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:

siwai@msu.edu

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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120 S. Iwai et al.

Supplementarymaterial

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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