R E S E A R C H L E T T E R
Developmentofanoligonucleotidemicroarray todetect di- andmonooxygenasegenes for benzene degradation in soilShoko Iwai1, Futoshi Kurisu2, Hidetoshi Urakawa3, Osami Yagi4, Ikuro Kasuga1 & Hiroaki Furumai2
1Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan; 2Research Center for Water Environment
Technology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan; 3Center for Advanced Marine Research, Ocean Research Institute,
The University of Tokyo, Tokyo, Japan; and 4Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan
Correspondence: Shoko Iwai, Center for
Microbial Ecology, Michigan State University,
A-540E Plant and Soil Sciences Building, MI,
48824, USA. Tel.: 11 517 355 0271, ext. 289;
fax: 11 517 353 2917; e-mail:
Received 4 February 2008; accepted 5 May
2008.
First published online 10 June 2008.
DOI:10.1111/j.1574-6968.2008.01223.x
Editor: Elizabeth Baggs
Keywords
benzene; dioxygenase; microarray;
monooxygenase; oligonucleotide probe.
Abstract
Diverse environmental genes have been identified recently. To characterize their
functions, it is necessary to understand which genes and what combinations of
those genes are responsible for the biodegradation of soil contaminants. In this
article, a 60-mer oligonucleotide microarray was constructed to simultaneously
detect di- and monooxygenase genes for benzene and related compounds. In total,
148 probes were designed and validated by pure-culture hybridizations using the
following criteria to discriminate between highly homologous genes: � 53-bp
identities and � 25-bp continuous stretch to nontarget sequences. Microarray
hybridizations were performed using PCR products amplified from five benzene-
amended soils and two oil-contaminated soils. Six of the probes gave a positive
signal for more than six soils; thus, they may represent key sequences for benzene
degradation in the environment. The microarray developed in this study will be a
powerful tool for the screening of key genes involved in benzene degradation and
for the rapid profiling of benzene oxygenase gene diversity in contaminated soils.
Introduction
Benzene is a common soil contaminant resulting from the
leakage of oil from oil production facilities. Thus, there is a
great deal of interest in the biodegradation of benzene, and
its pathways and enzymatic activities have been well char-
acterized using pure culture strains (Irie et al., 1987; Tan
et al., 1993; Johnson & Olsen, 1995; Kitayama et al., 1996;
Na et al., 2005). The first step in the aerobic degradation of
benzene is mediated by two oxygenases: benzene dioxygen-
ase and benzene monooxygenase. Although diversity among
the benzene oxygenase genes in environmental samples has
been reported (Witzig et al., 2006; Iwai et al., 2007), the
diversity among both oxygenase genes together in the
environment has not been discussed.
Microarray technology is a powerful tool for the analysis
of such diverse catabolic genes because it allows the simul-
taneous detection of large numbers of sequences. However,
microarrays are limited in that they can only be used to
detect known sequences. Regardless, microarray analyses
have revealed much broader gene diversity in soil compared
with conventional clone library techniques (Wagner et al.,
2007). Given that the amount of genetic information avail-
able in gene databases is expanding, microarray technology
will be of even greater use in the near future. Already,
microarray analyses have been conducted in several fields of
microbiology (Zhou, 2003; Bodrossy & Sessitsch, 2004;
Wagner et al., 2007), and some microarrays targeting cata-
bolic genes such as aromatic oxygenase genes (Denef et al.,
2003), which are known contaminants of biodegradation
genes and metal resistance genes (Rhee et al., 2004), and
benzene monooxygenase genes (Iwai et al., 2007) have been
constructed.
Though oligonucleotide microarrays have many advan-
tages, including probe design flexibility and independence
from the culturability of the target (Relogio et al., 2002),
difficulties remain in terms of probe specificity (Liu et al.,
2001; El Fantroussi et al., 2003; Wagner et al., 2007). Many
traditional DNA-shuffling experiments have demonstrated
that the mutation of a few amino acids can critically affect
the structure of individual enzymes, changing their sub-
strate utilization and degradation activities (Parales et al.,
2000; Suenaga et al., 2002; Bagneris et al., 2005; Vardar &
Wood, 2005). Therefore, it is necessary to distinguish
FEMS Microbiol Lett 285 (2008) 111–121 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
between genes that differ in only a few base pairs in order to
understand their biodegradation potential.
In this study, a microarray that includes both di- and
monooxygenase genes for benzene and related genes was
developed based on an analysis of genetic diversity. We
validated the specificity of each probe by pure-culture
hybridization and proposed probe design criteria to distin-
guish each gene. The microarray detected diverse oxygenase
genes in several benzene-amended soils and oil-contami-
nated soils. Moreover, the potential of the microarray for
identifying key sequences involved in biodegradation and
for profiling the degradation characteristics of contaminated
soils were discussed.
Materials and methods
Bacterial strains, plasmids, and soil samples
Pseudomonas pseudoalcaligenes KF707 was grown at 28 1C in
Luria–Bertani broth medium, whereas Pseudomonas aerugi-
nosa JI104 and Pseudomonas putida F1 (DSMZ 6899) were
grown in trypticase soy broth medium at 30 and 28 1C,
respectively. Plasmid pTB1 carrying bphA1 (Arai et al.,
1998), plasmid pB4E carrying bnzA1 (Na et al., 2005),
plasmid pTKScSI carrying bphA1 (Hiraoka et al., 2002),
and plasmid pIP103 carrying cumA1 (Aoki et al., 1996) were
used as PCR templates. Soils I–V were uncontaminated soils
collected in Japan from a lotus field in Tsuchiura, Ibaraki, an
agricultural field, the roadside in a residential area, the edge
of a pond, and the roadside in an area of heavy traffic in
Bunkyo-ku, Tokyo, respectively. In addition to these soils,
two oil-contaminated soil samples (Oil A and Oil B)
collected at different sites were used.
Genomic DNA extraction and whole-genomeamplification
Genomic DNA was extracted from the pure culture strains
using a Wizard Genomic DNA Purification Kit (Promega,
Madison, WI) as described previously (Iwai et al., 2007).
Benzene degradation was performed as described previously
(Iwai et al., 2007) using Soils I–V. Briefly, 500 mg of benzene
(Kishida Chemical, Osaka, Japan) was added to 5 g (wet
weight) of soil in a 35-mL vial and incubated aerobically at
25 1C. Near-complete degradation (4 99%) was observed
after 4, 12, 3, 6, and 4 days for Soils I–V, respectively.
Genomic DNA was extracted from 5 g of soil before and
after the experiment for Soils I and II, after the experiment
for Soils III–V, and directly for Oils A and B. The concentra-
tion of the extracted DNA was quantified and qualified using
a spectrophotometer. The DNA was then amplified using an
REPLI-g Midi Kit (Qiagen, Hilden, Germany) in accordance
with the manufacturer’s instructions. The amplified DNA
was stored at � 20 1C.
PCR primer design, amplification, andpurification
DNA and amino acid sequences of several toluene/biphenyl
family dioxygenase genes (Gibson & Parales, 2000) were
retrieved from the DDBJ/EMBL/GenBank database and
aligned using CLUSTALW version 1.83 (Thompson et al.,
1994). Using the conserved regions of these sequences, two
sets of PCR primers, BEDe (BEDemF and BEDeR) and
BEDm (BEDemF and BEDmR), were designed: BEDemF
(forward), 50-CAYGGVTGGGCBTAYGAYA-30; BEDeR (re-
verse), 50-GTTYTCDCCRTCRTCYTGCT-30; and BEDmR
(reverse), 50-GGAASGARCASGTDGGRAA-30. The unique-
ness of each target was confirmed by comparison to the
database. The reaction mixtures used for amplification were
prepared in 50mL as described previously (Iwai et al., 2007).
The conditions were optimized by changing the annealing
temperature, MgCl2 concentration, and each primer con-
centration. The parameters were finally fixed at 60 1C,
2.0 mM, and 0.6 mM for the BEDe primer set and 58 1C,
2.5 mM, and 0.6 mM for the BEDm primer set. Amplifica-
tion was performed as follows: 10 min at 95 1C followed by
30 cycles of 1 min at 95 1C, 1 min at the annealing tempera-
ture, and 1 min at 72 1C, with a final extension for 10 min at
72 1C. All experiments included a control tube that did not
contain DNA. The products from each pure-culture DNA
and BEDe- or BEDm-amplified soil sample were purified
using a QIAquick PCR Purification Kit (Qiagen).
Cloning, sequencing, and phylogenetic analysis
The purified products were cloned using a QIAGEN PCR
Cloning plus Kit (Qiagen) and sequenced using an ABI
PRISM 3100-Avant Genetic Analyzer (Applied Biosystems)
or an Applied Biosystems 3730xl DNA Analyzer with
BigDye version 3.1 sequencing chemistry. The sequences
were assembled and translated into amino acids then aligned
with reference sequences using CLUSTALW (Thompson et al.,
1994). Phylogenetic trees were constructed by neighbor-
joining analysis (Saitou & Nei, 1987) with 1000 bootstrap
replicates. Pairwise distances were calculated by PROTDIST
(Felsenstein, 1989) and input to DOTUR (Schloss &
Handelsman, 2005) to generate rarefaction curves.
Oligonucleotide probe design and microarrayconstruction
The phylogenetic software package ARB (Ludwig et al., 2004)
and the BLAST program (Altschul et al., 1997) were used to
design the probes and to predict their specificity. The GC%
value was set from 40 to 60. Continuous stretches between the
probes and targets were calculated using the PERL program as
described previously (He et al., 2005b). The Tm of each probe
was calculated using the nearest-neighbor method (Breslauer
FEMS Microbiol Lett 285 (2008) 111–121c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
112 S. Iwai et al.
et al., 1986; Sugimoto et al., 1996). A portion of two human
gene sequences used as control probes (BC008972 and
BC065005) by Moisander et al. (2006) were used as positive
controls, while two probes (Nonsense1 and Nonsense2)
modified from Loy et al. (2002) were used as negative controls
(Supplementary Table S1). Each probe was custom synthe-
sized and spotted in triplicate onto a Gene Slide (Toyo Kohan,
Tokyo, Japan) by Hokkaido System Science Co. (Hokkaido,
Japan) as described previously (Iwai et al., 2007).
Microarray hybridization
The purified products were labeled with Cy3 using a
BioPrime Array CGH Genomic Labeling Module (Invitro-
gen, Carlsbad, CA) as described previously (Iwai et al.,
2007). The labeled fragments were then purified using a
QIAquick PCR Purification Kit (Qiagen). The DNA con-
centration and labeling efficiency were determined by mea-
suring the OD260 nm and OD550 nm. Oligonucleotides
complementary to each of the positive control probes were
synthesized and labeled with Cy3 at the 50 end. A total of
20 ng of amplified and labeled pure-culture DNA or 500 ng
of amplified and labeled soil sample DNA was mixed with
the positive control oligonucleotides and 20mg of salmon
sperm DNA (BioDynamics Laboratory Inc., Tokyo, Japan).
Hybridization was performed using DIG Easy Hyb Buffer
(Roche Diagnostics, Basel, Switzerland) for 16 h at 60 1C,
and the slide was then washed and scanned as described
previously (Iwai et al., 2007). The signal to noise ratio (SNR)
was calculated based on the following formula: SNR = signal
intensity/SD of the local background (Rhee et al., 2004; He
et al., 2005a, b). A single slide was prepared for the pure-
culture DNA, whereas two slides were prepared for the soil
DNA. Those spots for which all replicate datasets (three
replicates for the pure-culture DNA hybridizations or six
replicates for the soil DNA hybridizations) had SNRs Z3
were considered positive signals.
Nucleotide sequence accession numbers
The nucleotide sequences described in this study have been
submitted to the DDBJ/EMBL/GenBank database under
the following accession numbers: AB274171–AB274198,
AB274200–AB274242, and AB294056–AB294105.
Fig. 1. Phylogenetic analysis of the large subunits of toluene/biphenyl family dioxygenase genes. The tree was constructed by the neighbor-joining
method with 1000 bootstrap replicates. The numbers at each branch point indicate the bootstrap percentages. dxnA1 from Sphingomonas sp. RW1
(X72850) was used as the outgroup to root the dendrograms. Those genes amplified using the BEDe or BEDm primer set are indicated. The scale bar
represents 5% estimated sequence divergence.
FEMS Microbiol Lett 285 (2008) 111–121 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
113An oligonucleotide microarray for oxygenase genes
Fig. 2. Phylogenetic analysis of the benzene oxygenase gene fragments from sampled soils on the basis of their deduced amino acid sequences and
specific probes. Cloned sequences were obtained using dioxygenase gene-specific primers (BEDe or BEDm) (a), and monooxygenase gene-specific
primers [BO12 (b) and RDEG (c)]. Phylogenetic trees were constructed from a CLUSTALW alignment of 180–190 aa (a), 131 aa (b), and 140–142 aa (c) by the
neighbor-joining method with 1000 bootstrap replicates. dbdCa from Xanthobacter polyaromaticivorans 127W (AB121977) (a), bupA4 from
Pseudomonas putida MT4 (AB107791) (b), and poxD from Ralstonia sp. E2 (AF026065) (c) were used as outgroups to root the dendrograms. The
scale bars represent the number of substitutions per amino acid. The clones were named in the following manner: (PCR primer set) – (name of soil) –
(days of incubation) – (clone no.). Those clones that were identical in sequence are indicated in parentheses after the clone name. The clones listed in
square parentheses were analyzed by PCR restriction fragment length polymorphism alone (Iwai et al., 2007). The probes were named based on the
target clone name and a letter. Each letter indicates a different probe for the same target.
FEMS Microbiol Lett 285 (2008) 111–121c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
114 S. Iwai et al.
Results and discussion
PCR primer design for benzene and relateddioxygenase genes
Several primer sets have been developed for dioxygenase
amplification (Taylor et al., 2002; Baldwin et al., 2003; Nı
Chadhain et al., 2006; Witzig et al., 2006); however, new
dioxygenase genes that cannot be amplified using these
previously reported primer sets are continually being dis-
covered. Therefore, new primer sets were designed based on
the updated dioxygenase dataset in the DDBJ/EMBL/Gen-
Bank database. Because none of the primer sets could cover
the Rieske nonheme iron dioxygenases of the toluene/
biphenyl family (Gibson & Parales, 2000), which includes
all reported benzene dioxygenase genes, two sets of primers
(BEDe and BEDm) were designed (Fig. 1). The BEDe and
BEDm primer sets successfully amplified 810- and 607-bp
fragments from reference strain genomic DNA or plasmids,
respectively (data not shown).
Diversity of the dioxygenase sequences inbenzene-degrading soils
DNA was extracted from Soils I and II before and after the
degradation of benzene. PCR products were obtained using
the BEDe primer set from Soil I after the degradation of
benzene only. In contrast, PCR products were obtained
using the BEDm primer set from all samples except Soil II
before degradation. Clone libraries were subsequently con-
structed for the amplicons. In total, 167 clones were
analyzed and 46 types of deduced amino acid sequences
were obtained. Forty-four of the clones had Asp230, His233,
and His239, which are conserved iron-binding sites among
Rieske nonheme iron dioxygenase genes (Furusawa et al.,
2004). No identical sequences were identified between the
two soils. Next, a phylogenetic tree was constructed that
included all reported toluene/biphenyl family dioxygenase
genes (Fig. 2a). The sequence of clone BEDm-II-12-20 (four
clones) was identical to ipbA1 from Rhodococcus erythropolis
BD2, while the sequence of clone BEDm-II-12-2 (eight
clones) was identical to tidA from Rhodococcus aetherivorans
I24 and bnzA1 from Rhodococcus opacus B-4. The rest of the
sequences were unique to each soil library and distributed in
many clusters.
Microarray probe design
Two types of monooxygenase genes for benzene degradation
were analyzed previously (Fig. 2b and c; Iwai et al., 2007).
Sixty-mer oligonucleotide probes were designed based on
the phylogenetic trees to cover all retrieved and reported
Fig. 2. Continued.
FEMS Microbiol Lett 285 (2008) 111–121 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
115An oligonucleotide microarray for oxygenase genes
di- and monooxygenase genes for the degradation of
benzene and related compounds (dioxygenase, Fig. 2a;
monooxygenase, Fig. 2b and c). Each probe had Z58-bp
identity (96.7% of 60-bp) except for probes BEDm_II-12-
11-a and -b, which had Z57-bp identity with the target
sequences. The rarefaction curve at the 3% difference level
shows a near plateau (Fig. 3), indicating that sufficient
coverage for an inclusive dioxygenase gene diversity study
Fig. 2. Continued.
FEMS Microbiol Lett 285 (2008) 111–121c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
116 S. Iwai et al.
was achieved using those probe sets. Previously, we designed
83 probes targeted to a subset of benzene monooxygenase
genes (Iwai et al., 2007). The design criteria used for those
probes (� 54 bp of the probe identity and � 20 bp of the
continuous stretch to the nontarget sequence) were strict
enough to successfully differentiate between each gene.
However, when we used those criteria to design probes for
each gene in the phylogenetic tree, they were too strict to
cover such highly homologous sequences. Thus, we relaxed
the criteria to � 56 bp of the probe identity and � 31 bp of
the continuous stretch to the nontarget genes in order to
cover all those genes. Moreover, we designed up to three
different probes for each gene using the new criteria to
confirm its presence (Loy et al., 2002; Bodrossy et al., 2003;
Iwai et al., 2007). For some targets, the probes (up to six
probes) used in our previous study met the new criteria;
thus, they were also used. In total, 53 and 95 probes were
designed for the di- and monooxygenase genes, respectively
(Fig. 2). The predicted Tm of each probe was between 72 and
80 1C. Detailed information concerning all 148 oligonucleo-
tides is listed in supplementary Table S1.
Microarray fabrication and validation
The specificity of each probe was evaluated by hybridization
with amplified DNA from seven pure cultures: BEDm-
amplified genomic DNA from P. putida F1, BEDm-ampli-
fied pTB1 (Arai et al., 1998), BEDe-amplified genomic DNA
from P. aeruginosa JI104, BEDe-amplified genomic DNA
from P. pseudoalcaligenes KF707, BEDe-amplified pB4E (Na
et al., 2005), BEDe-amplified pTKScSI (Hiraoka et al.,
2002), and BEDe-amplified pIP103 (Aoki et al., 1996). One
to three probes were available for the detection of these PCR
products using the microarray (target probes). All target
probes showed positive signals with SNRs between 191 and
1903; no false negatives were observed. Only nine of 1020
probes (all expected negative probes in the pure-culture
hybridizations) turned out to be false positives, and each of
these had a relatively low signal intensity (SNRs between 3
and 79). All positive control probes and negative control
probes had SNRs of Z3 and o 3, respectively. The calcu-
lated identities and continuous stretch of the sample DNAs
to the nontarget probes are shown in Fig. 4. When the
identity or continuous stretch of the sample sequence to the
probe was set at 4 53 or 4 25 bp, respectively, all probes
showed false-positive signals. Thus, we defined this bound-
ary as the mis-hybridization threshold (MHT). One of the
most critical problems in the application of microarray
technology to environmental samples is its lack of ability to
distinguish between highly homologous genes. In order to
detect a target gene specifically, probes with a similarity that
is lower than the MHT to the nontarget genes should be
designed.
Hybridization of the benzene-amended and oil-contaminated soils
The microarray profiles obtained from the benzene-
amended and oil-contaminated soils are shown in Fig. 5.
Because those probes with higher similarity than the MHT
to the nontarget sequences carried an increased risk of false-
positive signals, the positive signals for those probes are
indicated in parentheses [(1)] in Fig. 5. All sequences
detected by PCR-based cloning from Soils I and II were also
detected as positive signals by microarray analysis. Some
probes were positive only for Soil I or Soil II; in contrast,
even excluding our (1) results, BED_bnzA1-b was positive
Fig. 3. Rarefaction analysis of the dioxygenase clone library. Difference
levels of 3% are shown.
Fig. 4. Identity and continuous stretch of each probe to the sample
sequence in pure-culture hybridization. Only the results for the nontarget
probes (expected to be negative) are shown. Closed circles, false-positive
signals; open circles, expected negative signals.
FEMS Microbiol Lett 285 (2008) 111–121 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
117An oligonucleotide microarray for oxygenase genes
Fig. 5. Microarray profiles for the various soil
samples. 1, positive signal (all six replicate spots
had SNRs Z3); � , negative signal; NA, not
applicable (the sample was not amplified by the
primer set). Each ‘1’ or ‘� ’ corresponds to each
probe in alphabetical order. Positive signals
obtained using the probes with 4 53-bp identity
or 4 25-bp continuous stretch to the nontarget
sequences are indicated in parentheses. Cluster
analysis was performed by converting the
microarray profiles (positive signals = 1, negative
signals = 0). NA was considered a negative signal.
Probe RD_II-12-49 was previously shown to be
strongly positive (SNRs of 1030–1270; Iwai et al.,
2007) but was negative in the present study,
possibly because of a failure in spotting. Thus, we
excluded the results for this probe from further
analysis (gray shading).
FEMS Microbiol Lett 285 (2008) 111–121c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
118 S. Iwai et al.
in all seven soil samples while BEDm_II-12-5-a,b,c, BED_
bnzA1-c, and BEDm_II-12-10-a were positive in six sam-
ples. In particular, three probes designed to target clone
BEDm-II-12-5 (probe BEDm_II-12-5-a,b,c) were positive in
six soil samples, indicating the presence of the cloned gene
in those samples.
In addition to the probes that had higher similarity than
the MHT to the nontarget sequences, there were still some
probes that showed false-positive signals even within the
MHT (Fig. 4). However, these probes did not always show
false-positive signals (i.e. they showed negative signals in
other hybridizations). This suggests that these probes may
be used to discriminate between the broader group of
sequences even though they were not specific to the expected
target sequences. Thus, we can use these signals to create
microarray profiles for the characterization of soil samples.
Because the soil DNAs were amplified before the hybridiza-
tions, the SNRs may not reflect the relative quantity of
the genes in each sample. Thus, we converted the signal
profiles to a binary matrix (positive signals = 1; negative
signals = 0) and performed cluster analysis by the Ward
method (Ward, 1963; Fig. 5). The microarray profiles of
Soils III and IV were clustered together due to high
similarity. Although Soil V was a benzene-amended soil, its
microarray profile was close to those of Oils A and B rather
than those of Soils III and IV, which were also benzene-
amended soils. Because Soil V was collected from the road-
side in an area of heavy traffic, it may have been enriched
with hydrocarbons, making its oxygenase diversity similar to
that in oil-contaminated soil.
Although, we constructed our microarray based on only
two soil clone libraries, some genes were detected from all
soil samples examined. Updating the microarray by analyz-
ing additional soil samples will enable us to identify other
commonly occurring genes. Further analysis of the relation-
ship between those genes and biodegradation activity by
real-time PCR may help elucidate the roles of the genes in
bioremediation. Moreover, by producing microarray pro-
files for additional samples, we can construct databases of
hybridization profiles and degradation abilities. Such data-
bases will enable us to diagnose the degradation trend for
benzene and other contaminants in various soils based on
their microarray profiles.
Acknowledgements
We are grateful to Keishi Senoo for providing the soil
samples. We thank Kensuke Furukawa, Toshiya Iida, Junichi
Kato, Yasushi Kawakami, Kazuhide Kimbara, Atsushi
Kitayama, Toshiaki Kudo, and Hideaki Nojiri for providing
the strains and plasmids. We thank Robert D. Stedtfeld for
reading and correcting the English in our manuscript.
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FEMS Microbiol Lett 285 (2008) 111–121c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
120 S. Iwai et al.
Supplementarymaterial
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available online:
Table S1. Designed probes.
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FEMS Microbiol Lett 285 (2008) 111–121 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
121An oligonucleotide microarray for oxygenase genes
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