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Applied and Environmental Microbiology, November 2001, p. 5261-5266, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5261-5266.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Direct Detection by In Situ PCR of the
amoA Gene in Biofilm Resulting from a Nitrogen
Removal Process
Tatsuhiko
Hoshino,1
Naohiro
Noda,1
Satoshi
Tsuneda,1,*
Akira
Hirata,1 and
Yuhei
Inamori2
Department of Chemical Engineering, Waseda
University, Shinjuku-ku, Tokyo, 169-8555,1 and
National Institute for Environmental Studies, Tsukuba, Ibaraki
305-0053,2 Japan
Received 26 March 2001/Accepted 29 August 2001
 |
ABSTRACT |
Ammonia oxidation is a rate-limiting step in the biological removal
of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of
the gene encoding ammonia monooxygenase, is important for controlling
nitrogen removal. In this study, the amoA gene present
in Nitrosomonas europaea cells in a pure culture and
biofilms in a nitrifying reactor was amplified by in situ PCR. In this
procedure, fixed cells were permeabilized with lysozyme and subjected
to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon
was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA
gene in the biofilms was detected with an unavoidable nonspecific
signal when the former method was used for detection. On the other
hand, the amoA gene in the biofilms was detected without
a nonspecific signal, and the cells possessing the amoA
gene were clearly observed near the surface of the biofilm when Alexa
Fluor 488-labeled antidigoxigenin antibody was used for detection.
Although functional gene expression was not detected in this study,
detection of cells in a biofilm based on their function was demonstrated.
| |
INTRODUCTION |
The biological removal of
nitrogen compounds is an integral part of most modern wastewater
treatment facilities to preserve environmental water resources. In this
process, ammonia oxidation is a rate-limiting step, where autotrophic
ammonia conversion into hydroxylamine is catalyzed by ammonia
monooxygenase. Thus, analysis of microbial communities possessing the
amoA gene, which encodes ammonia monooxygenase, is important
for controlling nitrogen removal.
On the other hand, fluorescent in situ hybridization (FISH) (4,
9) and denaturing gradient gel electrophoresis (10, 17) based on 16S ribosomal DNA and rRNA for molecular analysis have been used in various fields to determine the genetic diversity of
a microbial community and to identify individual members. In particular, in situ hybridization with fluorescence-labeled
oligonucleotide probes has been widely used for in situ analysis of
microbial communities, such as a biofilm in a wastewater treatment
process (5, 18, 22, 29). This method relies on the
presence of many target sequences within an individual cell. Therefore,
bacterial cells containing insufficient rRNA cannot be detected by this approach. Moreover, this taxonomic identification approach cannot be
used to detect the presence of single-copy functional genes or their
expression at the single-cell level. Hence, in situ hybridization cannot estimate a specific metabolic activity such as ammonia oxidation.
Recently, in situ PCR was developed to amplify and detect functional
genes and their expression inside a single cell, thus making it
possible to detect a single copy of a functional gene. This
method was first developed to amplify and detect a DNA virus inside a
cell (11), and Nuovo et al. (21) and Bagasra
et al. (6) developed this method for molecular pathology.
In environmental microbiology, in situ PCR and in situ reverse
transcription-PCR protocols have been used to detect the presence and
expression of nahA (12) and todC1
(8) in Pseudomonas cells, lac in
Salmonella enterica serovar Typhimurium (28),
and slt-I and slt-II in Escherichia coli O157 (27). However, the in situ PCR protocol has
been applied only to a dispersed sample of a model microbial community
in seawater and river water and has never been used for the analysis of
a biofilm. For this reason, little is known about the distribution of a
functional gene in biofilms. To determine the distribution of
functional genes and their expression in a biofilm is a primary objective in the fields of microbial ecology and wastewater treatment. The purposes of this study were to establish an original in situ PCR
protocol for the detection of the amoA gene in a biofilm and to examine the distribution of a microbial community possessing the
amoA gene in a biofilm for nitrogen removal.
| |
MATERIALS AND METHODS |
Samples and cell fixation.
Nitrosomonas europaea
(IFO 14298) as a representative ammonia-oxidizing bacterium was
cultured in a nitrification medium according to the method of Watson
and Mandel (30) and Bock et al. (7) with
minor modifications. The culture was incubated at 28 to 30°C in the
dark, and then cells were collected by centrifugation and washed with
PBS solution (137 mM NaCl, 8.10 mM
Na2HPO4 · 12H2O, 2.68 mM KCl, 1.47 mM
KH2PO4; pH 7.4). Biofilms
were collected from a laboratory-scale aerobic up-flow nitrifying
reactor that treated inorganic-ammonia-rich wastewater when the ammonia
load was about 1.5 kg of N/m3/day. The
granule-like biofilms were collected through a sampling port and
were settled for a few minutes to separate them from the bioreactor
liquid phase. The samples were suspended in 4% paraformaldehyde in PBS
solution for 16 h (for the pure culture) and 20 h (for the
biofilm) at 4°C for fixation and were then washed with PBS solution.
The fixed biofilm samples were embedded in Tissu Mount (Chiba Medical,
Saitama, Japan) and rapidly frozen at 
25°C. Then, the biofilms were
cut with a cryostat (CM1850; Leica Microsystems, Nussloch, Germany)
into 10-µm-thick sections.
DNA extraction.
DNA extraction was carried out according to
the methods of Smalla (26) with minor modifications. DNA
was extracted from a 0.5-g (wet weight) biofilm pellet. The biofilms
were harvested by centrifugation at 10,000 × g for 10 min. The harvested cells were sonicated with an ultrasonic disrupter
(type UR-20P; probe diameter, 2 mm; TOMY Seiko Co., Ltd., Tokyo, Japan)
at 20 W and 28 kHz for 30 s in 5 ml of sucrose lysis buffer (0.3 M
sucrose, 0.7 M NaCl, 40 mM EDTA, 50 mM Tris-HCl) and then centrifuged
at 2,000 × g for 10 min. The supernatant was incubated
at 55°C in the presence of sodium dodecyl sulfate (0.5%), proteinase
K (0.1 mg/ml), and hexadecylmethylammonium bromide (1%). DNA was
purified by applying phenol, chloroform, and isoamyl alcohol and
precipitated by the addition of ethanol and sodium acetate.
Oligonucleotides.
The following three primers were used:
amoA-1F (5'-GGGGTTTCTACTGGTGGT-3'),
amoA-2R (5'-CCCCTCKGSAAAGCCTTCTTC-3' [K = G
or T; S = G or C]) (25), and amoA-1FF
(5'-CAATGGTGGCCGGTTGT-3'). These primers target stretches
corresponding to positions 332 to 349, 802 to 822, and 187 to 203 of
the open reading frame published previously for the amoA
gene sequence of N. europaea, respectively. The
amoA-1FF primer was designed specifically for
ammonia-oxidizing bacteria belonging to the 
subclass of
Proteobacteria. Specificity was examined using FASTA
(24) and BLAST (1) programs to compare the
primers with the complete sequence data registered in GenBank. As for
FISH analysis, the oligonucleotide probe Nso190 (16) labeled with CY3 was used for in situ detection of ammonia-oxidizing bacteria. Nso190 is a probe specific for a region in the 16S rRNA of
ammonia-oxidizing bacteria.
Cell wall permeabilization.
Cell wall permeabilization and
in situ PCR were carried out according to the method of Hodson et al.
(12) with minor modifications. The washed samples were
resuspended in PBS solution. A 30-µl aliquot was spotted onto amino
alkylsilane-coated slides (Applied Biosystems, Foster City, Calif.) and
dried in an oven at 50°C for 5 min. Prior to cell wall
permeabilization, the fixed samples were dehydrated sequentially in 50, 80, and 100% ethanol. The dehydrated samples were treated with the
lysozyme solution (0.5 mg [for the pure culture] and 1.0 mg [for the
biofilm] of lysozyme per ml, 100 mM Tris-HCl [pH 8.2], and 50 mM
EDTA) for 30 min at room temperature. Lysozyme was removed by
consecutive washes with PBS solution. Further permeabilization was
carried out by treatment with proteinase K at a final concentration of
0.1 µg/ml for 10 min at room temperature, followed by heat
inactivation of proteinase K at 94°C for 3 min. Then, the samples
were washed with the PBS solution and dehydrated sequentially in 50, 80, and 100% ethanol. The samples on the slides were ready for the
addition of PCR mixture.
In situ PCR procedures.
A GeneAmp in situ PCR core kit
(Applied Biosystems) was used for amplification of the amoA
gene in cells. Then, the following seminested PCR protocol was used.
First, a 50-µl aliquot of PCR buffer (10 mM Tris-HCl, 50 mM KCl; pH
8.3) containing 0.6 µM (each) amoA-1FF and
amoA-2R, 2.5 mM MgCl2, 0.6 mM (each)
deoxynucleoside triphosphates, and 10 U of AmpliTaq DNA polymerase,
IS (Applied Biosystems), was added to each sample spot on the
slide and covered with an AmpliCover disk and AmpliCover clip (Applied
Biosystems). The concentrations of polymerase and
MgCl2 were higher than those used in the
conventional PCR because polymerase and MgCl2
tend to adhere to a glass slide or a coverslip (20). PCR
was performed with the following temperature profile using a GeneAmp in
situ PCR system 1000 (Applied Biosystems): initial denaturation at 94°C for 90 s and then 20 cycles of denaturation at 94°C for
40 s, annealing at 53°C for 30 s, and extension at 72°C
for 40 s. The slide was then washed with 0.5× SSC (750 mM NaCl,
75 mM trisodium citrate; pH 7.0) at 45°C for 10 min. In the second
PCR, the reaction mixture was the same as that for the first
amplification except that amoA-1FF was replaced by
digoxigenin-labeled amoA-1F.
Detection of the amplified gene with Alexa Fluor 488-labeled
antidigoxigenin antibody.
A digoxigenin-labeled amplicon was
detected with an Alexa Fluor 488 (Molecular Probes Inc., Eugene,
Oreg.)-labeled antidigoxigenin antibody. Antidigoxigenin Fab fragments
(Roche Diagnostics, Mannheim, Germany) were labeled with an Alexa Fluor
488 protein labeling kit (Molecular Probes) according to the
manufacturer's instructions. First, 50 µl of a blocking buffer (30 µg of bovine serum albumin per ml in PBS solution) was spotted onto
the samples and incubated for 30 min at room temperature. Then, 25 µl
of the buffer was replaced by 25 µl of alkaline
phosphatase-conjugated antidigoxigenin Fab fragments (Roche
Diagnostics) diluted to 68 nM (for biofilm) or 0.27 µM (for pure
culture) in a dilution buffer (100 mM Tris-HCl [pH 7.5], 150 mM
NaCl), and incubated at 37°C for 1 h. After that, the slides
were washed with buffer I (100 mM Tris-HCl [pH 7.5], 150 mM NaCl,
0.05% Tween 20) three times and then washed with buffer II (100 mM
Tris-HCl [pH 8.0], 100 mM NaCl, 10 mM MgCl2) twice. Then, the samples were counterstained with TO-PRO-3 (Molecular Probes) for 5 min. For preparation of a working solution, TO-PRO-3 was
diluted with double-distilled water (ddH2O)
1,000-fold. After washing, the samples were mounted in FluoroGuard
antifade reagent (Bio-Rad, Richmond, Calif.) for observation under a
confocal laser scanning microscope (TCS4D; Leica Lasertechnik,
Heidelberg, Germany) equipped with an Ar-Kr ion laser (488, 568, and
647 nm). Confocal images were obtained with a PL FLUOTAR ×10/0.30
objective (total magnification, ×100) and PL APO ×63/1.40 oil
objective (total magnification, ×630). Figures were composed using
Adobe Photoshop 6.0 (Adobe Systems, San Jose, Calif.) on a Macintosh
PowerPC (Apple Computer Co., Cupertino, Calif.).
Detection of the amplified gene with alkaline phosphatase-labeled
antidigoxigenin antibody.
The detection of the amplicon with an
alkaline phosphatase-labeled antidigoxigenin antibody and
2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP), which was
combined with Fast Red TR according to the methods of Yamaguchi et al.
(31) with minor modifications. HNPP is converted to HNP by
alkaline phosphatase. Then, HNP combines with Fast Red TR and produces
a bright red fluorescent material, HNP-TR (13). The
procedure was almost the same as that for detection using the Alexa
Fluor 488-labeled antidigoxigenin antibody. After blocking, 25 µl of
the buffer was replaced by 25 µl of alkaline phosphatase-labeled
antidigoxigenin antibodies diluted to 60 in the dilution buffer and
incubated at 37°C for 1 h. After the slides had been washed, the
samples were counterstained with TO-PRO-3 for 5 min. After being
washed with buffer II, the samples were incubated in HNPP-Fast Red TR
(Roche Diagnostics) solution (100 µg of HNPP per ml and 250 µg of
Fast Red TR per ml in buffer II) for 15 min at room temperature, and
the slides were washed with ddH2O. The samples
were mounted in Macllavaine buffer (53.2 mM citric acid and 93.6 mM Na2HPO4 [pH 4.5]) for
observation under a confocal laser scanning microscope.
FISH targeting 16S ribosomal DNA.
Hybridization was
performed according to the standard protocol described by Amann
(2) at 46°C for 2.5 h in a hybridization buffer
containing NaCl (0.9 M), formamide (40%) (5, 16), Tris-HCl (20 mM, pH 7.4), and sodium dodecyl sulfate (0.01%). The
probe concentration was 0.5 ng/µl. Hybridization was followed by a
stringent washing step at 48°C for 20 min in a washing buffer containing Tris-HCl (20 mM, pH 7.4), NaCl (35 mM) (5, 16), and sodium dodecyl sulfate. The washing buffer was removed by rinsing
the slides with ddH2O. Then, the samples were
counterstained with TO-PRO-3, mounted in FluoroGuard antifade reagent
(Bio-Rad), and observed under the confocal laser scanning microscope.
PCR and agarose gel electrophoresis.
To confirm specificity,
extracted DNA was amplified by PCR with amoA-1F,
amoA-2R, and amoA-1FF. Extracted DNA was added to 50 µl of a PCR buffer (5 mM Tris-HCl, 5 mM KCl, 10 µM EDTA, 0.1 mM
dithiothreitol, 0.0001% Tween 20, 0.0001% Nonidet P-40) containing 0.5 µM (each) primers, 200 µM deoxynucleotide triphosphate, 1 mM
MgSO4, and 1 U of KOD DNA polymerase
(Toyobo Co., Ltd., Osaka, Japan). PCR was carried out in a GeneAmp PCR
system 9700 (Applied Biosystems) using the same cycle program as the in
situ PCR cycle program. PCR products were electrophoresed in a 2%
agarose gel and visualized by ethidium bromide staining.
| |
RESULTS |
Amplification of amoA from extracted DNA.
Amplification of the amoA gene from extracted DNA of the
biofilms using the oligonucleotide primer set amoA-1FF and
amoA-2R resulted in a 636-bp DNA fragment with a slight
smear (Fig. 1, lane 2). The 636-bp
amplicon was reamplified using the seminested primer set
amoA-1F and amoA-2R, and as a result, only a
491-bp amplicon was generated (Fig. 1, lane 1).
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FIG. 1.
Gel electrophoresis of amoA gene products
amplified from extracted DNA of the biofilms. Lane 1, 491-bp
amoA gene fragment amplified by primers
amoA-1F and amoA-2R; lane 2, 636-bp
amoA gene fragment amplified by primers
amoA-1FF and amoA-2R
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|
amoA gene detection with in situ PCR.
To
establish the in situ PCR protocol, the amoA gene in
N. europaea was amplified by in situ PCR. At first,
detection of the amplicon generated by amoA-1F and
amoA-2R was attempted by in situ hybridization with the
fluorescein isothiocyanate-labeled probe. However, no signal was
observed under the confocal laser scanning microscope. An insufficient
amount of amplicon or problems related to hybridization were thought to
be the cause of this failure. To solve these problems, the seminested
PCR protocol has been used to increase the specificity and sensitivity
of in situ PCR (12, 17, 28). The amoA-1F and
amoA-2R primers were used in the first PCR, and
amoA-1F and a fluorescein isothiocyanate-labeled primer that
was complementary to a region internal to amoA-1F and
amoA-2R were used in the second PCR. As a result, although the level of sensitivity was certainly increased, the level of signal
intensity was still very low. To increase the sensitivity further, a
digoxigenin-labeled primer was used in the second PCR, and the amplicon
was detected with the alkaline phosphatase-labeled antidigoxigenin
antibody and HNPP combined with Fast Red TR. The level of sensitivity
was increased significantly, and thus, this method was applied to the
subsequent in situ PCR procedure.
First, the
amoA gene in an
N. europaea cell was
amplified and detected. In situ PCR requires that the polymerase be
able to
penetrate into the cells and act on the nucleic acid in cells
without the PCR amplicon diffusing away from the cells.
Therefore,
to determine the optimal pretreatment conditions, the time
of
fixation with 4% paraformaldehyde was varied from 4 to 20 h
and
the lysozyme concentration was varied from 0.05 to 5.0 mg/ml.
As a
result, the
amoA gene was successfully detected inside
intact
N. europaea cells that had been fixed for 16 h
and permeabilized
with 0.5-mg/ml lysozyme buffer (Fig.
2A). Moreover, the protocol
was applied
to the biofilm that was collected from the up-flow
aerobic nitrifying
reactor. The fixation time was varied from
4 to 32 h, and the
lysozyme concentration was varied from 0.05
to 5.0 mg/ml. The
amoA gene was detectable in the sample fixed
for 20 h
and permeabilized with 1.0 mg of lysozyme per ml. However,
an
unavoidable nonspecific signal was particularly observed in
the case of
the biofilm (Fig.
2B and C).
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FIG. 2.
Detection of ammonia-oxidizing bacterial cells
possessing amoA gene in pure culture (A) and in the
biofilm (B and C), with alkaline phosphatase-labeled antidigoxigenin
antibody. Panels B and C show the same biofilm. The yellow color
indicates positive cells, while the green shows negative cells (arrows
indicate the nonspecific signal). The portion of panel B that was
magnified to produce panel C is indicated with a white box.
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|
To reduce the intensity of the nonspecific signal, the
digoxigenin-labeled amplicon in the cell was detected with
antidigoxigenin
antibodies labeled with Alexa Fluor 488. As a result,
the
amoA gene was detected inside
N. europaea
cells without the nonspecific
signal (Fig.
3A). Moreover, the
amoA gene
in the biofilm was also
detected with a weak nonspecific signal, as
shown in Fig.
3B and
C. The green fluorescence in Fig.
3C (upper right)
is the supposed
nonspecific signal caused by adsorption of the
antibodies. However,
it was clear that cells possessing the
amoA gene were found near
the surface of the biofilm.
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FIG. 3.
Detection of the cells possessing amoA in
pure culture (A) and in the biofilm (B and C), with Alexa Fluor
488-labeled antidigoxigenin antibody. Panels B and C show the same
biofilm. The yellow color indicates positive cells, while the red shows
negative cells. The portion of panel B that was magnified to produce
panel C is indicated with a white box.
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|
FISH.
To confirm the validity of microbial communities
detected by in situ PCR, ammonia-oxidizing bacteria in the biofilm were
visualized by FISH analysis (Fig. 4). The
yellow shows ammonia-oxidizing bacteria, and the green shows other
bacteria. It was observed that ammonia-oxidizing bacteria were the
dominant population on the surface of the biofilm.
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FIG. 4.
Detection of ammonia-oxidizing bacteria with a
CY3-labeled Nso190 probe. Panels A and B show the same biofilm. The
yellow color indicates ammonia oxidizers, while the green shows other
bacteria. The portion of panel A that was magnified to produce panel B
is indicated with a white box.
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| |
DISCUSSION |
At first, the amoA gene in the N. europaea
cells (pure culture) and the biofilm (mixed culture) was detected with
an alkaline phosphatase-labeled antidigoxigenin antibody; however, an
unavoidable nonspecific signal was observed, which has also been
reported by Tani et al. (27). This was probably due to the
fluorescent HNP-TR diffusing out of the cells.
To reduce the intensity of the nonspecific signal, a new strategy of
detecting the amplicon in the cells with an Alexa Fluor 488-labeled
antidigoxigenin antibody was proposed. First, detection of the
amoA gene in the cell was conducted completely under the same conditions as for the alkaline phosphatase-labeled antidigoxigenin antibody. However, a sufficient signal intensity was not obtained, which might be due to the lower penetration efficiency and/or the lower
sensitivity of the antibody compared with the alkaline phosphatase-labeled antidigoxigenin antibody. Therefore, several concentrations (0.035, 0.07, and 0.14 µM) were attempted to increase signal intensity. In consequence, when the concentration of the antibody was increased to 0.14 µM, for the N. europaea
cells, the amoA gene was detected without the nonspecific
signal. As for the biofilm, the amoA gene was successfully
detected with a low background signal, although the concentration of
the antibody was increased to 35 nM. These results indicate that the
use of Alexa Fluor 488-labeled antibody enables the construction of a promising detection system that can be applied to in situ PCR.
The images obtained both by in situ PCR analysis with the Alexa Fluor
488-labeled antibody and by FISH analysis show that the
ammonia-oxidizing bacteria were found near the surface of the biofilms.
This result suggests that the bacteria possessing the amoA
gene were successfully detected by in situ PCR. Although FISH analysis
is a very important tool in microbial ecology, cells with an
insufficient ribosomal content cannot be detected by this method.
Practically, a fluorescent signal was observed only near the surface of
the biofilms by FISH analysis with EUB338 (a probe targeting all
bacteria except Archaea), which was probably due to the low
activity of the bacteria living in a deep part of the biofilm (data not
shown). In spite of extensive attempts to increase the intensity of the
fluorescent signal from FISH by sample enrichment (25) and
using a single probe with multiple fluorochromes (4) and
multiple probes (3), low-activity cells are difficult to detect. In contrast, a few copies of the amoA gene in the
cells were successfully detected by in situ PCR. This indicates that cells possessing a functional gene can be detected by in situ PCR
regardless of its activity. Actually, when the biofilm cultivated without ammonia for a month was examined by FISH and in situ PCR, ammonia-oxidizing bacteria were not detected by FISH at all, whereas the cells possessing the amoA gene were detected near the
surface of the biofilms by in situ PCR (data not shown).
However, the images obtained by in situ PCR analysis are less distinct
than those obtained by FISH analysis. This was probably because cells
were damaged by permeabilization and/or thermal cycling, as well as (i)
diffusion of the labeled amplicon and its adhesion to negative cells,
(ii) adhesion of the digoxigenin-labeled primer to negative cells, and
(iii) adhesion of the Alexa Fluor 488-labeled antibody to negative cells.
Cases 2 and 3 are practically inconceivable because no signal was
detected when the negative control without polymerase was examined. As
for case 1, this diffusion artifact has been reported as a problem of
in situ PCR (14, 20). On the other hand, Nuovo (19) reported that the movement of DNA segments was
restricted not by the pore size but rather by biochemical forces, such
as hydrogen bonding and ionic charges. Therefore, under optimal
fixation and permeabilization conditions, there is minimal migration of the amplicon from its site of origin, and diffusion artifacts are
significantly reduced by reduction of the PCR cycle (
30), generation
of longer PCR products, or incorporation of biotinylated nucleotides to
generate bulkier and thus less diffusible PCR products (15,
23). In this study, to increase the level of sensitivity, the
seminested PCR protocol was used, and the total number of thermal
cycles was 40 cycles. This led to the destruction of the cell
morphology, which probably caused the diffusion artifacts. However,
taking into account that fewer cycles caused a very low signal, it is
important to optimize these parameters very carefully and to simplify
the PCR protocol to the furthest extent possible.
In this study, the cells possessing the amoA gene
were successfully detected by in situ PCR with the Alexa Fluor
488-labeled antibody. As a result, bacteria could be detected by in
situ PCR regardless of its rRNA content and could be detected
based on their function. On the other hand, problems that are not
observed when in situ PCR is applied to dispersed samples were revealed when in situ PCR was applied to biofilms. However, the fact that the
spatial distribution of a functional gene in the biofilm becomes detectable represents significant progress in this field. This in situ
PCR is expected to be a valuable method in cases such as those in which
the ammonia oxidation rate has dropped although the number of
ammonia-oxidizing bacteria is not changed, and it will be essential for
explaining such phenomena. It is anticipated that in future studies,
functional gene expression (mRNA) will be detected by in situ PCR, and
consequently the spatial distribution of the "actually functioning"
bacteria in a biofilm from the natural environment or from a wastewater
treatment system will be elucidated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku,
Tokyo, 169-8555, Japan. Phone: 81-3-5286-3210. Fax: 81-3-3209-3680. E-mail: stsuneda{at}mn.waseda.ac.jp.
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Applied and Environmental Microbiology, November 2001, p. 5261-5266, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5261-5266.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
