ARTICLE IN PRESS

Microbiological Research 162 (2007) 335340

www.elsevier.de/micres

Detection and quantication of phnE gene from

oil-contaminated soil samples by competitive
quantitative PCR
Peng Lui, Chang Kai Zhang
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China
Accepted 26 January 2006

KEYWORDS
Competitive quantitative PCR;
Catechol 2,3-dioxygenase;
Quantication;
Oil-contaminated
soil

Summary
The phnE gene encoding catechol 2,3-dioxygenase belonging to the meta-cleavage
pathway was selected as the marker gene and was detected and quantied from soil
samples by competitive quantitative PCR. A PCR primer pair was designed based on
the phnE gene to amplify the target DNA bands and competitor DNA bands. The phnE
gene was detected in two samples of three. In samples S1 and S2, the phnE gene
copy number was 6.2  107/g soil and 5.8  107/g soil, respectively. But no phnE
gene was detected in sample S3. The target DNA bands were extracted and
expressed. The results conrmed that the target DNA bands were the native
phnE genes.
& 2006 Elsevier GmbH. All rights reserved.

Introduction
Modern industry produced a variety of aromatic
compounds and resulted in the environmental
pollution. Bacteria expressing the enzymes of the
pathways for the degradation of aromatic compounds were used to cleaning up contaminated
environments (van der Meer et al., 1992). Catechol
is one of the most important and few key
intermediates in the degradation of toxic polluCorresponding author.

E-mail address: ckzhang@sdu.edu.cn (C.K. Zhang).

tants (Gibson and Subramanian, 1984). Enzymatic

cleavage of catechol is catalyzed by two categories
of catechol dioxygenases, referred to as intradiol
and extradiol dioxygenases. In the extradiol dioxygenases, catechol 2,3-dioxygenases (C23O) form a
superfamily of extradiol dioxygenases (Harayama
and Rekik, 1989). So the C23O genes are good
targets for monitoring the organisms involved in
meta-cleavage of aromatics.
Competitive quantitative PCR techniques are
initially developed to enumerate viral loads, and
are used widely in enumerating the biodegradation genes, such as naphthalene catabolic genes

0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2006.01.013

ARTICLE IN PRESS
336

P. Lui, C.K. Zhang

(Herrick et al., 1993), methane monooxygenase

gene (Mohan and Walia, 1994), and 2,4-dichlorophenoxyacetic acid degradation genes (Neilson et
al., 1992). The C23O genes belong to ve subfamilies I.2.AI.2.E (Eltis and Bolin, 1996) and a few
of C23O-specic primers have been used to detect
and enumerate the C23O genes (Junca and Pieper,
2003; Mesarch et al., 2000, 2004). The reported
primers were only specic to certain C23O genes
such as the C23O genes belong to the I.2.A
subfamily, and C23O genes belong to other subfamilies can not be detected. It is necessary to
develop primers outside of these reported C23Ospecic primers. The I.2.C subfamily of the C23Os is
a notable subfamily, and may have arisen in
response to low concentrations of substrates (Kukor
and Olsen, 1996). A feature of this I.2.C phylogenetic group is the presence of an additional 24 bp in
these C23O genes, which is absent from all other
subclasses (Laurie and Lloyd-Jones, 1999). Burkholderia cepacia L68 is a phenol degradation bacterium isolated from the oil-contaminated water
discharged by an oil renery in Jinan (PR China).
The dioxygenase region genes were sequenced and
analyzed (GenBank accession number DQ191058).
The phnE gene, which encodes C23O, belongs to
the I.2.C subfamily of the C23Os dened by Eltis
and Bolin (1996). In this paper, the phnE gene was
selected as the marker gene, and competitive
quantitative PCR methods were established to
detect and enumerate the phnE gene from the
environmental samples.

Materials and methods

Environmental samples
The soil samples were collected from three oilcontaminated sites in Jinan, PR China. All the
samples were transferred to the laboratory and
stored at 20 1C within 2 h after sampling. The
Table 1.

standard direct photometric method, based on the

oxidative coupling of phenols with 4-aminoantipyrine (4-AAP) in alkaline medium (Ettinger et al.,
1951; Gordon, 1960), was carried out to determine
the concentrations of total phenols in the samples.

Bacterial strains and plasmids

Burkholderia cepacia L68 was isolated for its fast
phenol catabolism from the oil-contaminated water
discharged by an oil renery in Jinan (PR China),
and grow on a modied minimal medium (Santos
et al., 2003). Escherichia coli DH5a was used as the
host strain for pUC18 plasmid. The strains and
plasmids used in this study are described in Table 1.

DNA manipulation
DNA used for PCR amplication was extracted
from soil samples following a modied repeated
blending protocol (Gabor et al., 2003). Fifty-gram
environmental samples were dispersed in 100 ml
blending buffer (100 mM TrisHCl, 100 mM sodium
EDTA, 0.1% SDS, 1% CTAB, pH 8.0). Coarse particles
were collected at 1000g for 10 min at 10 1C,
resuspended in 100 ml blending buffer, and subjected to another two blendingcentrifugation
cycles. Supernatants obtained during the three
rounds of cell extraction were pooled. Supernatants were centrifuged at 10,000g for 30 min at
4 1C to collect the microbial cell fraction, which
was subsequently washed in 150 ml of 0.1% sodium
pyrophosphate. After a second wash in 100 ml
Chrombach buffer (0.33 M TrisHCl, 1 mM EDTA,
pH 8.0), pellets were resuspended in 8 ml lysis
buffer (100 mM TrisHCl, 100 mM sodium EDTA,
1.5 M NaCl, 1% CTAB, pH 8.0), 160 ml lysozyme
(50 mg/ml), and 40 ml proteinase K (10 mg/ml)
solution and incubated at 37 1C for 30 min. Lysis
was completed chemically by adding 1 ml of 20%
SDS and incubation for 2 h at 65 1C with rotary
shaking (225 rpm). The combined supernatants

Bacterial strains and plasmids used in this study.

Strains or
plasmid

Relevant characteristic(s)

Sources

B. cepacia L68
E. coli DH5a

Degrades phenol
deoR endA1 gyrA96 hsdR17 (rK mK+) recA1 relA1 supE44
thi-1 D(lacZYAargFV169)F80dlacZDM15 Fl
General cloning vector
Recombined plasmid (phnE gene from L68 inserted into
pUC18)
Competitor plasmid (partial phnE gene from L68 inserted
into pUC18)

This study
Clontech

pUC18
pE
pE-C

Pharmacia Biotech
This study
This study

ARTICLE IN PRESS
Detection and quantication of phnE gene from oil-contaminated soil samples
were extracted with an equal volume of chloroform
before precipitating the DNA from the recovered
water phase by addition of 0.6 volumes of
isopropanol and overnight incubation at 4 1C. The
precipitates were collected by centrifugation at
16,000g, washed with 70% ethanol, and suspended
in a total volume of 250 ml TE buffer. The DNA
dissolved in TE buffer was rened using DNA
Fragment Purication Kit from TaKaRa (PR China).
The total DNA of B. cepacia L68 was extracted
using UNIQ-10 Column Bacterial Genomic DNA
Isolation Kit from Sangon (PR China). Plasmid DNA
purication was performed using E.Z.N.A. Plasmid
Miniprep Kit I from Omega (USA). PCR products
extraction was performed using Sephaglas BandPrep Kit from Pharmacia Biotech (USA). Gene
manipulation and bacterial transformation were
carried out according to Sambrook et al. (Sambrook
et al., 1989). DNA was quantied by spectrophotometer calibrated with calf thymus DNA.

Construction of competitor
Two PCR primer pairs were constructed based on
the sequence of phnE gene (Table 2). The phnE
gene was amplied from B. cepacia L68 using
primers BCO1 and BCO2, and was ligated with
pUC18 vector at EcoR I and Sal I endonuclease sites.
Then two primers DCO1 and DCO2 were used to
amplify a 3299 bp sequence containing the 50
partial sequence of phnE gene, the pUC18 gene
and 30 partial sequence of phnE gene from the pE.
The 3299 bp sequence was self-cycled at the Kpn I
endonuclease site and formed the competitor
plasmid (Fig. 1).

337

denaturation step at 97 1C for 10 min followed by 30

cycles of denaturation at 94 1C for 60 s, annealing
at 58 1C for 30 s, and extension at 72 1C for 45 s,
then a nal extension step at 72 1C for 10 min. All
reaction mixtures were held at 4 1C until analyzed.
Ten microliters of each PCR mixture was run on a
1.5% agarose gel in 0.5  TBE buffer stained with
ethidium bromide (0.0001%) and visualized under
UV light. Target and competitor band intensities
were analyzed and compared using Gene Tools
software. Each experiment was repeated three
times and the means values are reported.

Enzyme assay of phnE gene isolated from

environmental samples
The competitive quantitative PCR products were
separated on 1.5% agarose gels. Then the assumed
phnE genes were extracted and ligated with pUC18
vector at EcoR I and Sal I endonuclease sites and
transformed to E. coli DH5a. The white transformants were routinely cultured on LB medium with
ampicillin (100 mg/ml). The cells were harvested by
Sal I
Ap
phnE

pE

EcoR I
PCR (DCO1+DCO2)
Sal I

Ap

Kpn I
pE
Kpn I
EcoR I

PCR amplication
The PCR amplication mixture consisted of
10 mM TrisHCl (pH 8.3), 2 mM MgCl2, 0.4 mM each
dNTP, 1 mM each primer, 0.04 U/ml pfu DNA polymerase and 0.4 ng/ml template DNA (0.2 ng/ml
target DNA and 0.2 ng/ml competitor DNA for
competitive quantitative PCR experiments). The
PCR amplication conditions included an initial

Table 2.

Ap

Sal I
pE-C

Kpn I
EcoR I

Figure 1. Construction of competitor.

Primers used in this study.

Primer

Sequence

Position on phnE

Endonuclease site

BCO1
BCO2
DCO1
DCO2

50 -GTCGAATTCATGGGTGTGATGCGA-30
50 -ACTGTCGACTCAGGTGTAAACCTC-30
50 -AGAGGTACCTGGGCTTTTATCTGG-30
50 -TACGGTACCTTGTAGGCGACATG-30

115
945931
530544
206193

EcoR I
Sal I
Kpn I
Kpn I

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338

Results and discussion

PCR amplication
The competitor was constructed as described in
the methods. When equal concentrations of total
DNA of B. cepacia L68 and competitor were used as
templates and BCO1 and BCO2 were used as
primers, the expected 963 and 646 bp DNA fragments were amplied. The logarithms of the ratios
of L68 DNA to competitor band intensities were
approximately zero when various cycle number
were tested (data not show), which showed that
the amplication efciencies of the L68 DNA and its
competitor pE-C DNA were the same and allowed
that the quantication of phnE gene copy number
was only based on the ratio of target DNA and
competitor (Lee et al., 1996).
High concentration of nonamplifying DNA relative
to target DNA may inhibition the PCR, so the effects
of nonamplifying DNA were examined. The total
DNA of E. coli DH5a or the calf thymus was used as
the nonamplifying DNA. When the E. coli DH5a DNA
or the calf thymus DNA was used as the template,
no PCR products were detected. Various nonamplifying DNA/L68 DNA ratios were tested, and quantication of phnE gene copy number was not affected
by the ratios as high as 1000:1.

0.002 ng/ml to 2 ng/ml) were used as templates

(Fig. 2).
The competitive quantitative PCR assay was
applied to detect and quantitate the phnE gene
using DNA extracted from various samples (Fig. 3).
In sample S1 and S3, the phnE gene copy number
was 6.2  107/g soil and 5.8  107/g soil, and the
concentrations of total phenols was 1.522 and

0.2

y = 0.1129x - 0.0409
R2= 0.9934

0.15
log[pE/pE-C]

centrifugation at 10,000g for 10 min and washed

twice with ice-cold sonication buffer (10 mM
potassium phosphate containing 10% acetone, pH
7.5). Then the cells were resuspended and disrupted by sonication in an ice-bath. After centrifugation at 10,000g for 30 min, the supernatant was
used as the enzyme solution to detect the C23O
activity.
The activity of C23O was assayed by measuring
the increase in absorbance at 375 nm due to the
formation of 2-hydroxymuconate semialdehyde
(2-HMS) at 25 1C in an air-saturated buffer of
1 mM potassium phosphate (pH 7.5). The reaction
mixture (3.0 ml) contained 1 mmol catechol and
0.2 ml enzyme solution. One unit is dened as the
amount of the enzyme that produces 1 mmol of
2HMS per minute under the standard assay conditions (Kojima et al., 1961).

P. Lui, C.K. Zhang

0.1
0.05
0
-0.05

0.5

-0.1

1.5

pE DNA (ng/l)

Figure 2. Calibration curve of competitive quantitative

PCR of phnE gene. The relative masses of the bands of
amplication products corresponding to pE and competitor of the gel were used to construct this calibration
curve. The averages of three determinations were
plotted.

2000bp

1000bp
750bp
500bp

Detection of phnE gene by competitive

quantitative PCR
Calibration curve of competitive quantitative
PCR of phnE gene was drawn when the mixture of
pE-C DNA (0.2 ng/ml) and pE DNA (ranged from

Figure 3. Results of competitive quantitative PCR to

quantitate the phnE gene using DNA extracted from
various samples. Lane 13, samples S1S3; lane 4, DNA
marker DL2, 000.

ARTICLE IN PRESS
Detection and quantication of phnE gene from oil-contaminated soil samples
gene copy number (/g soil)
concentrations of total phenols (mg/g soil)
6.2107

5.8107

3.181
2.078
1.522
0
sample S1

sample S2

sample S3

Figure 4. Relationships of phnE gene copy number and

concentrations of total phenols in the three soil samples.

2.078 mg/g, respectively (Fig. 4). The concentrations of total phenols in sample S2 was higher
than those in samples S1 and S3, reaching
3.181 mg/g soil, but no phnE gene was detected
in sample S2.

Enzyme assay of phnE gene isolated from

environmental samples
The 963 bp PCR products were ligated into the
pUC18 vector and transformed to E. coli DH5a. The
recombinants were routinely cultured on LB medium with ampicillin (100 mg/ml). After induced by
5 mM IPTG, the lysis of the transformants showed
specic C23O activities. The SDSPAGE results also
showed that the protein was induced by IPTG and
was similar to the C23O puried from L68 in size.
All the results conrmed that the 963 bp PCR
products were the phnE genes.
In this report, a competitive quantitative PCR
was used to detect and quantify the phnE gene
from soil samples. This technology may be useful in
the study of soil microbial ecology of bioremediation. Many factors such as the nonamplifying DNA
and humic acids may affect the accuracy of
competitive quantitative PCR. We have proved that
high concentrations of E. coli DH5a DNA and calf
thymus DNA did not render the results of PCR
inaccurate. Humic acids may be coextracted with
DNA (Tsai and Olson, 1992), then interfere with PCR
reaction components (Wilson, 1997). The DNA
Fragment Purication Kit was used to rene the
DNA and remove the humic acids. The nal DNA
extracts should be free of humic material.
The primers used in this study are specic for the
phnE gene. When coupled with the quantitative
competitive PCR approach, the phnE gene copy
number can be accurately quantied. This is a valid
method for detection and quantication of the

339

phnE gene. In the soils polluted with the aromatic

compounds, the microorganisms harboring the phnE
gene multiplied and began to utilize these aromatic
compounds. The results showed that the phnE gene
copy numbers did not differ signicantly among the
soil sample S1 and S3, which indicated that the
organisms in these soils might be similar. It is
notable that the phnE gene was not detected in
high total phenols concentrations soil sample S2.
Other subfamily of C23O or other aromatic degradation pathway may be active in this sample, but
there is another possibility. Kukor and Olsen
suggested that the phnE gene may have arisen in
response to low concentrations of substrates (Kukor
and Olsen, 1996). The concentration of total
phenols in sample S2 was higher than those in the
other tested samples, which may be the main
reason of the absence of phnE gene in S2.
Native phnE gene was amplied from the soil
samples, ligated to the vector and expressed. The
colorless catechol was turned to yellow by formation of 2-HMS when the lysis of the transformants
was added, which conrmed that the target amplied DNA bands were the amplied phnE genes.
But there still are some questions waiting to be
solved. The presence of phnE gene cannot ensure
that the entire meta-cleavage pathway will be
present, and cannot ensure that the phnE gene is
active. Moreover, the primers used in this study are
specic solely for the phnE gene belonging to the
meta-cleavage pathway and will not amplify genes
in other degradation possessing. So mRNA extraction techniques may be used to detect the gene
activity. More primers for other degradation genes
should be developed to determine whether other
degradation pathway genes are present.

Conclusions
The phnE gene encoding catechol 2,3-dioxygenase belonging to the meta-cleavage pathway was
selected as the marker gene and was detected and
quantied from two of three soil samples by
competitive quantitative PCR. And the absence of
phnE gene in soil sample with high concentrations
of substrates may provide the evidence that the
phnE gene has arisen in response to low concentrations of substrates.

Acknowledgments
This work was supported by the National Fund Of
Nature Sciences (30370014).

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