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Applied and Environmental Microbiology, December 2005, p. 7933-7940, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7933-7940.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Fumito Maruyama,* Takehiko Kenzaka, Nobuyasu Yamaguchi, Katsuji Tani, and Masao Nasu*

Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, Japan

Received 12 May 2005/ Accepted 24 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Specific detection of DNA or RNA sequences inside bacterial cells at the single cell level allows determination of the abundance of target bacteria, together with analysis of the ecophysiology of individual cells without cultivation (5). Fluorescence in situ hybridization (FISH) and in situ nucleic acid amplification, followed by fluorescent labeling of the amplicon, have been used for these purposes.

Many microbial ecology studies have used conventional FISH with 16S or 23S rRNA-targeted oligonucleotide probes (41). The limitation of conventional FISH is that low copy numbers of targets such as chromosomal or plasmid DNA or unstable mRNA cannot be detected (1, 44). In order to improve the sensitivity, multiple fluorochrome-labeled polynucleotide probes were used to target low-copy-number genes (chromosomal painting [20, 27, 40]; RING-FISH [44]).

The most popular application of in situ nucleic acid amplification is in situ PCR (34, 39). In situ PCR can use FISH to detect amplicons as well as the incorporation of fluorescently labeled deoxynucleoside triphosphate (dNTP) during PCR. Alternative in situ nucleic acid amplification techniques such as cycling primed in situ labeling (cycle PRINS [18]) and in situ loop-mediated isothermal amplification (LAMP [25]) have improved specificity and sensitivity. Cycle PRINS uses only one primer for gene amplification. This causes the linear amplification of several target genes, and amplicons are labeled via the incorporation of fluorescently labeled dNTP during target amplification. The key feature of in situ LAMP is the mild isothermal conditions used for gene amplification. This causes less cell damage than in situ PCR, allowing the use of a fluorescent antibody staining after gene amplification, which was used for detection of E. coli O157:H7 cells in the bacterial mixture (25).

In situ nucleic acid amplification has considerable potential for microbial ecology including studies of lateral gene transfer (18, 35). Some bacterial strains, including Escherichia coli cells, enter a nonculturable state despite sustaining respiratory activity or substrate responsiveness under starvation stress (2, 4, 13, 31). Thus, the green fluorescent protein gene (GFP), gfp, which can be detected by fluorescence microscopy at the single cell level without cultivation, is an obvious choice for use in lateral gene transfer studies (9). However, a suitable promoter sequence that can generate sufficient levels of gfp gene expression detectable by fluorescence microscopy is necessary. Levels of expression as well as physiological states might differ among bacterial species. In addition, GFP will not function in an anaerobic environment (14), and it can only be applied to investigate microorganisms that can be genetically manipulated. Thus, the detection and quantification of target bacteria based on DNA sequences can be valuable in studies of lateral gene transfer (33).

Improvement of in situ nucleic acid amplification requires specific test characteristics. A shorter target sequence than those of polynucleotide probes in FISH (140 mer [44]) or in situ LAMP (90 mer [25]) would facilitate primer or probe design, resulting in more specific detection. A larger amplicon might prevent false-positive or false-negative results because the extracellular leakage of short amplicons produced by PCR causes such results (28). The conditions for cell permeabilization should be optimized to maintain sufficient fluorescent intensity of target cells independently of their physiological state, which affects these conditions for in situ PCR (38). The concentration of target bacteria on membrane filters would facilitate reliable enumeration because the reproducibility and reliability of counts on glass slides is limited (24, 26).

In the present study, rolling circle amplification (RCA [7, 21, 37]) was applied to in situ nucleic acid amplification (in situ RCA) to meet these test characteristics. In situ RCA has been successfully used to determine gene copy number and detect single base mutations in eukaryotic cells (7, 21) and has not been developed for prokaryotic cells. This technique requires one short target sequence (less than 40 mer) and generates large single-stranded and tandem repeats of target DNA as amplicons. Monitoring of plasmid uptake by in situ RCA in nonculturable E. coli cells for 149 days was carried out, and compared the results with those determined by conventional culture method.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacterial strains and plasmids.
The following strains were used: E. coli K-12 W3110 with or without plasmid pGFPuv (vector size, 3.3 kbp; Clontech, Palo Alto, CA) or pHSG299 (copy number, 30 to 200 copies/cell; vector size, 2.7 kbp; Takara Bio Co., Shiga, Japan), E. coli K-12 HB101 with or without plasmid pGFPuv, E. coli O157:H7 ATCC 43888 (42) with plasmid pGFPuv, and E. coli O157:H7 RIMD 0509952 (16). E. coli O157:H7 RIMD 0509952 has one Shiga-like toxin 1 (stx1) gene on the chromosome, whereas the other strains do not harbor the gene.

Target genes for in situ RCA.
The target sequences for in situ RCA were the replication origin (ori [37]) region on plasmid pGFPuv, the ampicillin resistance (Apr) and gfp genes on the plasmid, and the chromosomal Shiga-like toxin 1 (stx1) gene.

Each gene was a single copy on the plasmid or on the chromosome of E. coli O157:H7. The copy number of the plasmid and chromosome varied from 30 to 200 and from 1 to 5 copies/cell, respectively, depending on the growth stage of the cells.

Circularizable probes, RCA primers, and detector probes.
Table 1 summarizes the primers and probes used for in situ RCA, and Fig. 1 describes the general principle of RCA. All primers and labeled probes with a phosphate group or Cy3 were supplied by Espec Oligo Service Co. (Tsukuba, Japan). A circularizable probe and an RCA primer were used for RCA, and a fluorescently labeled probe (detector probe) detected the intracellular amplicon by FISH.


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  		TABLE 1. Primers and probes used for in situ RCA

 


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  		FIG. 1. Summary of RCA reaction. See the text for details.

 
The circularizable probes (ori-RCA [37], amp-RCA, gfp-RCA, and stx1A-RCA) were 89 to 93 mers, comprising a 19 to 21 mer and a 15 to 18 mer of the complementary sequence of the target DNA at the 3' and 5' ends, respectively (total, 35 to 39 mer), with an arbitrary sequence in the middle (Fig. 1). These probes were labeled with a phosphate group at 5' end and circularized by ligation when hybridized to the target sequences.

The19-24 mer RCA primers (amporif, gfpf, and stx1Af) amplified the complementary sequence of the circularized probe by hybridization to a specific region of the probe. The amplicons were single-stranded tandem repeats of the circularized probe sequence (Fig. 1).

The 18 to 20 mer detector probes (ampori-d and gfpstx-d) were labeled with Cy3 for visualization of amplicons inside cells by FISH. The detector probes consisted of complementary sequences of a specific region of the RCA amplicon (Table 1).

The circularizable probes for the ori on pUC19 derivative, plasmid pGFPuv have been described (37), and all other probes and RCA primers were designed in the present study. The general principles to design a circularizable probe were basically as described by Faruqi et al. (11). In brief, the 5' target-specific region of the probe was 20 to 30 bp in length, while the 3' target-specific region was 12 to 20 bp. The Tm of the 5' target specific region was ~5°C above the ligation temperature, whereas that of the 3' target specific region was ~15°C below the ligation temperature.

Culture condition and cell fixation.
All E. coli strains were cultured at 37°C in Luria-Bertani medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl [pH 7.0]). Overnight cultures of the strains were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4 [pH 7.2]), and suspended in freshly prepared paraformaldehyde (4% in PBS) for 16 h at 4°C. After fixation, the cells were washed twice with PBS and suspended in 50% ethanol with PBS.

Sample preparation.
Fixed cells were filtered onto gelatin-coated white polycarbonate membrane filters (25 mm2. pore size, 0.2 µm; Advantec, Tokyo, Japan), washed twice with sterile distilled-deionized water, dehydrated with 100% ethanol for 3 min, and dried thoroughly. To avoid cell loss (destruction and/or detachment from the membrane filter) during procedures such as cell permeabilization, bacterial cells were embedded in Metaphor agarose (0.2% [wt/vol] in sterilized distilled-deionized water (FMC Bioproducts, Rockland, ME), dried on glass slides, and dehydrated with 100% ethanol as described previously (29). Both embedded and unembedded cells in agarose were processed to the following experimental steps. All samples were prepared in triplicate.

Cell permeabilization.
Cells on the membrane filters were permeabilized under various conditions. These included (i) no enzymatic digestion; (ii) incubation with 0.5 mg of lysozyme (100 mM Tris-HCl [pH 8.2], 50 mM EDTA; Nacalai Tesque, Inc., Kyoto, Japan) ml–1 at 37°C for 5, 15, 30, 50, and 70 min, followed by 0.1 µg of proteinase K (Roche Diagnostics; 100 mM Tris-HCl [pH 8.2], 50 mM EDTA) ml–1 at 37°C for 5 min; (iii) incubation with 0.5 mg of lysozyme ml–1 at 37°C for 50 min, followed by 0.1 µg of proteinase K ml–1 at 37°C for 0, 5, 15, and 30 min; (iv) incubation with 10 mg of lysozyme ml–1 at 37°C for 15, 30, 60, and 90 min, followed by 0.1 µg of proteinase K ml–1 at 37°C for 45 min; and (v) incubation with 10 mg of lysozyme ml–1 at 37°C for 60 min, followed by 0.1, 1, 10, 100, and 1,000 µg of proteinase K ml–1 at 37°C for 45 min.

After enzymatic digestion, the membrane filters were washed twice in sterilized distilled-deionized water, dehydrated in a graded ethanol series (50, 80, and 100%) for 1 min, and then dried thoroughly.

Nuclease digestion.
Removal of RNA from permeabilized cells was carried out by using DNase-free RNase A (Sigma) at a final concentration of 0.5 mg ml–1 for 20 min at room temperature. The membrane filters were rinsed with sterile distilled-deionized water, dehydrated in a graded ethanol series for 1 min, and dried thoroughly.

RCA inside bacterial cells.
In situ RCA required ligation and amplification of the circularized probe by the RCA primer (Fig. 1). Before ligation, the membrane filters were cut into eight sections with sterilized scissors. Samples embedded in agarose or not were ligated under the following conditions. The membrane filters were immersed in 100 µl of ligation mixture containing 1x Ampligase buffer with (i) 0.25 µM circularizable probe and 5 U of Ampligase (Epicenter, Madison, WI) for unembedded samples or (ii) 0.80 µM circularizable probe and 25 U of Ampligase for embedded samples in 0.2-ml PCR tubes.

The ligation mixtures containing the membrane filters were incubated on ice for 15 min to allow the reagents to permeate the cells, and then circularized probes were ligated by an incubation at 94°C for 10 min, followed by 60°C for the ori, Apr, and gfp genes, and at 50°C for the stx1 gene for 80 min in a thermocycler.

The membrane filters were rinsed twice with PBS containing 0.01% Nonidet P-40 (Sigma) at 50°C for 10 min, twice with sterilized water at room temperature for 5 min, and then dehydrated with a graded series of ethanol for 1 min and dried thoroughly.

After ligation of the circularizable probe, RCA proceeded in 100 µl of reaction mixture containing 0.2 mM dNTP mixture, 1 M Betain (Sigma), and 1x ThermoPol buffer with (i) 0.5 µM RCA primer and 8 U of Bst DNA polymerase (New England Biolabs) for unembedded samples or (ii) 1 µM RCA primer and 40 U of Bst DNA polymerase for samples embedded in agarose in PCR tubes.

The RCA mixture containing the membrane filters was incubated on ice for 15 min to allow permeation of the reagents, and then RCA proceeded for 90 min at 63°C for the ori, Apr, and gfp genes and at 55°C for the stx1 gene. The membrane filters were then rinsed once with PBS containing 0.01% Nonidet P-40 at 50°C for 15 min and twice with sterilized water at room temperature for 5 min, dehydrated in a graded ethanol series for 1 min, and dried thoroughly.

Detection of amplicons inside bacterial cells.
The intracellular amplicons were detected by FISH. After intracellular RCA, the membrane filters were immersed in 100 µl of prewarmed hybridization solution (900 mM NaCl, 5 mM EDTA, 20 mM Tris-HCl [pH 7.5], 0.01% sodium dodecyl sulfate, 30% formamide) containing 1 ng of Cy3-labeled detector probe µl–1 at 46°C for 16 h in a thermal cycler. The membrane filters were washed with PBS containing 0.01% Nonidet P-40 at 48°C for 15 min twice and then again with sterilized water and counterstained with 1 µg of DAPI (4',6'-diamidino-2-phenylindole) ml–1 for 10 min at room temperature.

The specificity of in situ RCA was examined by simultaneous cell identification. Cells on membrane filters were stained with 2 µg of fluorescein isothiocyanate (FITC)-labeled anti-E. coli O157:H7 antibody (Goat, polyclonal; Kirkegaard and Perry Laboratories, Inc.) ml–1 in PBS, including 30 mg of bovine serum albumin ml–1 at 37°C for 30 min before counterstaining when E. coli O157:H7 cells were used as the positive or negative controls.

Microscopic examination.
Counterstained membrane filters were placed on glass slides and mounted in nonfluorescence immersion oil. Cells on the slides were observed by using an epifluorescence microscope (E-400; Nikon, Tokyo, Japan) with the Nikon filter sets UV-2A (30-350, DM400, and BA420), B-2A (Ex450/490, DM505, and BA520), and HQ-Cy3 (G535/50, FT565, and BP610/75) for UV, blue, and green excitations, respectively. Images were obtained by using a cooled charge-coupled device camera (Sensys 1401; Photometrics, Tucson, AZ) and stored as digital files. At least 1,000 cells from 20 different fields were counted per sample.

Plasmid uptake.
Plasmid DNA uptake by E. coli K-12 HB101 was examined by using the modified method described by Baur et al. (3). E. coli K-12 HB101 cultured at 37°C in LB medium overnight was inoculated into fresh LB medium and incubated at 37°C until the cells reached the early stationary phase. The cells were sedimented by centrifugation at 10,000 x g for 10 min, washed twice, resuspended in a 10 mM CaCl2 solution (pH 7.5) at a final concentration of 2 x 109 cells ml–1, and incubated at 4°C for 149 days.

DNA uptake was assayed as described previously (3). Three parallel samples of E. coli cells incubated in a CaCl2 solution were collected on days 0, 1, 3, 6, 10, 17, 27, 47, 71, 101, and 149, and concentrated by centrifugation at 10,000 x g for 5 min and resuspended in the CaCl2 solution at 2 x 1010 cells ml–1. One nanogram (about 5 x 108 copies) of the pGFPuv plasmid carrying the gfp and Apr genes was added to a 50-µl cell suspension (~109 cells) in 10 mM CaCl2 solution, incubated for 30 min at 4°C, and heat shocked at 42°C for 2 min. The samples were transferred to 4°C for 10 min, inoculated into 1 ml of warm LB medium, and incubated at 37°C for 45 min.

Plasmid uptake by E. coli cells was then examined by in situ RCA and by spreading the cell suspension on LB agar plates containing 50 µg of ampicillin ml–1. Total E. coli cells were counted after staining with 1 µg of DAPI ml–1 at room temperature for 10 min. Total culturable E. coli cells were counted by spreading the cell suspension on LB agar plates without ampicillin.

The gfp gene was the target for detecting plasmid uptake by E. coli cells using in situ RCA. After the incubation with LB medium at 37°C for 45 min, the E. coli suspension was washed with PBS twice and fixed with freshly prepared paraformaldehyde (4% in PBS) for 16 h at 4°C. Subsequent steps proceeded as described above. In brief, fixed cells were washed with PBS, filtered onto the membrane filter and embedded in agarose. The membrane filters were incubated in 0.5 mg of lysozyme ml–1 at 37°C for 30 min and 0.1 µg of proteinase K ml–1 at 37°C for 5 min. Intracellular RNA was removed, and the membrane filters were immersed in a ligation mixture that included 0.8 µM circularizable probe, 1x Ampligase buffer, and 25 U of Ampligase. Ligation proceeded at 94°C for 10 min, followed by 60°C for 80 min. The RCA step proceeded in the reaction mixture (1 µM RCA primer, 1 M Betain, 1x ThermoPol buffer, and 40 U of Bst DNA polymerase) at 63°C for 90 min. Thereafter, the membrane filters were immersed in hybridization solution containing Cy3-labeled detector probe at 46°C for 16 h, washed at 48°C for 15 min twice, and counterstained with DAPI. The number of E. coli cells carrying the gfp gene was counted under fluorescence microscopy.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Circularizable probe, RCA primer, and detector probe.
The targets for in situ RCA were the ori, gfp, Apr, and stx1 genes (Table 1). Figure 1 shows that RCA yielded a single-stranded DNA as the amplicon. The required lengths of the target for in situ RCA to detect the genes were 35 to 39 mer.

Targets with a low copy number have been detected by FISH using multiple fluorochrome-labeled polynucleotide probes (30). The shortest polynucleotide probe which can target genes with low copy numbers by FISH is 140 mers of the target sequence (44), which is about 3.5 times longer than that required for in situ RCA in the present study. LAMP requires 90 mers of the target sequence, which is about twice as long as that required for RCA (25). Shorter target sequence of RCA can, therefore, increase the specificity of gene detection.

Applicability of RCA for in situ nucleic acid amplification.
RCA reaction (ligation and amplification) condition was firstly examined with extracted DNA using the ori-RCA probe, which was reported previously (37), the amporif primer, and the ampori-d detector probe (Table 1). The ligation step, which is to bring the two ends of the single-strand circularizable probe together and permit ligation, is critical for the specificity and reliability of the RCA reaction. The RCA reaction conditions were therefore examined by testing different ligation and amplification temperatures. An aliquot of the amplicon was digested with an appropriate restriction enzyme according to the method of Thomas et al. (37). Then the ligation and amplification conditions, under which the size of the digested RCA products was in good agreement with the predicted size in agarose gel electrophoresis, was selected for in situ RCA (data not shown). The RCA reaction conditions for the Apr, gfp, and stx1 genes using extracted DNA were also optimized before in situ RCA (data not shown).

The ligation and amplification conditions were optimized, and then the in situ RCA conditions were initially investigated targeting the ori region on plasmid pGFPuv under the same cell permeabilization conditions (0.5 mg of lysozyme/ml for 15 min and 0.1 µg of proteinase K/ml for 5 min) as those used for in situ PCR targeting of a specific sequence inside E. coli (34). Controls to examine specificity of in situ RCA were bacterial cells (i) without a target sequence (Fig. 2); (ii) without enzyme for ligation of the circularizable probe or extension of the RCA primer (data not shown); or (iii) without circularizable probe, RCA primer, or detector probe (data not shown). The controls did not produce false-positive or false-negative results, or nonspecific extracellular fluorescence.



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  		FIG. 2. In situ RCA detection of ori region on plasmid pGFPuv in E. coli using circularizable probe. Under UV excitation, all DAPI-stained bacterial cells were visualized (A and C). Under green excitation, cells harboring amplified products of the target gene emitted red fluorescence of Cy3-labeled detector probe (B and D). The same microscope fields are shown with UV and green excitation. A and B, E. coli K-12 W3110 carrying plasmid pGFPuv (positive control); C and D, E. coli O157:H7 ATCC 43888 without plasmid (negative control).

 
Cell permeabilization.
Lysozyme and proteinase K were used to optimize and determine the range of permeabilization conditions for in situ RCA of samples with or without agarose embedding (Table 2). Non-mild enzymatic cell permeabilization caused the uneven distribution of fluorescent cells after FISH even among positive control cells. In turn, some target cells failed to be detected by in situ RCA, followed by FISH. The mildest permeabilization conditions for both embedded and unembedded samples under which all positive cells were detectable were 0.5 mg of lysozyme ml–1 for 15 min and 0.1 µg of proteinase K ml–1 for 5 min at 37°C (Fig. 3A to D). These were identical to the conditions required for in situ PCR in E. coli cells (34). In addition, permeabilization with 0.5 mg of lysozyme ml–1 for 50 min, followed by 0.1 µg of proteinase K ml–1 for 15 min allowed target gene detection without altering the cell morphology (Fig. 3E to H). However, the number of cells on the membrane filter unembedded in agarose was decreased to 85% of the initial number under these conditions. More rigorous permeabilization distorted the cell morphology.


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  		TABLE 2. Effects of various cell permeabilization conditions on the specificity of in situ RCA

 


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  		FIG. 3. In situ RCA detection of Apr gene on plasmid pGFPuv in E. coli cells under two different cell permeabilization conditions. Cell permeabilization conditions: 0.5 mg ml–1 lysozyme for 15 min then 0.1 µg of proteinase K ml–1 for 5 min at 37°C (A, B, C, and D) and 0.5 mg of lysozyme ml–1 for 50 min and 0.1 µg of proteinase K ml–1 for 15 min at 37°C (E, F, G, and H). Excitation with UV visualized all DAPI-stained bacterial cells (A, C, E, and G). Under green excitation, cells harboring amplified products of Apr gene emitted red fluorescence of Cy3-labeled oligonucleotide probe (B, D, F, and H). Same microscope fields are shown with UV and green excitation. A and B, E. coli O157:H7 ATCC 43888 carrying plasmid pGFPuv with Apr gene (positive control); C and D, E. coli K-12 W3110 carrying plasmid pHSG299 without Apr gene (negative control); E and F, E. coli K-12 W3110 carrying plasmid pGFPuv (positive control); G and H, E. coli O157:H7 ATCC 43888 without carrying Apr gene (negative control).

 
Target genes inside bacterial cells embedded in agarose were visualized after permeabilization with 10 mg of lysozyme ml–1 for 15 to 60 min, followed by 0.1 µg of proteinase K ml–1 for 45 min and 10 mg of lysozyme ml–1 for 60 min, followed by 0.1 to 100 µg of proteinase K ml–1 for 45 min. The morphology of cells permeabilized more rigorously became distorted. In situ RCA could detect target sequences inside bacterial cells that were permeabilized under harsher conditions than those used for in situ PCR (34). Unless the cell morphology was altered, none of the permeabilization conditions affected the fluorescence intensity of positive cells (data not shown).

The amplification of nucleic acids inside bacterial cell has been hampered by the balance between sufficient permeability of the cell membrane for the polymerase or other reagents to enter the cell and enough cell integrity to prevent amplicon leakage (44). The optimal permeabilization conditions for in situ PCR differed depending on the physiological state of the cell (38). Furthermore, fluorescence intensity considerably varied with slight modifications to cell permeabilization (19). Although in situ RCA also requires permeabilization so that the enzymes and reagents can enter cells, the amplicon generated by RCA might be long enough to remain inside permeabilized cells. This entire optimized permeabilization might be useful only for E. coli cells grown at 37°C in LB medium. Therefore, although milder cell permeabilization (0.5 mg of lysozyme/ml for 15 min and 0.1 µg of proteinase K/ml for 5 min) of in situ PCR than those of in situ RCA in the present study was reported to work for all of the bacteria in a ground water (35), modification of the permeabilization condition will be required to examine the applicability of in situ RCA depending on bacterial species and their physiological state.

Specificity and sensitivity.
Apr and gfp gene were selected as a target for in situ RCA because these were used as a marker for gene transfer studies, and stx1 gene was targeted to confirm whether in situ RCA could detect a single-copy gene. The specificity of in situ RCA was further examined using E. coli O157:H7 as the positive or negative control and FITC-labeled anti-E. coli O157 polyclonal antibody (Fig. 4). When E. coli O157:H7 was not the target for in situ RCA, specificity was examined by changing the ratios of positive and negative control bacterial cells (data not shown). These control experiments showed that the target DNA sequence was specifically detected inside bacterial cells by in situ RCA without nonspecific extracellular fluorescence.



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  		FIG. 4. In situ RCA detection of the gfp gene on plasmid pGFPuv (A, C, and E) or stx1 gene on chromosome (B, D, and F) in E. coli HB101 carrying plasmid pGFPuv mixed with E. coli O157:H7 RIMD 0509952, followed by fluorescent anti-E. coli O157 antibody staining. All DAPI-stained bacterial cells were visualized under UV excitation (A and B). Under blue excitation, E. coli O157:H7 cells emitted green fluorescence of FITC-labeled anti-E. coli O157:H7 antibody (C and D). Under green excitation, cells harboring amplified products of the gfp (E) or stx1 (F) genes emitted red fluorescence of Cy3-labeled oligonucleotide probe. The same microscope fields are shown under UV, blue, and green excitation.

 
Three DNA sequences (ori, Fig. 2B; Apr gene, Fig. 3B and F; gfp gene, Fig. 4E) on a plasmid and one DNA sequence (stx1, Fig. 4F) on a chromosome were detected at the single cell level by in situ RCA. The results showed that the probe, primer design, and copy number of the intracellular target gene did not affect the fluorescence intensity of the target cell. Although the copy number of a target gene amplified inside a cell by in situ RCA was not examined, the signal intensity of positive cells was not affected, and positive and negative cells were discriminated visibly in all experiments without dependence on the target sequences.

Investigation of target genes with low copy numbers by in situ nucleic acid amplification have been carried out on cells fixed on coated glass slides because the in situ PCR might cause high background fluorescence with bacterial samples filtered onto a membrane filter (6). This limits the concentration of bacterial samples and reliable enumeration of target bacteria (26). For conventional 16S rRNA targeted FISH, the detachment of bacterial cells was prevented by coating the membrane filter with poly-L-lysine (24). In the present study, samples were trapped onto gelatin-coated filters and embedded in agarose, which was found to be more effective than poly-L-lysine coating (data not shown). Previous studies of in situ LAMP, an alternative to in situ PCR, suggests that mild isothermal gene amplification can decrease nonspecific background fluorescence (25). In situ RCA selectively detected target bacterial cells with low background fluorescence, which might be because thermal cycling was not applied.

Quantification of bacteria taking up plasmid by in situ RCA and conventional culture.
Plasmid uptake by E. coli at different physiological states was determined by both in situ RCA and conventional selective plating to clarify the difference of detection level between culture-dependent and DNA-based methods (Fig. 5). The total cell number slowly decreased and the number of culturable cells rapidly decreased and fell below the detection limit (10 CFU ml–1) after day 101. Plasmid uptake by E. coli cells determined by selective plating was detected until day 27 (<10 CFU ml–1). The ratio of E. coli cells taking up plasmid determined by selective plating to the culturable cells increased 10-fold at day 27 compared to that at day 0 (Fig. 5B).



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  		FIG. 5. Number and ratio of E. coli cells taking up plasmid during prolonged incubation in 10 mM CaCl2 solution. (A) Number of E. coli cells taking up plasmid determined by in situ RCA (crosses) and selective plating on LB agar plate with 50 µg of ampicillin ml–1 (triangles). Total cells were stained with DAPI and counted under fluorescence microscopy (diamonds), and culturable cells on LB agar were enumerated (squares). Samples were taken at days 0, 1, 3, 6, 10, 17, 27, 47, 71, 101, and 149. Error bars represent standard deviations of three replicates of each sample. (B) Comparison of ratio of E. coli cells taking up plasmid determined by in situ RCA (numbers of E. coli cells taking up plasmid/total cells) and selective plating (numbers of E. coli cells taking up plasmid/culturable cells).

 
The number of E. coli taking up plasmid determined by selective plating was consistent with previous findings on LB medium, in which the ratio of E. coli cells taking up plasmid to culturable cells increased (3). The ratio was approximately 102 lower than that in the previous study, perhaps because 102 fewer plasmids (5 x 108 copies) were used in this study.

The number of E. coli cells taking up plasmid determined by in situ RCA was 103-fold higher than that determined by selective plating at day 0. This number increased to 102-fold of the initial number and was 106 higher than that by selective plating at day 10. After day 10, the number of E. coli cells taking up plasmid determined by in situ RCA gradually decreased but remained detectable at day 149. During the experimental period, the number of E. coli cells identified by in situ RCA was 103- to 106-fold higher than that determined by selective plating.

An increase in number of bacteria taking up donor plasmid determined by in situ RCA during the incubation until day 10 might be explained by development of the competence in nonculturable E. coli cells even though such development has been reported only in their culturable state thus far (3, 8). Under starved conditions, E. coli cells are reported to retain substrate responsiveness, respiratory activity, and membrane integrity, even if nonculturable (4). In addition, E. coli cells have competence gene homolog and can take up free DNA in their stationary phase (12). After day 17, the number determined by in situ RCA decreased till day 149, which might be due to an increased number of dead E. coli cells in the suspension (4).

Genetic transformation is characterized by the uptake of free DNA by a recipient bacterium, its chromosomal integration or extra-chromosomal stabilization, and its expression, which leads to a new phenotype (10, 22). Because donor DNA used in the present study was a replicative plasmid in the recipient, all of the E. coli cells that taking up plasmids detected by in situ RCA have potential to become transformants if the plasmid exists stably in a cell and genes on the plasmid are expressed. To exclude the possibility of plasmids being absorbed by dead E. coli cells during in situ RCA, E. coli cells were killed by heating them at 65°C for 30 min before plasmid uptake on each sampling day. In situ RCA did not detect plasmids absorbed by killed E. coli cells in any of the samples (data not shown). This finding suggests that most E. coli cells detected by in situ RCA were nonculturable but retained an intact cell membrane.

In the present study, E. coli cells were incubated in 10 mM CaCl2 solution. E. coli competence for free DNA uptake have been known to be induced by existence of calcium ion. In addition, other reports showed the competence development occurred in river, spring, mineral water, and foodstuffs, in which calcium ion concentrations of two mineral waters were higher than 10 mM (3, 23, 43). A potential future work, therefore, might be to examine DNA uptake by E. coli in aquatic environments using in situ RCA.

The gfp gene is used as a marker in recent lateral gene transfer studies because it can detect bacteria that express the encoded protein under fluorescence excitation without depending on culturability. In a previous study (15), the conjugation frequency using this protein was 103-fold higher than that determined by selective plating or by a culture-based assay. Using expression of the gfp gene, Acinetobacter sp. transformation was detectable at a 106-fold-lower DNA concentration than those reported in other studies (17). To analyze lateral gene transfer in bacteria in different physiological states, detection and quantitation of target bacteria based on DNA sequence using in situ RCA might be useful because any DNA sequence can be targeted without depending on expression or a promoter sequence for the specific gene. In order to apply in situ RCA to indigenous bacteria within an ecosystem, further modification of the permeabilization condition will be required using other bacterial species and starved cells in natural environments. Although insufficient permeabilization conditions might cause false-negative results as shown in Table 2, culture-independent approach described here has described above advantages prior to conventional methods. Combining green fluorescent protein expression with in situ RCA might allow analysis of the environmental and physiological factors that determine the fate of replicative plasmids upon free DNA uptake.

Conclusions and future perspectives.
Independently of the physiological state of E. coli cells carrying a target gene or its promoter sequence, in situ RCA enabled simultaneous visualization of specific genes and surface antigen at the single-cell level.

Although the method reported here used only E. coli as the host microorganisms, our approach is of potential value for microbial ecology research. For example, in situ RCA may be combined with FISH with rRNA-targeted probes to assign environmentally retrieved bacterial artificial chromosome fragments to phylogenetically defined populations. Therefore, further modification of the permeabilization should be carried out to examine the applicability of in situ RCA to bacterial species, including bacterial communities in the natural environment, for enzyme penetration without destruction or detachment of target cells (30, 32, 36).

In situ RCA could differentiate cells harboring a plasmid at much higher efficiency than conventional selective plating. Because the donor DNA was a replicative plasmid in the recipient E. coli, the number of bacteria taking up plasmid determined by in situ RCA was the possible maximum number of transformants if all of the E. coli cells that harbored the plasmid expressed the marker gene. Future studies on gene transfer using plasmid uptake will include a comparison of in situ RCA with expression of the gfp gene as a marker instead of selective plating. Such studies should help improve understanding of free DNA uptake in starved bacteria.


   ACKNOWLEDGMENTS
 
The study was supported by The Development of Monitoring Methods for Microorganisms in Environment Project of New Energy and Industrial Technology Development Organization, Tokyo, Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8170. Fax: 81-6-6879-8174. E-mail: nasu{at}phs.osaka-u.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, December 2005, p. 7933-7940, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.7933-7940.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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