) Relative abundance of19F-fluorine to 12C-carbon after hybridization with probe CF319a. Arrows in panels B and C indicate probe-identified microorganisms that haveincorporated the labeled substrate. Color bars indicate the relative isotopic/elemental enrichment/abundance in the image.


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that the use of a background control probe (NON338) resultedin about 40-fold lower intracellular fluorine abundance and nodetected halogen background (Table 1; see also Fig. S1 in thesupplemental material).

We also demonstrated that combined EL-FISH/NanoSIMSprovides information on metabolic activity of single cells andoffers insights into the distribution of microbial activities in andamong individual cells of probe-identified populations. Ourmethod will facilitate studies of the phenotypic response ofindividual cells to environmental perturbations and enable thequantitative description of cellular behavior.

EL-FISH has the potential to be extended to mRNA andprotein detection. This would enable the simultaneous visual-ization and quantification of multiple cellular characteristics when information on transcriptional and translational activityis integrated with carbon/nitrogen distribution and cellularidentification. Key enzymes of metabolic pathways can reveal acells principle mode of energy conservation (e.g., sulfate re-duction and methanogenesis) or allow identification of theassimilatory pathway that is used for the synthesis of new

biomass.Given the high spatial resolution of NanoSIMS (50 nm), it

should be possible to study intracellular distribution and local-ization of cellular activity (cytoplasmic, membrane bound, andperiplasmic) by isotope tracer experiments or labeling of cel-lular features through EL-FISH or immunostaining. The de-tection of multiple cell features or different 16S rRNA phylo-types at the same time may require additional elemental labels.We have successfully established the use of bromine and flu-orine. Li et al. report the application of iodine (19). Selenium,boron, gold, and silver might be other suitable elements forcellular labeling because of their relatively low natural abun-dance in biomass (10). Subsequent application of different

probes and substrates has been demonstrated for CARD-FISH, and a combination of the technique with standard FISHis feasible (4, 5, 46). Further experiments will evaluate the useof multiple elements for simultaneous identification of differ-ent microbial populations or labeling of different intracellularfeatures, such as mRNA and proteins, based on EL-FISH/NanoSIMS.

EL-FISH/NanoSIMS combines features of high-resolutionmicrobial imaging techniques, stable isotope probing, and mi-crobial identification methods that rely on molecular biomar-kers. This combination of techniques will facilitate the study ofhitherto uncultivated microorganisms in diverse environmentalcommunities and will reveal insights into the interrelationshipsof individual microbial community members (15, 16).

ACKNOWLEDGMENTS

We thank Larry Nittler (Carnegie Institute of Washington) for soft-ware development.

Work was funded in part by the U.S. Department of Energy Officeof Biological and Environmental Research Genomics: GTL researchprogram (J.P.-R. and P.K.W.) and grants from NIH (DP1OD000964 toD.A.R.). This work was performed under the auspices of the U.S.Department of Energy by Lawrence Livermore National Laboratoryunder contract DE-AC52-07NA27344. T.L. was supported by a Stan-ford Deans Postdoctoral Fellowship. D.A.R. is a recipient of an NIHDirectors Pioneer Award and a Doris Duke Distinguished ClinicalScientist Award. This work was supported by grants from NSF (Mi-crobial Genetics) and SERDP to A.M.S.

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