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rRNA probe-based cell fishing of bacteria

Marion Stoffels, †* Wolfgang Ludwig andKarl Heinz SchleiferLehrstuhl fur Mikrobiologie,Technische Universitat Munchen, Arcisstraße 16,D-80290 Munich, Germany.

Summary

We have developed a new, cultivation-independent,fast and flexible method for the rRNA-targeted probe-based enrichment of bacteria. The target cells werelabelled by in situ hybridization with biotinylated poly-ribonucleotide probes. These probes were generatedby in vitro transcription of amplified rDNA of a vari-able region in domain III of the 23S rRNA molecules.The probes were about 300 nucleotides in lengthand were labelled by incorporation of biotin-UTP dur-ing the transcription. Probes were hybridized withbacterial cells and incubated with paramagneticstreptavidin-coated particles. The labelled targetcells can be separated in a column filled with steelwool inserted into the field of a permanent magnet.Unlabelled, non-target cells pass through the column,whereas labelled cells are retained. They were elutedfrom the column after removal of the magnetic field.Up to now, the method has been tested with mixturesof different pure cultures. For the first time, transcriptprobes have been used for the labelling of the targetcells and for their specific separation. The enrichmentof the target cells can be monitored by a streptavidin–fluorescein staining of the biotinylated target cellsand/or by a subsequent in situ hybridization withfluorescently labelled oligonucleotide probes. Enrich-ment rates of up to 90-fold, depending on the originalabundance of the cells of interest, could be deter-mined. To demonstrate that the sorted cells wereamenable to molecular analysis, we amplified andsequenced a part of the tuf gene of enriched Acineto-bacter calcoaceticus cells.

Introduction

The analysis of the structure and function of microbialcommunities is at the centre of interest of microbial ecology.Therefore, the identification and characterization of bac-teria play an important role. Given that only 1% of bacteriain ecosystems such as soil are culturable (Amann et al.,1995), it is necessary to develop new culture-independentmethods to obtain more information about the contributionof microorganisms to the different ecosystems.

Today, the rRNA approach is a powerful tool for analys-ing the structure of microbial communities in a cultivation-independent way (Amann et al., 1995). This approachconsists of two parts: in the first step, 16S rRNA genesof an environmental sample are selectively amplified bypolymerase chain reaction (PCR) with universal or con-served oligonucleotide primers (Springer et al., 1993).The gene fragments in the resulting mixture are separatedby cloning, the sequences are determined and a compara-tive analysis of the retrieved sequences is performed. Inthe second phase, sequence-specific hybridization probesare designed for identification, quantification and visualiz-ation of the spatial distribution of the whole fixed cells inthe original sample. Nowadays, the combination of thesetwo rRNA-based techniques is commonly used for theinvestigation of different environments, such as activatedsludge (Snaidr et al., 1997), soil (Ludwig et al., 1997),lake snow (Huber, 1997) and bacterial endosymbionts(Amann et al., 1991).

The cyclic rRNA approach offers the potential to eluci-date the phylogenetic positions of so far unculturablemicroorganisms and to analyse their distribution overspace and time in complex ecosystems, but it is not usuallypossible to gain information about the physiological poten-tial of the organisms and their role and function in thesystem. A helpful way out of this dilemma would be a cul-ture-independent enrichment of the target organism. Asubsequent genome analysis would offer the possibilityof gaining insights into the genetic properties and physio-logical potential of hitherto unculturable microorganisms.

For the selective enrichment and/or isolation of bac-teria, different methods are available. Besides the classi-cal enrichment on selective cultivation media, molecularmethods have become more and more applicable.

Immunomagnetic separation has been shown to be anefficient and sensitive method for the enrichment and iso-lation of cells in different medical (Luk and Lindberg, 1991;Drancourt et al., 1992; Islam and Lindberg, 1992), food

Environmental Microbiology (1999) 1(3), 259–271

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Received 25 November, 1998; revised 4 February, 1999; accepted 8February, 1999. †Present address: Institut fur Bodenokologie, GSF-Forschungszentrum fur Umwelt und Gesundheit GmbH, IngolstadterLandstraße 1, D-85764 Neuherberg, Germany. *For correspondence.E-mail [email protected]; Tel. (þ49) 89 3187 3415; Fax (þ49) 893187 3376.

(Skjerve et al., 1990; Vermunt et al., 1992; Cudjoe et al.,1993) and environmental samples (Christensen et al.,1992; Mitchel et al., 1994). Paramagnetic beads coatedwith antibodies against the target bacteria are used tolabel the cells. After the incubation, the labelled targetcells can be separated from the unlabelled cells in a strongmagnetic field. However, the technique is limited by therequirement for pure cultures of target organisms for theproduction of specific antibodies.

Flow cytometry (FCM) is another method that has beenapplied successfully for the enrichment of target cells.This technique allows the rapid analysis (more than 103

cells s¹1) and sorting of single cells (Wallner et al., 1995).Various FCM criteria, such as cell size, morphology,DNA content and specific staining conferred by fluorescentantibodies (Porter et al., 1993) or rRNA targeted oligonu-cleotide probes, allow the deliberate sorting of cell popula-tions of interest from highly diverse systems. In a recentstudy (Wallner et al., 1997), this approach was used to col-lect cells selectively from activated sludge, lake water andlake sediment for molecular analysis.

The applicability of optical trapping and manipulation ofbacterial cells and viruses with infrared lasers has beendemonstrated by Ashkin and Dziedzic (1987). Micro-manipulation has also been applied to collecting individualcells from Epulopiscium fishelsoni, an extremely largebacterium from ethanol-fixed gut contents of a surgeon-fish, for phylogenetic analysis (Angert et al., 1993).

As all these methods were either not suitable for theenrichment of uncultured bacteria or require expensiveequipment, the aim of this study was to develop a newenrichment method. This method should combine theadvantages of in situ hybridizations with those of magneticseparations. The underlying idea was to label target cellsby an in situ hybridization with biotinylated transcriptprobes targeted against a hypervariable region of the23S rRNA and then to separate the cells in a strongmagnetic field after incubation with streptavidin-coatedparamagnetic beads. In this study, it is shown that thisprobe-based cell fishing (PCF) concept can be usedspecifically to enrich bacterial cells for subsequentmolecular analysis and genomic investigations.

Results

In vitro transcription and specificity of thetranscript probes

By using T3 RNA polymerase biotin-labelled transcriptprobes for Acinetobacter calcoaceticus and Burkholderiacepacia were produced. The yields of transcript probeswere about 10–27mg for a template concentration of 1–2mg.The labelling and specificity of the probes were determinedby whole-cell hybridizations of paraformaldehyde-fixed

cells immobilized on glass cover slides. The hybridized tar-get cells were detected with fluorescein-labelled streptavidinand epifluorescence microscopy. As observed by Trebesiuset al. (1994) for in situ hybridizations with digoxygenin- ordirect fluorescently labelled polyribonucleotide probes, thefluorescence was unevenly distributed over the hybridizedcells after biotin detection. Brightly fluorescent rings markedthe cell peripheries, whereas the cell centres were onlyweakly fluorescent. Probe specificity was strongly depen-dent on the hybridization conditions and was checked byvarying the formamide concentration in the hybridizationbuffer as well as the hybridization temperature, while keep-ing the NaCl concentration in the hybridization buffer con-stant. Table 1 shows the specificity of probe BcepDIII,generated by using B. cepacia DNA at stringent hybridiza-tion conditions (95% formamide, 588C). By applying theseconditions, the transcript probe hybridized to B. cepaciaand B. vietnamiensis, which share the same targetsequence within domain III (M. Stoffels, unpublished results)and, in addition, to B. cocovenenans, B. plantarii and B. gla-dioli. The specificity of the probe Aca23III, generated byusing A. calcoaceticus DNA, was demonstrated by Ludwiget al. (1994) using radioactive dot blot hybridizations.

Separation of the hybridized target and non-targetcells using the Dynal system

The first experiments for the magnetic separation ofhybridized target cells were performed with the magnetic

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Table 1. Specificity of the transcript probe DIIIBcep (biotin-labelled,detection with streptavidin–fluorescein) under stringent hybridizationconditions (95% formamide in the hybridization buffer, hybridizationtemperature 588C).

Strain FISHBurkholderia cepacia LMG 1222T þ

Burkholderia vietnamiensis LMG 10929T þ

Burkholderia cocovenenans LMG 11626T þ

Burkholderia plantarii LMG 9035 þ

Burkholderia gladioli LMG 2216 þ

Burkholderia caryophyli LMG 2155T –Burkholderia glumae LMG 2196T –Burkholderia andropogonis LMG 2129T –Alcaligenes faecalis LMG 1229T –Alcaligenes ruhlandii DSM 653 –Ralstonia solanacearum LMG 2299T –Acidovorax temperans LMG 7169T –Acidovorax avenae LMG 2117T –Hydrogenophaga palleronii LMG 2366T –Hydrogenophaga taeniospiralis LMG 7170T –Aquaspirillum metamorphum DSM 1837T –Neisseria canis LMG 8383T –Neisseria elongata LMG 5124T –Chromobacterium violaceum LMG 1267T –Brevundimonas diminuta DSM 1635 –Sphingomonas paucimobilis LMG 1227T –Acinetobacter calcoaceticus ATCC23055T –Escherichia coli ATCC 11775T –Bacillus sphaericus DSM 28T –Microbacterium imperiale DSM 20530T –

260 M. Stoffels, W. Ludwig and K. H. Schleifer

cell sorting system of Deutsche Dynal. The biotinylatedtarget cells were labelled with streptavidin-coated para-magnetic Dynabeads (2.8 mm diameter). Then the beadsand bead–cell complexes were bound to the wall of atube using a magnetic particle concentrator (Dynal MPC).The supernatant was removed carefully, and the cell–particle complexes were washed and concentrated twice(using the Dynal MPC). Microscopic investigation of thedifferent fractions did not show a significant enrichmentor depletion of the target cells. No cells were attached tothe surface of the beads. The DynaBeads (2.8 mm diameter)used are larger than the bacteria. Microscopic examina-tion was required at several levels of focusing. Further-more, the detection/observation of cells that werehybridized with fluorescently labelled oligonucleotide probeswas hampered by the strong autofluorescence of the beads.

Separation of the hybridized target andnon-target cells using the MiniMACS(magnetic cell separation system)

Using the MiniMACS (Fig. 1), the labelled target cells could beenriched successfully (Table 2). The enrichment was per-formed as described in Experimental procedures. In contrastto the Dynabead system, the MiniMacs microbeads did notdisturb the analysis by autofluorescence. Given their smalldiameter (50 nm), they are not visible by light microscopy.

Effectiveness of the enrichment

The enrichment experiments were performed with tran-script probes of domain III of the 23S rRNA of B. cepacia

and A. calcoaceticus and different mixtures of pure cul-tures. To test the general applicability of the method, theinitial concentrations of the target cells were varied in arange between 6% and 95%. The target cells could be


Fig. 1. Separation of the hybridized target cells using the Miltenyicell separation system (MiniMACS).

Table 2. Results of the probe-based cell fishing (PCF) experiments.

Fraction 1 Depletion Fraction 2 Enrichment Fraction 7Experiment Organisms (initial fraction) rate (depletion of target cells) rate (enrichment of target cells)

Enrichment of B. cepaciaA B. cepacia 38.0% 25 2.4% 19 92.0%

N. canis 62.0% 97.6% 8.0%B1 B. cepacia 6.0% 32 0.2% 21 56.7%

N. canis 94.0% 99.8% 43.3%B2 B. cepacia 6.0% 128 0.05% 23 59.4%

N. canis 94.0% 99.95% 40.6%C B. cepacia 95% 127 13.0% 26 99.8%

B. sphaericus 5% 87.0% 0.2%D B. cepacia 6.1% 32 0.2% 87 85.0%

N. canis 88.1% 95.1% 12.5%B. sphaericus 5.8% 4.7% 2.5%

Enrichment of A. calcoaceticusE A. calcoaceticus 24.6% 19 1.7% 90 96.7%

B. cepacia 26.7% 47.1% 3.3%B. diminuta 48.7% 51.2% 0%

F A. calcoaceticus 20.8% 52 0.5% 18 82.5%B. diminuta þ M. imperiale 79.2% 99.5% 17.5%

Initial concentrations of target and non-target cells as well as depletion and enrichment are given for target and non-target cells respectively.Fraction 1, fraction before performing the magnetic separation of the labelled target cells.Fraction 2, fraction containing the cells that were not retained in the magnetized column.Fraction 7, fraction containing the cells that were eluted from the column after it was removed from the magnetic field.

rRNA probe-based cell fishing of bacteria 261

reasonably enriched in all cases, indicating the effective-ness of the new method. The fractions of target cells ineach sample before and after the separation as well asthe depletion and enrichment rates are given in Table 2.The magnetic separations of the target cells were gener-ally performed with a recovery for fraction 7 of about20–30%.

Enrichment of B. cepacia

Figures 2 and 3 show the results of one typical enrichmentexperiment (experiment A). For this experiment, an initialconcentration of 38% B. cepacia and 62% Neisseria caniswas chosen. After MACS separation, the unretained frac-tion (fraction 2) contained only 2.4% of B. cepacia cells(Fig. 2). The target cells were nearly completely retainedon the magnetized separation column, resulting in a deple-tion rate of 128-fold. The retained fraction, eluted from thecolumn after four washing steps (fractions 3–6), contained92% target cells and only 8% N. canis cells (Fig. 2). Thiscorresponds to an enrichment rate of 19-fold. In the nega-tive control without transcript probe, no significant deple-tion or enrichment of cells could be detected. Figure 3shows the number of cells counted in 10 microscopic fieldsfor the different fractions. It is clearly visible that, in thenegative control (Fig. 3B), hardly any cells are elutedfrom the column after the first washing step, even whenit was removed from the magnetic field. However, in thesample with the hybridized target cells (Fig. 3A), the cellnumber of eluted cells increased significantly in fractions7–9.

For experiments B1 and B2 (Table 2), the initial concen-tration of B. cepacia target cells for the MACS separationwas reduced to 6% of the total number of cells. To showthe reproducibility of the separation step and the stabilityof the labelling, the hybridized sample in experiment B

was split into two samples after incubation of the cellswith the paramagnetic beads. One of the samples wasseparated in the magnetic field after an incubation withthe paramagnetic beads for 10 h (experiment B1); theother after an incubation for 7 days (experiment B2). Theenrichment rates for both experiments agreed well. Inexperiment B1, the target cells were enriched from aninitial concentration of 6.0% to a concentration of 56.7%and, in experiment B2, to 59.4% (fraction 7), resulting inenrichment rates of 21- and 23-fold. In fraction 2, the targetcells were nearly completely depleted to 0.05% or 0.5% ofthe total number of cells. Therefore, incubation for 10 h issufficient for effective binding of the beads.

In experiment C (Table 2), B. cepacia cells wereseparated from the significantly larger B. sphaericuscells and enriched from 95% initially to 99.8% in fraction7. More impressive is the cell percentage in fraction 2.Here, the concentration of B. sphaericus was enrichedfrom 5% to 80% by retaining the labelled B. cepacia cellsin the magnetized column.

Figure 4 (experiment D) shows the enrichment ofB. cepacia with an initial concentration of 6.1% from a mix-ture with cocci (N. canis, 88.1%) and large rods (Bacillussphaericus, 5.8%). A comparison of the cell concentrationbefore and after the separation showed that the percen-tage of target cells increased from 6.1% in fraction 1 to85% in fraction 7 by MACS separation. This correspondsto an enrichment rate of 87-fold.

Enrichment of Acinetobacter calcoaceticus

The efficacy of cell sorting was also tested with A. calco-aceticus labelled with the biotinylated transcript probeDIIIAc. In comparison with the enrichment of B. cepacia,only three instead of four washing steps were performedfor these experiments. The results are shown in Table 2.


Fig. 2. Enrichment of Burkholderia cepaciawith the transcript probe DIIIBcep from amixture with Neisseria canis (Table 2;experiment A). Concentration of target andnon-target cells in the sample with transcriptprobe. Fraction 1, fraction containing the cellsbefore the magnetic separation is performed;fraction 2, fraction containing the cells thatwere not retained in the column inserted intothe magnetic field; fraction 3, washing stepwith flow reducer; fractions 4–6, washingsteps without flow reducer; fraction 7,enriched target cells eluted from the columnafter it was removed from the magnetic field;fractions 8 and 9, enriched target cells (elutedfrom the column with pressure).


For experiment E, an artificial mixture of A. calcoaceticus,B. diminuta and B. cepacia was used. Acinetobacter targetcells could be enriched from an initial frequency of 24.6%to 96.7%, resulting in an enrichment rate of 90-fold.

B. cepacia cells, which were the target cells in the preced-ing experiments, were not retained in the magnetizedcolumn, indicating that the cell separation was reallymediated by the labelling of the target cells with the


Fig. 3. Enrichment of Burkholderia cepaciawith the transcript probe DIIIBcep from amixture with Neisseria canis (Table 2;experiment A).A. Cell counts for 10 microscopic fields in thesample with transcript probe.B. Cell counts for 10 microscopic fields in thesample without transcript probe (control).Fraction 1, fraction containing the cells beforethe magnetic separation is performed; fraction2, fraction containing the cells that were notretained in the column inserted into themagnetic field; fraction 3, washing step withflow reducer; fractions 4–6, washing stepswithout flow reducer; fraction 7, enrichedtarget cells eluted from the column after itwas removed from the magnetic field;fractions 8 and 9, enriched target cells(eluted from the column with pressure).

Fig. 4. Enrichment of Burkholderia cepaciawith the transcript probe DIIIBcep from amixture with Neisseria canis and Bacillussphaericus (Table 2; experiment D).Concentration of target and non-target cells inthe sample with transcript probe. Fraction 1,fraction containing the cells before themagnetic separation is performed; fraction 2,fraction containing the cells that were notretained in the column inserted into themagnetic field; fraction 3, washing step withflow reducer; fractions 4–6, washing stepswithout flow reducer; fraction 7, enrichedtarget cells eluted from the column after itwas removed from the magnetic field;fractions 8 and 9, enriched target cells(eluted from the column with pressure).


biotinylated transcript probes. In a further experiment(experiment F), A. calcoaceticus was enriched 18-foldfrom 20.8% to 82.5% from a mixture with B. diminutaand M. imperiale.

Identification of the enriched target cells

The paramagnetic beads are too small (50 nm) to be vis-ualized by light microscopy and thus do not interfere withthe optical analysis of the cell they are bound to. So, ifthe target cells are distinguishable from the non-targetcells by phase contrast microscopy, the enrichment canbe observed directly without further preparation of thesample.

Independently of morphological identification, theenrichment of target cells can also be observed by astreptavidin–fluorescein detection of the biotinylated tar-get cells in combination with DAPI staining of all cells.Therefore, it was possible to quantify the different cell pro-portions in the fractions before, during and after the MACSseparation steps by epifluorescence microscopy. Again,the rRNA probes seem to be very stable. The identificationof the hybridized target cells via the biotinylated transcriptprobes could be performed successfully, although thecells were pelleted by centrifugation and resuspendedbefore the streptavidin–fluorescein detection was carriedout. In addition, it was possible to perform an in situhybridization with fluorescently labelled oligonucleotideprobes (Fig. 4). This hybridization can also be performedbefore or after the magnetic separation. Even a com-bination of the streptavidin–fluorescein detection of thebiotinylated transcript probes and in situ hybridizationwith fluorescently labelled oligonucleotide probes is possi-ble and allows a clear-cut distinction between target andnon-target cells. This detection of the cells only works ifthe washing buffer, recommended by the manufacturer,is replaced by binding buffer. Apparently, the BSA in thewashing buffer disturbed the probe-conferred fluorescence.

The enrichment effect was also demonstrated by a PCRamplification of a part of the hypervariable domain III ofthe 23S rRNA (experiment F). For this experiment, A. cal-coaceticus was enriched from a mixture with Brevundi-monas diminuta and Microbacterium imperiale. Thesebacteria belonging to different phyla and/or subclasses dif-fer in the length of the 23S rRNA domain III regions, which

are homologous to positions 1366–1617 of E. coli (Rolleret al., 1992; Ludwig et al., 1994). B. diminuta is phylogen-etically a member of the alpha-subclass of Proteobacteriaand has a deletion (in comparison with E. coli) of about 80nucleotides within the domain III, whereas M. imperiale asa member of the Gram-positive bacteria with a high DNAG þ C content has an insertion of about 100 nucleotides.This variability in domain III size was used to demonstratethe enrichment of the target cells. A hot-start PCR wasperformed with the primer pair 1900V and 317R and0.1 ml of cell suspensions before and after the enrichment,as well as positive and negative control samples. The data(obtained by counting the cell fractions before and after theseparation) for this experiment are given in Table 2.Figure 6 shows the PCR fragments obtained from the dif-ferent samples after gel electrophoretic separation in a 3%agarose gel. Amplification of the domain III region from theinitial sample (before the enrichment) clearly resulted(Fig. 6, lane 3) in three typical fragments for the domainIII of alpha- (B. diminuta, about 172 bp) and gamma-subclass Proteobacteria (A. calcoaceticus, about 275 bp)as well as for Gram-positive bacteria with a high DNAG þ C content (M. imperiale, about 375 bp), whereasafter the separation, only the amplicon for A. calcoaceticuscan be recognized (Fig. 5, lane 4). In the negative control,which did not contain a transcript probe, no PCR productscould be detected after performing the whole procedure.

PCR amplification and sequence analysis of the tufgene of enriched cells

To demonstrate that, after the enrichment of target cells,the complete genome is available for a subsequentsequence analysis, we amplified and sequenced part ofthe tuf gene from the enriched A. calcoaceticus cells(experiment F). The resulting sequence was identicalwith that obtained from DNA preparation of an A. calcoa-ceticus pure culture.

Discussion

Idea and concept

In previous studies, the enrichment of bacteria usingimmunomagnetic separation (Luk and Lindberg, 1991;


Fig. 5. Enrichment of Acinetobacter calcoaceticus cells with the transcript probe DIIIAc from a mixture with cultures of Brevundimonasdiminuta and Microbacterium imperiale (Table 2, experiment F). The cells are detected by an in situ hybridization with the FLUOS-labelled23S rRNA-targeted oligonucleotide probe Aca23a, which is specific for A. calcoaceticus, and DAPI staining. For each picture identical fieldswere viewed by epifluorescence microscopy. The left and right photomicrographs viewed identical fields (left, DAPI staining; right,Aca23a-FLUOS).A. Cells before performing the cell fishing (fraction 1).B. Cells after passing the separation column placed in the magnetic field (depletion of the target cells; fraction 2).C. Cells eluted from the separation column after it was removed from the magnetic field (fraction 6).


Islam and Lindberg, 1992; Cudjoe et al., 1993) or flowcytometry (Spring et al., 1993; Jacobsen et al., 1997;Wallner et al., 1997; Snaidr et al., 1998) and micromanipu-lation (Skerman, 1968; Ashkin et al., 1987) has beendescribed. Here, a new separation concept combiningthe methods of in situ hybridization and magnetic separa-tion is introduced.

The possibility of labelling target cells by an in situ hybri-dization with biotinylated transcript probes and streptavidin-coated particles for a subsequent magnetic separation hasbeen shown. For the first time, it has been demonstratedthat transcript probes can act as an anchor between thehybridized target cells and the paramagnetic beads. Forthis probe-based physical enrichment of whole fixedcells, it is decisive that, although the paramagneticbeads cannot penetrate into the hybridized target cells,they can nevertheless bind to the biotinylated transcriptprobes (Fig. 7). Furthermore, the transcript probes haveto withstand the shearing forces generated during theseparation.

The idea of using transcript probes for the physicalseparation of target cells was suggested for the first timeby Trebesius et al. (1994). They made the following

observations. After a hybridization of A. calcoaceticuscells with digoxygenin (DIG)-labelled transcript probesand the detection of the hapten with FLUOS-labelled anti-body fragments, brightly fluorescent rings marked the cellperipheries, whereas the cell centres were only weaklyfluorescent. This halo appearance, which is characteristicof the identification of cells using fluorescently labelledantibodies binding to surface epitopes, indicated that thepolynucleotide probes bound preferentially to target struc-tures in the periphery of the cells. A lysozyme treatmentbefore hybridization permeabilized the cell walls, resultingin evenly stained target cells. Target cells that werehybridized with DIG-labelled transcript probes could bedetected specifically with gold-labelled anti-DIG antibodiesfollowed by silver enhancement, although complete anti-DIG antibodies are too big to penetrate into paraformalde-hyde-fixed bacterial cells (Zarda et al., 1991). Theseobservations indicated that the polynucleotide probesbound preferentially to target structures in the peripheryof the cells and that non-hybridizing parts of themextended into or beyond the cell wall.

Separation of the hybridized target cells

With the Dynal and the MiniMACS separation systems,two different approaches for the magnetic labelling andseparation of the hybridized target cells were tested, butonly the MiniMACS system worked successfully. Animportant difference between these two cell separationsystems is the size of the paramagnetic beads. The diam-eter of the Dynabeads (Dynal) used for this study is


Fig. 6. Diagnostic PCR based on different fragment lengthsresulting from in vitro amplification of a part of domain III of the23S rRNA before and after the enrichment of the Acinetobactercalcoaceticus (about 250 nucleotides) target cells (experiment F;Table 2; Fig. 5) from a mixture of Brevundimonas diminuta (about180 nucleotides) and Microbacterium imperiale (about 350nucleotides).1. 123 bp DNA ladder.2. Negative control without cells.3. Sample hybridized with the transcript probe DIIIAc beforethe enrichment.4. Sample hybridized with the transcript probe DIIIAc afterthe enrichment.5. Sample without transcript probe before the enrichment(control experiment).6. Sample without transcript probe after the enrichment(control experiment).7. Positive control: PFA-fixed cells of A. calcoaceticus,B. diminuta and M. imperiale.8. 1 kb DNA ladder.

Fig. 7. Labelling of bacteria with 23S rRNA-targeted, biotinylatedpolynucleotide probes and streptavidin-coated paramagnetic beads.


2.8 mm, corresponding to a bead volume of 11 490 nm3 perbead. The paramagnetic MiniMACS beads (0.05 mm diam-eter, volume 0.065 nm3) are significantly smaller than theDynabeads (176 769 times smaller volume). From thesevalues, it can be estimated that, in the case of the Dyna-beads, only two or three beads can bind to the surfaceof a ‘standard’ bacterium (estimated diameter 1 mm,volume 0.52 mm3). In contrast, there is enough room forabout 1380 MiniMACS particles on the surface of thesame bacterium. The number of binding particles couldprobably be increased further by binding of severalbeads to one probe molecule. The target cells are con-nected via the single-stranded RNA molecules with theparamagnetic beads. These thin ‘nucleic acid strings’have to withstand all the shearing forces arising duringthe separation. It is unlikely that two RNA strings, asexpected in the case of the use of the Dynabeads, maycompensate for the mechanical forces during the separa-tion in the magnetic field. Obviously, in the case of Mini-MACS, many more bound rRNA molecules are present,supporting a stable linkage between the target cells andthe paramagnetic beads. Moreover, in the MiniMACS sys-tem, the separation distance for the magnetized cell ismuch shorter (only 100–200 mm; Miltenyi et al., 1990)than in the Dynal system.

The magnetic separation in combination with thebiotinylated transcript probes enables the fast and efficientenrichment of target cells. To show the general applicabil-ity of this method, the initial concentrations of the targetcells were varied over a wide range. The observed varia-tions in enrichment rates and recovery depended not onlyon the initial concentration of the target cells but also ontheir efficiency of binding to the beads and several physicalparameters of the separation, i.e. capacity of the ferro-magnetic matrix of the separation column and the buffervolume and flow rate during washing. In general, increas-ing the number of washing steps leads to a higher loss oftarget cells. Consequently, the procedure can be directedeither at maximizing the recovery of target cells andaccepting a higher level of contamination or at obtaininga smaller population of pure target cells.

To demonstrate that the separation of target cells wasreally mediated by the biotinylated transcript probes,negative controls without transcript probes were performed.Nounspecific binding was observed. In all cases, most of thenon-target cells were eluted already during the first wash-ing step (Fig. 3B). The target cells were nearly completelyretained in the magnetized separation columns, indicatingtheir effective and almost quantitative labelling. This offersthe possibility for the negative selection of cells. For thisapproach, all cells that should not be enriched are labelledwith transcript probes and beads and, therefore, areretained in the magnetized separation column. The un-labelled target cells can then be found in the first eluate.

This negative selection should be useful for enriching bac-teria present only in low numbers in the original sample.The only limitation is that transcript probes for all non-target organisms must be produced. Depending on thespecificity of the probes, a more or less comprehensiveset of probes would be necessary. For this application, itmight be advantageous for the specificity of the transcriptprobes to be adjusted to different phylogenetic levels(Trebesius, 1995).

In comparison with the immunomagnetic separation(IMS; Morgan et al., 1991; Christensen et al., 1992; Olsviket al., 1994), the enriched cells are not viable, because it isnecessary to fix the cells with paraformaldehyde beforethe in situ hybridization can be performed. However, theIMS method requires specific antibodies that are onlyavailable for culturable bacteria. In contrast, the probe-based cell fishing (PCF) has the great advantage that itdoes not require pure cultures. For the preparation of theprobes, only a part of domain III of the 23S rDNAsequence must be available. This transcript probe targetsite comprises the largest variable stretch within rRNA pri-mary structures (Ludwig et al., 1992). There is no othercomparable region within the 23S or 16S rRNAsequences. As outlined above, the specificity of the tran-script probes can be varied over a wide range by changingthe hybridization conditions. In this respect, the techniqueis most similar to the flow sorting of bacteria, labelled withfluorescently labelled oligonucleotide probes (Wallneret al., 1997).

Future perspectives

The great potential of this method for the analysis ofcomplex systems is that it should provide access to thegenomic information of uncultured bacteria. For the con-struction of the transcript probes, only an rDNA sequenceof the target organism must be available. Therefore, thistechnique will be an ideal complement to the cyclic rRNAapproach (Amann et al., 1995) to increase our knowledgeof uncultured bacteria. After performing the cyclic rRNAapproach, interesting clone sequences can be chosenfor the production of biotinylated transcript probes. Theapplicability of biotinylated transcript probes for in situhybridizations in complex systems has been shown forthe specific detection of B. cepacia, in samples of a bio-reactor degrading aromatic compounds and activatedsludge (unpublished data). After the enrichment, the tar-get cells can be subjected to various methods for furtherinvestigations. First tests have already shown that thewhole genome of the PFA fixed and enriched target cellsis amenable for molecular analysis such as PCR andgenome sequencing. Therefore, the new technique shouldprovide information about the genetic and metabolic prop-erties of uncultured bacteria. Moreover, the sorted cells



could also be used for electron microscopic studies. Usingthis technique, not only the ultrastructure but also the ele-mental composition of single cells can be studied.

Experimental procedures

Organisms and growth conditions

Sources and strain numbers of the bacteria used in this studyare given in Table 1. All strains were cultured as described inthe relevant catalogues of strains.

Purification of nucleic acid

Genomic DNA of Acinetobacter calcoaceticus and Burkholderiacepacia were prepared according to the method of Marmur(1961).

Cell fixation

Cells from exponentially growing cultures were fixed with 3%paraformaldehyde and stored in 50% ethanol in PBS (130 mMNaCl, 10 mM sodium phosphate buffer, pH 7.2) as describedpreviously (Amann, 1995). Cell suspensions were immobil-ized on precleaned six-well glass slides (Paul Marienfeld),dried at room temperature and dehydrated in 50%, 80% and96% ethanol (3 min each).

Fluorescent-oligonucleotide probes

Fluorochrome-labelled oligonucleotide probes were purchasedfrom Interactiva. The following oligonucleotides were usedunder standard conditions: EUB338 (58-GCTGCCTCCCGT-AGGAGT-38) specific for Bacteria (Amann et al., 1990);ACA23a (58-ATCCTCTCCCATACTCTA-38) specific for Aci-netobacter spp. (Wagner et al., 1994); and Bcv13b (58-GCTCATCCCATTTCGCTC-38) specific for Burkholderiacepacia and Burkholderia vietnamiensis (Stoffels et al.,1998).

Preparation and labelling of polynucleotide probes

Purified genomic DNAs of A. calcoaceticus and B. cepaciawere used for PCR amplification of 23S RNA gene fragmentsencoding the variable region of domain III. Purified geno-mic DNA (0.1–1 mg) was used for in vitro amplificationwith the modified primer pair 1900V and 317RT3 (Ludwiget al., 1994). The nucleotide sequences of the primerswere 58-MADGCGTAGBCGAWGG-38 (1900V; E. coli 23SrDNA position 1366–1381; Brosius et al., 1981) and 58-ATAGGTATTAACCCTCACTAAAGGGACCWGTGTCSGTT-THBGTAC-38 (317RT; E. coli 23S rDNA position 1602–1617;Brosius et al., 1981). The latter contained the T3-RNA polymer-ase promoter sequence (underlined) needed for the in vitrotranscription. Amplification was performed with a HybaidOmniGene temperature controller (MWG-Biotech) as follows.One microlitre (0.1–1 mg) of genomic DNA, 50 pmol each ofthe appropriate primers, 200 mmol of each deoxyribonucleoside

triphosphate, 10 ml of 10 ×PCR buffer (500 mM KCL, 100 mMTris-HCl, pH 8.3, 15 mM MgCl2, 0.1% w/v gelatine), 10 ml of25 mM MgCl2 and 1.5 U of thermostable Taq polymerase(Promega) were added to a 0.5 ml reaction tube. The totalvolume was adjusted to 100 ml with sterile water. The mixturewas overlaid with 70 ml of mineral oil (Sigma). After initial heat-ing to 948C for 3 min, 35 cycles consisting of denaturation at948C for 30 s, annealing at 588C for 60 s and extension at728C for 90 s were performed, followed by a final elongationstep at 728C for 10 min. The resulting rDNA fragments wereanalysed by electrophoretic separation in 2% (w/v) agarosegel and staining with ethidium bromide. Subsequently, thefragments were recovered and purified with the Magic Prepkit (Serva). The amplicons were used as templates for thein vitro transcription.

In vitro transcription

The transcription of the 23S rDNA fragments was performedby applying an RNA transcription kit (Boehringer Mannheim)as recommended by the manufacturer. For labelling of thetranscripts biotin-16-UTP (Boehringer Mannheim) was usedat a biotin-16-UTP to UTP ratio of 6.5:3.5. The yield of thetranscription product was analysed spectrophotometricallyon a Beckmann DU650 (Beckmann Instruments).

In situ hybridization with oligonucleotide probes

Whole-cell hybridization of cells immobilized on microscopicslides was performed as described previously by Snaidret al. (1997). The slides were examined with an Axioplanmicroscope (Zeiss) with filter sets 01 (for DAPI staining), 09and 15. Photomicrographs were taken with a Kodak TMAX400 black-and-white film. Exposure times were between0.01 s and 0.06 s for phase contrast micrographs and 0.5–30 s for epifluorescence micrographs.

Oligonucleotide hybridizations in liquid samples were per-formed in 30 ml of hybridization buffer in 1.6 ml tubes. Afterhybridization at 468C for 90 min, the cells were washed in300 ml of prewarmed (488C) washing buffer for 15 min at488C, pelleted by centrifugation (8000 g for 2 min) and resus-pended in 50 ml of PBS solution. For detection, the hybridizedcells were immobilized on microscope slides, air dried andexamined as described above.

In situ hybridization with polynucleotide probes

In situ hybridizations with polynucleotide probes were per-formed with cells immobilized on glass cover slides and withliquid samples. When the hybridization was performed withPFA-fixed cells immobilized on glass cover slides, each wellwas covered with 10 ml of hybridization buffer containing100 mM NaCl, 0.01% SDS, 20 mM Tris-HCl (pH 8.0), 5 mMEDTA (pH 8.0), variable amounts of formamide (80–95%)and 150 ng of transcript probe. For the incubation steps, theslides were stored in isotonically equilibrated chambers.After incubation at 808C for 30 min, the chambers were imme-diately transferred to an incubator with the determinedhybridization temperature between 538C and 688C. The



hybridization was carried out for 4 h and terminated by immer-sing slides in distilled water. The proper stringency for thespecificity of the approach was determined by in situ hybridiz-ations of target and non-target reference cells and varying theformamide concentration and the hybridization temperature,while the NaCl concentration in the hybridization buffer waskept constant.

For the enrichment of cells with polynucleotide probes,hybridizations were performed in solution. Therefore, thehybridization procedure was modified as follows: a mixtureof PFA-fixed cells was washed in 200 ml of PBS buffer and pel-leted at 8000 g for 2 min. The pellet was resuspended in 30 mlof hybridization buffer and 200 ng of transcript probe wasadded per 10 ml of hybridization buffer. The sample was incu-bated for 30 min at 808C to denature the polynucleotide probe,followed by a hybridization for 4 h at temperatures between538C and 688C. After 4 h, 60 ml of cold (48C) binding buffer(130 mM NaCl, 10 mM Na2HPO4/NaH2PO4, 0.5 M EDTA,pH 7.2) was added, and the sample was stored at 48C.

In situ detection of biotin-labelledpolynucleotide probes

Biotin was detected using fluorescently labelled streptavidin(FLUOS-Streptavidin; Boehringer Mannheim). Hybridized cellsimmobilized on microscope slides were covered with 30 mlof detection buffer (136 mM NaCl, 2.7 mM KCl, 8 mM Na2H-PO4 × 2H2O, 1.5 mM KH2PO4, pH 8,5) containing 5 mg ml¹1

streptavidin–fluorescein. The incubation was performed for1 h at 278C in an isotonically equilibrated chamber in thedark. Subsequently, the slides were washed for 15 min atthe same temperature in detection buffer and finally rinsedin distilled water. The slides were air dried, embedded inanti-bleaching agent and examined by epifluorescencemicroscopy.

Staining with DAPI

Staining of the hybridized cells with 48,6-diamidino-2-phenylindole (DAPI) was performed as described previously(Wagner et al., 1993).

Combination of in situ hybridization with fluorescentlylabelled oligonucleotide and biotin-labelledpolynucleotide probes

For the combination of oligonucleotide and polynucleotideprobes, no simultaneous hybridization was possible because ofdifferent stringencies. Therefore, the hybridization and wash-ing for the polynucleotide probes were performed first, fol-lowed by subsequent hybridization and washing with theoligonucleotide probe requiring lower stringency than thepolynucleotide probes. Then the streptavidin–fluoresceindetection of the biotinylated transcript probes was performed.

Separation of the hybridized target cells

Cell separation with the Dynal system. For the enrichmentof the biotinylated target cells with the Dynal cell separation

system (Deutsche Dynal) streptavidin-coated DynabeadsM280 with a diameter of 280 mm were used. Before use,the Dynabeads were washed as recommended by themanufacturer. After the hybridization with the biotin-labelledtranscript probes, 5 ml of prewashed Dynabeads were addeddirectly to 15 ml of the sample, or the cells in the sample werepelleted for 3 min at 8000 g and then resuspended in 5 ml ofwashing buffer (130 mM NaCl, 10 mM Na2HPO4/NaH2PO4,0.5 M EDTA, 0.1% BSA, pH 7.4) before the beads wereadded. Subsequently, the samples were incubated for 30–45 min in a hybridization oven at 258C. The separation of thebeads and the bead–cell complexes was performed in amagnetic particle concentrator. After placing the sample inthe magnetic particle concentrator (Dynal), the beads andbead–cell complexes were attracted to the wall of the 1.5 mltest tube. The solution was removed, and the pellet wasresuspended in 100 ml of washing buffer (130 mM NaCl,10 mM Na2HPO4/NaH2PO4, 0.5 M EDTA, 0.1% BSA,pH 7.4). Then, the magnetic separation was repeated, andthe pellet was resuspended in 30 ml of washing bufferfor subsequent microscopic analysis. To determine cellattachment, bead samples were inspected by phasecontrast microscopy or after a hybridization with fluorescentlylabelled oligonucleotide probes by epifluorescencemicroscopy.

Cell separation with the Miltenyi Mini-MACS. The Miltenyicell separation system (Miltenyi Biotec) consists of strepta-vidin-coated paramagnetic beads (MicroBeads, diameter50 nm) and MSþ separation columns filled with steel wool,flow resistors and a strong magnet. After the hybridizationwith the biotinylated transcript probes, 60 ml of binding buffer(PBS: 130 mM NaCl, 10 mM Na2HPO4/NaH2PO4, 0.5 M EDTA,pH 7.2) and 10 ml of streptavidin-coated paramagnetic beadswere added to 30 ml of the samples. The sample wasincubated after gentle mixing for 4 h or overnight at 48C.For cell separation, the separation column was placed in themagnetic field and treated further according to the manu-facturer’s instructions. The column with the attached flowresistor was washed twice with 600 ml of binding buffer. Tenmicrolitres of the sample was retained as fraction 1; the rest(90 ml) was passed through the column three times. Theeluate was collected as fraction 2. The column waswashed with 600 ml of washing buffer (PBS þ 0.5% BSA)(fraction 3). Then, the flow resistor was removed and twoor three further washing steps were performed (fractions4–6). Afterwards, the column was removed from themagnetic field. The retained, labelled cells were elutedwith 600 ml of washing buffer (fraction 7). For twoadditional elution steps, 2 × 600 ml of washing buffer waspressed through the column with a plunger and collectedas fractions 8 and 9. All fractions and 10 ml of the originalsample (fraction 1) were collected and made up to 1000 mlwith binding buffer. The cells were pelleted at 8000 g for3 min, resuspended in 20 ml of PBS and 20 ml of EtOHabs

and stored at ¹208C.To examine the effectiveness of the cell enrichment, 10–

20 ml of each sample was fixed on glass cover slides, andthe cell fractions were counted in 10–20 randomly chosenmicroscopic fields. The control sample without transcriptprobe was analysed in the same way.



For quantification, the enrichment rate E and depletion rate1/E were defined according to Kato and Radbruch (1993) as:

E ¼ð% positive cells after separation/% negative cells after separationÞ

(% positive cells before separation/% negative cells before separation)

In vitro amplification of tuf and 23S rDNA fragmentsfrom enriched cells

The paraformaldehyde-fixed cell suspensions were usedwithout prior treatment for PCR amplifications. The amplifica-tion of the tuf gene fragment was performed with the primerpair 280 V and 1441R (Neumaier, 1996). The nucleotidesequences of the primers are 58-GCDCAGATGGACGG-38(E. coli tuf gene position 289–302; An and Friesen, 1980)and TCNCCNGGCATNACCAT (E. coli tuf gene position1048–1064; An and Friesen, 1980). The PCR was performedas described above with the following modifications. Cell sus-pension (1 ml) was used instead of purified DNA, and the Taqpolymerase was added after incubation of the complete PCRmixture for 10 min at 958C. The following amplification pro-gramme was run: after initial heating to 948C for 3 min, 35cycles consisting of denaturation at 948C for 30 s, annealingat 488C for 60 s and extension at 728C for 120 s were per-formed, followed by a final elongation step at 728C for10 min. For the amplification of domain III of the 23S rDNAfrom the cell suspensions before and after the enrichment,the same PCR programme and primers were used asdescribed for the preparation of the polynucleotide probes.Again, the PCR sample was heated for 10 min before theTaq polymerase was added.

The amplified tuf and rDNA fragments were analysed byelectrophoretic separation using 1% and 3% agarose gelsrespectively. For subsequent sequence analysis, the tuf genefragment was purified using the Qiagen direct purification kit.

Sequencing of the tuf gene fragment

Sequencing of the amplified part of the tuf gene was per-formed with a direct blotting electrophoresis system (GATC1500; MWG Biotech). The cycle sequencing protocolbased upon the chain termination technique (Chen andSeeburg, 1985) was applied according to the manufac-turer’s instructions (Boehringer) with the primer 755V(Neumaier, 1996; GCCNRTBGARGAYGT, E. coli tuf geneposition 636–653; An and Friesen, 1980) and an annealingtemperature of 428C.

Acknowledgement

This work was supported by a grant from the DeutscheForschungsgemeinschaft (LU421). The authors thankR. Amann for valuable discussions concerning the designof this method.

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