RESEARCH ARTICLE
Cytochromes P450-mediated degradation of fuel oxygenates by
environmental isolates
Cédric Malandain1,2, Françoise Fayolle-Guichard1 & Timothy M. Vogel2
1
IFP, Rueil-Malmaison, France; and 2Environmental Microbial Genomics Group, Laboratoire AMPERE, Ecole Centrale de Lyon, Université de Lyon, Ecully,
France
Correspondence: Françoise FayolleGuichard, IFP, 1-4, avenue de Bois-Préau,
F-92852 Rueil-Malmaison, France. Tel.: 133 1
47 52 68 64; fax: 133 1 47 52 70 58;
e-mail: [email protected]
Received 3 July 2009; revised 22 January 2010;
accepted 22 January 2010.
Final version published online 10 March 2010.
DOI:10.1111/j.1574-6941.2010.00847.x
MICROBIOLOGY ECOLOGY
Editor: Max Häggblom
Keywords
cytochrome P450; ethyl tert-butyl ether (ETBE);
biodegradation; eth genes distribution;
substrate specificity; inducer.
Abstract
The degradation of fuel oxygenates [methyl tert-butyl ether (MTBE), ethyl tertbutyl ether (ETBE) and tert-amyl methyl ether (TAME)] by Rhodococcus ruber IFP
2001, Rhodococcus zopfii IFP 2005 and Gordonia sp. IFP 2009 (formerly Mycobacterium sp.) isolated from different environments was compared. Strains IFP 2001,
IFP 2005 and IFP 2009 grew on ETBE due in part to the activity of a cytochrome
P450, CYP249. All of these strains were able to degrade ETBE to tert-butyl alcohol
and are harboring the CYP249 cytochrome P450. They were also able to degrade
MTBE and TAME, but ETBE was degraded in all cases most efficiently, with
degradation rates measured after growth on ETBE of 2.1, 3.5 and 1.6 mmol
ETBE g1 dry weight h1 for strains IFP 2001, IFP 2005 and IFP 2009, respectively.
The phylogenetic relationships between the different ethR (encoding the regulator)
and ethB (encoding the cytochrome P450) genes were determined and showed
high identity between different ethB genes (4 99%). Only ETBE was able to induce
the expression of ethB in strains IFP 2001 and IFP 2005 as measured by reverse
transcriptase quantitative PCR. Our results are a first indication of the possible role
played by the ethB gene in the ecology of ETBE degradation.
Introduction
Fuel oxygenates, methyl tert-butyl ether (MTBE), ethyl tertbutyl ether (ETBE) and tert-amyl methyl ether (TAME),
were incorporated into gasoline over the last 20 years in
order to boost the octane index. Their incorporation
improved the air quality in urban areas by reducing hydrocarbon and carbon monoxide emissions. As a result of its
extensive use in the USA, MTBE was frequently detected as a
groundwater pollutant and several states decided to ban its
use as an additive in gasoline (Johnson et al., 2000; Schmidt
et al., 2004), but in other parts of the world, MTBE is still
used and is present in the environment (Klinger et al., 2002;
Schirmer et al., 2003; Rosell et al., 2006). In several European
countries (France, Spain, Germany, Belgium, the Netherlands), ETBE is now added to gasoline (8–15% v/v) to
replace MTBE. According to Mirabella & den Hertzog
(2008), ETBE is produced in several European plants for a
total installed capacity of 5750 kt year1. Both MTBE and
ETBE are highly water soluble, and generally recalcitrant to
biodegradation. Moreover, the biodegradation is generally
FEMS Microbiol Ecol 72 (2010) 289–296
slow when it does occur. As a consequence, the natural
attenuation of MTBE in contaminated sites has not been
frequently observed. Information about the possible release
of ETBE into the environment and the state of groundwater
contamination by ETBE in the European countries that use
it as an additive is, to the best of our knowledge, poorly
documented (Rosell i Linares, 2006; Van Wezl et al., 2009).
Thus, the degradation capacity of microorganisms and the
genes involved in the ETBE degradation pathway needs to be
studied in order to have tools for determining their distribution in the environment when contamination occurs.
The first monooxygenase system able to degrade ETBE and
characterized at the physiological and genetic level was a
cytochrome P450 monooxygenase (encoded by the
ethRABCD genes) induced after growth on ETBE in Rhodococcus ruber IFP 2001 (Chauvaux et al., 2001; Hernandez-Perez
et al., 2001). This cytochrome P450 was classified as
CYP249A1 and was the first member of its family. The activity
of CYP249A1 in R. ruber IFP 2001 was responsible for the
degradation of ETBE and growth occurred at the cost of the
C2-moiety released by the cleavage of the ether bond. At the
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C. Malandain et al.
same time, tert-butyl alcohol (TBA), an ETBE degradation
intermediate, accumulated in the culture. In the case of R.
ruber IFP 2001, the cometabolic biodegradation of MTBE and
TAME was shown after induction of the cytochrome P450
system during growth on ETBE (Hernandez-Perez et al.,
2001). Highly similar clusters containing eth genes were also
detected in Rhodococcus zopfii IFP 2005 and Gordonia sp. IFP
2009 (formerly Mycobacterium sp.), two strains also isolated
for their capacity to grow on ETBE (Fayolle et al., 1998;
Béguin et al., 2003). Rhodococcus ruber IFP 2001, R. zopfii IFP
2005 and Gordonia sp. IFP 2009 were obtained from activated
sludge collected at different wastewater treatment plants in
France. The different eth clusters belonging to these different
strains (1) were all under the control of an ethR gene encoding
a putative positive transcriptional regulator of the AraC/XylS
family (Chauvaux et al., 2001; Béguin et al., 2003), (2) all
clusters include an ethD gene encoding a 10-kDa protein
expressed in R. ruber IFP 2001 when demonstrating ETBEdegrading activity, although its function was not known
(Chauvaux et al., 2001), and (3) all clusters were located on
transposons, which are easily lost under nonselective culture
conditions. This loss led to the isolation of an ETBE(minus)
mutant from R. ruber that was used to demonstrate the role of
the eth cluster in ETBE degradation (Béguin et al., 2003). After
isolation of the eth gene cluster in R. ruber IFP 2001, a search
for sequence similarities showed that the cluster displayed
remarkable similarity to the Rhodococcus erythropolis NI86/
21’s thc system encoding a cytochrome P450 system that
catalyzes the S-dealkylation of the thiocarbamate herbicide
S-ethyl dipropylthiocarbamate (Nagy et al., 1995a, b).
Recent data emphasized the possible ecological role of the
EthB cytochrome P450 in the natural attenuation of MTBE/
ETBE contaminated sites: similar genes were amplified using
nondegenerate primers (designed from the ethB sequence of
R. ruber IFP 2001) and template DNA extracted from several
ETBE- or MTBE-degrading microcosms obtained from aquifer
or soil samples from different sites contaminated by MTBE
(mainly) or ETBE (Babé et al., 2007). Moreover, Breuer et al.
(2007) identified a cluster of ethABCD genes in the new strain
Aquincola tertiaricarbonis L108. These genes were highly similar to those from R. ruber IFP 2001. Aquincola tertiaricarbonis
L108 had been isolated previously from the MTBE-contaminated groundwater of the Leuna site (Germany) due to its
capacity to grow on MTBE (Lechner et al., 2007). Kim et al.
(2007) reported that one strain, Rhodococcus sp. PEG604, from
among 17 linear alkyl ether-utilizing rhodococci isolated from
activated sewage sludge, generated an 877-bp PCR product using
primers designed from the ethB sequence of R. ruber IFP 2001.
The objective of the present study was to determine the role
of different environmental strains, all possessing the ethB gene.
We (1) compared the ETBE-, MTBE- and TAME-degrading
activity of the cytochrome P450 in the different strains
(R. ruber IFP 2001, R. zopfii IFP 2005 and Gordonia sp.
IFP 2009) isolated for their capacity to grow on ETBE, (2)
determined the phylogenetic relationships between the different strains and their eth genes (ethR and ethB) and (3) studied
which ether(s) induced the cytochrome P450 systems using
reverse transcriptase quantitative PCR (RT-qPCR).
Materials and methods
Microorganisms and preservation
The strains used in our study are presented in Table 1. Stock
cultures of all strains were kept frozen at 80 1C in 20%
glycerol (v/v).
Table 1. Bacterial strains
Strain
Description
R. ruber IFP 2001
Wild type, growth on ETBE
R. ruber IFP 2006
Method of isolation
Culture on MM with
ETBE as the sole carbon
and energy source
Mutant unable to grow on ETBE Serial cultures on a
ETBE (minus) phenotype
rich medium (LB)
Origin
Source (reference)
Activated sludge (waste water
treatment plant, Achères, 78, France)
IFP (Hernandez-Perez
et al., 2001)
Serial transfer of R. ruber IFP 2001
on LB broth
Deletion of the eth cluster
R. zopfii IFP 2005
Wild type, growth on ETBE
Culture on MM with
Activated sludge (waste water
ETBE as the sole carbon treatment plant, Achères, 78, France)
and energy source
R. zopfii IFP 2004
Mutant unable to grow on ETBE Serial cultures on
Serial transfer of R. zopfii IFP 2005
ETBE (minus) phenotype
a rich medium (LB)
on LB broth
Deletion of the eth cluster
Gordonia sp. IFP 2009 Wild type, growth on ETBE
Culture on MM with
Activated sludge (waste water
ETBE as the sole carbon treatment plant, Valenton, 94, France)
and energy source
IFP (Chauvaux et al., 2001)
IFP (Fayolle et al., 1998)
IFP (Béguin et al., 2003)
IFP (Béguin et al., 2003)
No PCR product was obtained for these two strains when using the primer pairs (ethRfor/ethRrev) and (ethBfor/ethBrev) described in Materials and
methods used for amplification of the ethR and ethB genes, respectively.
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FEMS Microbiol Ecol 72 (2010) 289–296
291
Cytochromes P450-mediated degradation of fuel oxygenates
Growth medium and culture conditions
All strains were grown at 30 1C in Luria–Bertani (LB)
medium or in the following mineral medium (MM):
KH2PO4 1.40 g L1; K2HPO4 1.70 g L1; MgSO4, 7H2O
0.5 g L1; NH4Cl 1.5 g L1; CaCl2, 2H2O 0.04 g L1; FeSO4,
7H2O 1 mg L1. A vitamin solution was added as described
previously (Piveteau et al., 2001). MM was supplemented
before inoculation with the required substrate: ETBE
(250 mg L1). Because ETBE is volatile, the cultures were
grown in flasks closed with a cap equipped with an internal
Teflon septum to avoid any loss of substrate either by
volatilization or by adsorption. The headspace volume was
sufficient to prevent any O2 limitation during growth.
Growth was followed by measuring the A600 nm.
Degradation assay using resting cells
Cells of the different strains grown on LB or on MM
containing ETBE as the carbon source were harvested by
centrifugation (23 000 g for 15 min), washed twice and
suspended in phosphate buffer (20 mM, pH 7). Cells were
suspended in 30 mL of the phosphate buffer containing the
test substrate (ETBE, MTBE or TAME) in 135-mL flasks
closed with a butyl rubber stopper and sealed to obtain an
initial OD600 nm of c. 0.4. After inoculation, the flasks were
incubated at 30 1C on an orbital shaker. All experiments
were performed in triplicate. Filtered samples were analyzed
by GC. Substrate concentration was measured at 0, 3, 24, 120
and 192 h. The dry weight of the cells was measured by
filtration through a 0.22-mm filter at the end of the experiment. The filters were dried and weighed to calculate the
biomass (dry weight) concentration. The rate of ether
degradation (mmoles of ether degraded g1 biomass dry
weight h1) was calculated from the residual substrate concentration measured after 3 h.
Analytical procedures
ETBE, MTBE, TAME, TBA or tert-amyl alcohol (TAA) was
quantified by flame ionization detection on a Varian 3300
gas chromatograph (Varian, France) equipped with a
0.32 mm 25 m Porabond-Q capillary column (J&W Scientific, Chromoptic, Auxerre, France), using a two-step temperature gradient ranging from 105 to 210 1C at
10 1C min1, and then maintained at 210 1C for 20 min.
Helium (1.6 mL min1) was used as the carrier gas. Samples
were filtered through 0.22-mm filters (Prolabo, Fontenaysous-Bois, France) and injected without further treatment.
Nucleic acid extraction
The preparation of genomic DNA and total mRNA was
carried out using the Nucleospin RNAII kit (MachereyNagel). The digestion of the bacterial membrane was
FEMS Microbiol Ecol 72 (2010) 289–296
performed with 2 mg mL1 of lysozyme and 0.05 mg mL1
of lysostaphin for 20 min. To elute the genomic DNA, we
used the complementary buffer ‘DNA elute’ following the
supplier’s protocol. RNA and DNA were quantified using the
Qbit (Qiagen) and the respective kit.
Amplification of eth genes
Primers were designed from the ethR and ethB sequences of
R. ruber IFP 2001. The primers used are ethRfor: CCA CAG
ATA TGA CAT CGG TCA C and ethRrev: GTC GGC ATC
GAG AGG AGA for the amplification of the ethR gene;
ethBfor: GCA CCT TTC ACC GAC ACA C and ethBrev:
TTG GGA GAA GGT GAT CTT GG for the amplification of
the ethB gene. The amplifications were performed using the
Phusion High Fidelity Master Mix (New England Biolabs)
according to the manufacturer’s protocol. PCRs were carried
out on a Piko thermocycler (Finnzyme) using the following
program: initial denaturation: 98 1C for 30 s; 35 cycles with
denaturation at 98 1C for 10 s, annealing at 60 1C for 10 s
and extension at 72 1C for 35 s; and a final extension at 721 C
for 5 min. The PCR products (1122 bp for ethR and 1270 bp
for ethB) were then cloned using the TOPO TA cloning kit
for sequencing (Invitrogen). The sequencing was performed
by GTAC Biotech using the M13for and M13rev primers.
RT-qPCR
The RT-qPCRs were performed on a Rotorgene 6000
(Corbett) and using the Quantitect SyBr RT-PCR kit (Qiagen). The program was: 50 1C for 30 min, 95 1C for 15 min, 55
cycles of 95 1C for 15 s, 60 1C for 30 s and 721 for 30 s. The
specificity of the amplification was verified using the melt
function. The specificity of the primers was tested first by
PCR. The primers used were ethBfor (5 0 -GGT GTC CAA CAC
CGA GAT G-3 0 ) and ethBrev (5 0 -CGG ATG AGG TTG TTG
ATG TG-3 0 ). The 16S rRNA genes and their corresponding
mRNA were amplified using the specific primers 16SRhodofor
(5 0 -GTA CTG CAG GGG AGA CTG GA-3 0 ) and 16SRhodorev
(5 0 -GGT CCG GTG TAG TCA AAC C-3 0 ). 1 ng of total RNA
was used in RT-qPCR.
Bioinformatics
The sequences were analyzed using the software BIOEDIT
(Hall, 1999).
Phylogenetic trees were built using the neighbor-joining
method of Saitou & Nei (1987), and evolutionary distances
were calculated according to the model of Tamura & Nei
(1993). Bootstrap analysis with 1000 replicates was performed
to determine the statistical significance of the branching order.
Only the branches yielding a bootstrap superior at 70% were
considered as relevant and the percentage of occurrence over
70% for a node in the phylogenetic tree is indicated on the
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292
C. Malandain et al.
figures. To construct the trees we selected, by BLAST, the genes
that showed at least 35% of identity with the eth genes. The
sequences used to construct the trees were retrieved from the
NCBI database. The accession numbers or the locus numbers
are given in parentheses in the corresponding figures.
2005 and Gordonia sp. IFP 2009 after growth on LB, whereas
higher degradation rates of these two compounds were obtained
when the strains were cultivated on ETBE. ETBE was degraded
in all cases at the highest rate after induction. Moreover, the
degradation of ETBE to TBA was total, which was not the case
for MTBE and TAME (results not shown). No degradation of
ETBE, MTBE or TAME was observed with the mutant strains,
R. ruber IFP 2006 and R. zopfii IFP 2004, where the eth cluster
was deleted and they were unable to grow on ETBE.
Nucleotide sequence accession numbers
The ethR and ethB nucleotide sequences of R. ruber IFP 2001
(AF333761 and gi: 16551188, respectively), R. zopfii IFP
2005 (FJ481916 and FJ481919, respectively) and Gordonia sp.
IFP 2009 (FJ481918 and FJ481921, respectively) have been
deposited in the GenBank nucleotide sequence database.
Relationships between strains and genes
The DNA sequences encoding two Eth polypeptides, i.e. the
regulator (ethR gene) and the cytochrome P450 (ethB gene),
were amplified and sequenced from the strains showing an
activity towards ETBE, MTBE and TAME, i.e. R. ruber IFP
2001, R. zopfii IFP 2005 and Gordonia sp. IFP 2009. No
amplification was obtained in the case of R. ruber IFP 2006
and R. zopfii IFP 2004, which are unable to grow on ETBE.
Phylogenetic trees were constructed for comparison with
genes showing at least 35% of identity with ethR (Fig. 1) and
ethB (Fig. 2) genes. Each of the eth genes isolated from IFP
2005 and IFP 2009 had a high identity (4 99%) with those
of IFP 2001 as shown in the figures.
Results
ETBE, MTBE and TAME degradation capacities
The capacity to degrade ETBE, MTBE and TAME was tested
using LB-grown resting cells of the different wild-type strains
R. ruber IFP 2001, R. zopfii IFP 2005 and Gordonia sp. IFP
2009 and of the ETBE (minus) mutants, R. ruber IFP 2006
and R. zopfii IFP 2004. The residual concentrations of ETBE,
MTBE and TAME were measured by GC and the initial
degradation rates were calculated after 3 h of incubation,
during which most of the degradation occurred (Table 2).
TBA was produced from ETBE and MTBE degradation
and TAA was produced from TAME (data not shown).
Rhodococcus ruber IFP 2001, R. zopfii IFP 2005 and Gordonia
sp. IFP 2009 were able to degrade ETBE at a low level after
growth on LB, but the ether-degrading capacity was clearly
induced after growth in the presence of ETBE, the ETBE
degradation rates being, respectively, 10-, 6- and 11-fold
higher after growth on ETBE.
No degradation or degradation at a low level was observed
for MTBE or TAME by strains R. ruber IFP 2001, R. zopfii IFP
Expression level of eth genes by RT-qPCR
The ethB gene regulation in strains R. ruber IFP 2001, R.
zopfii IFP 2005 and Gordonia sp. IFP 2009 was investigated
by RT-qPCR. Experiments showed an induction of the ethB
genes in response to the presence of ETBE in MM. In the
case of R. ruber IFP 2001, the first increase in the number of
mRNA copies for the ethB gene was detected after three
hours of induction (contact with ETBE) and this number
increased through the 9 h of the experiment (Fig. 3).
However, the addition of the other ethers, MTBE or TAME,
Table 2. Degradation rates of ETBE, MTBE and TAME by the different strains
Initial degradation rate expressed in mmoles of ether degraded g1 dry weight h1 on
ETBE
MTBE
w
Strains
No induction
R. ruber IFP 2001
R. ruber IFP 2006
R. zopfii IFP 2005
R. zopfii IFP 2004
Gordonia sp. IFP 2009
210.3 14.8
0
267.6 7.3
0
124.6 4.4
Induction
z
2118.6 94.1
ND
3508.5 20.2
ND
1621.6 5.3
No induction
0
0
0
0
34.7 1.6
TAME
w
Induction
z
195.2 29.0
ND
49.5 3.7
ND
97.2 2.9
No inductionw
Inductionz
96.9 3.4
0
49.0 1.8
0
25.9 3.1
255.1 1.7
ND
78.6 1.9
ND
133.7 5.9
The residual concentrations of ETBE, MTBE and TAME were measured after 3 h of incubation. These results are expressed in mmoles of ether
degraded g1 dry weight h1. The average values are based on three replicates.
The degradation rates were measured using resting cells of the different strains:
w
without induction (i.e. growth on LB) or
z
after induction (i.e. growth on MM in the presence of ETBE).
ND, not determined.
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FEMS Microbiol Ecol 72 (2010) 289–296
293
Cytochromes P450-mediated degradation of fuel oxygenates
0.05
Escherichia coli E24377A (gi:157154711)
Mycobacterium sp. JLS (gi:126432613)
Rhodococcus jostii RHA1 plasmid pRHL1 (gi:111024785)
93
Rhodococcus jostii RHA1 (gi:111017022)
100
100 Rhodococcus erythropolis NI86 / 21 (gi:576669)
Rhodococcus erythropolis NI86 / 21 (gi:576667)
99
100
Nocardia sp. C-14-1 (gi:82548047)
100
Rhodococcus erythropolis CCM2595 (AJ973228)
Rhodococcus jostii RHA1 plasmid pRHL3 (gi:111026827)
Rhodococcus ruber IFP 2001 (AF333761)
100
Rhodococcus zopfii IFP 2005 (FJ481916)
88 Gordonia sp. IFP 2009 (FJ481918)
Fig. 1. Phylogenetic tree of the different ethR genes. The accession numbers for each sequence are indicated after the name of the strain. Branch
lengths are shown to scale, indicating relatedness.
0.05
Pseudomonas putida P450 CAM (gi:216870)
Nocardioides sp. (gi:2647406)
97
Streptomyces refuineus ssp.thermotolerans (gi:158530270)
Xanthomonas campestris pv. vesicatoria str. 85-10 (gi:78045556)
Rhodococcus jostii RHA1 (gi:111017022)
100
100
Streptomyces sp. TP-A0274 (gi:27753570)
77
Rhodococcus erythropolis NI86 / 21 (U17130.1)
100
Rhodococcus ruber DSM44319 (gi:62869556)
100
Rhodococcus sp. NCIMB9784 (gi:21622606)
Thermobifida fusca YX (gi:71914138)
100
100
Gordonia sp. IFP 2009 (FJ481921)
Rhodococcus ruber IFP 2001 (gi:16551188)
99 Rhodococcus zopfii IFP 2005 (FJ481919)
Fig. 2. Phylogenetic tree of the different ethB genes. The accession numbers for each sequence are indicated after the name of the strain. Branch
lengths are shown to scale, indicating relatedness.
did not induce expression during the 9-h experiment. When
the induction test was carried out in the presence of both
ETBE and LB, the expression level was similar to that on
MM (control).
The effect of ETBE on the expression level in R. zopfii IFP
2005 was tested and compared with that of R. ruber IFP 2001
(Fig. 4). Different levels of transcription were observed by
comparing these two strains and, for a better visualization of
these variations, the variations are expressed in percentage.
The ethB gene expression of R. zopfii IFP 2005 was induced
in the presence of ETBE and the response was greater than in
the case of R. ruber IFP 2001. Under similar conditions,
Gordonia sp. IFP 2009 did not show any induction at the
transcriptional level (data not shown).
FEMS Microbiol Ecol 72 (2010) 289–296
Discussion
Among the oxygenates, ETBE was degraded the most
efficiently by all of the strains (R. ruber IFP 2001, R. zopfii
IFP 2005 and Gordonia sp. IFP 2009) and therefore appeared
to be the preferred substrate of these strains (Table 1),
with initial degradation rates of 2.1, 3.5 and 1.6 mmol
ETBE g1 dry weight h1, respectively. Gordonia sp. IFP 2009
was the least efficient strain regarding ETBE degradation. In
all cases, the total degradation of ETBE to TBA was achieved
as shown by extending the incubation time (results not
shown). The lack of degradation by the ETBE (minus)
mutants supports the idea that the cytochrome P450
CYP249 is responsible at least in part for the degradation.
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294
Fig. 3. Level of ethB mRNA of Rhodococcus ruber IFP 2001 in response
to the presence of ETBE and other oxygenates. The test was performed in
MM containing ETBE, MTBE and TAME at 100 mg L1 or in LB containing
ETBE at 100 mg L1. , t = 0 h; , t = 3 h; , t = 6 h; , t = 9 h.
Fig. 4. Variations in the expression level for the gene ethB of Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005 in response to ETBE.
Relative copy number is the result of the following calculation: copy
number of ethB gene/copy number of 16S rRNA gene. The test was
performed in MM (control) or in MM1ETBE (ETBE) at 100 mg L1. One
hundred percent represents the control level at t = 0. , t = 0 h;
,
t = 3 h; , t = 6 h; , t = 9 h.
The cytochrome P450 encoded by ethB was more effective
towards ETBE than towards MTBE or TAME. The accession
of MTBE or TAME to the active site of the cytochrome P450
EthB could be hampered due to structural differences with
ETBE. Moreover, the total degradation of MTBE and TAME
to TBA and TAA, respectively, was not achieved in resting
cell systems and this could be attributed to a lack of reduced
cofactors that are not produced by these strains when a C1moiety is released during the cleavage of the ether bond.
ETBE was partially mineralized by all the strains tested
(4 25%), generating reduced cofactors, whereas MTBE and
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C. Malandain et al.
TAME were not mineralized at all (data not shown). Moreover, the addition of ethanol to the resting cell system of R.
ruber IFP 2001 was previously shown to be beneficial by
allowing the biodegradation of MTBE or TAME to resume
(Hernandez-Perez et al., 2001).
The degradation rates of MTBE by the different strains
IFP 2001, IFP 2005 and IFP 2009 could be compared with
that measured previously for Pseudomonas putida CAM
using camphor-induced cells where the P450 CAM was
active (Steffan et al., 1997). The rate of MTBE degradation
was 0.4 nmol g1 protein min1, i.e. 12 mmol of MTBE degraded g1 biomass dry weight h1 (Steffan et al., 1997). This
value was considerably lower than that measured with
induced cells of R. ruber IFP 2001, R. zopfii IFP 2005 and
Gordonia sp. IFP 2009 (Table 1).
The EthB proteins have a high amino acid identity
(4 70%) and belong to the same ‘new’ family of CYP249s
(results of alignment not shown). All of the primary
structure of the different EthB cytochromes P450 studied
here exhibit the P450 signature motif: Phe-XX-Gly-Xb-XXCys-X-Gly (polypeptides alignment deduced from the nucleotide sequences, data not shown). This motif includes the
invariant cysteine that ligates the heme iron to the protein.
The genes encoding EthR and EthB isolated from the
strains IFP 2001, IFP 2005 and IFP 2009 clustered together
on the two different trees showing the high conservation of
these genes. An 877-bp amplicon that was 99% similar to
ethB from R. ruber IFP 2001 was obtained from Rhodococcus
sp. PEG604 isolated for its capacity to grow on linear alkyl
ethers and also able to degrade ETBE (Kim et al., 2007). In
the case of Rhodococcus aetherivorans, which is a new species
able to degrade ethers, the enzymatic system responsible for
the oxidation was not elucidated (Goodfellow et al., 2004).
Microorganisms other than rhodococci can harbor the
eth system as demonstrated by the presence of highly
conserved eth genes in A. tertiaricarbonis L108 (Breuer
et al., 2007). The ethA, ethB, ethC and ethD genes from A.
tertiaricarbonis L108 have 98%, 98%, 97% and 99% identity,
respectively, with the corresponding eth genes of R. ruber
IFP 2001. Interestingly, the ethR gene was not detected in
this strain, which degrades MTBE and ETBE and that was
isolated from a heavily MTBE contaminated site.
The inducibility of the cytochrome P450 system by ETBE
was shown during degradation experiments after growth on
ETBE vs. LB in the case of R. ruber IFP 2001, R. zopfii IFP
2005 and Gordonia sp. IFP 2009 and the ETBE degradation
rates were, respectively, 10-, 6- and 11-fold higher after
growth on ETBE.
EthR, responsible for the regulation of the expression, has
been classified as an AraC/XylS-positive transcriptional
regulator type. This is supported by the predicted secondary
structure deduced from the nucleotide sequences (data not
shown). These are the typical family domains for binding to
FEMS Microbiol Ecol 72 (2010) 289–296
295
Cytochromes P450-mediated degradation of fuel oxygenates
DNA composed of two helix-turn-helix motives and a
nonfamily domain involved in the heteromerization and
recognition of the substrate.
Because it was not possible at a physiological level to
determine whether MTBE or TAME were also possible
inducers of the eth system due to the fact that the strains
did not grow on these two compounds whereas they grew on
ETBE; RT-qPCR experiments were performed to evaluate
the potential inducers of EthR at a molecular level. The
results obtained by RT-qPCR (Fig. 3) showed an induction
of the transcription of the ethB gene in strain IFP 2001 in
response to the presence of ETBE in the medium. TAME and
MTBE did not show any induction at the transcriptional
level. Rhodococcus zopfii IFP 2005 also showed induction by
ETBE. The induction process appeared to be more efficient
in R. zopfii IFP 2005 as the transcription rate increased more
in the presence of ETBE than it did in R. ruber IFP 2001 (Fig.
4). Thus, the transcription system might be more efficient in
R. zopfii IFP 2005 in comparison with that of R. ruber IFP
2001. This question deserves a separate study evaluating the
regulation mechanism in addition to the interaction of the
substrate and the regulator as described here.
The presence of an easily degradable substrate (e.g.
incubation on ETBE in presence of LB) inhibited the
induction process. Hence, in contrast to what was observed
in other Rhodococcus strains with hydrocarbons and chlorinated compounds (Warhurst & Fewson, 1994; TomasGallardo et al., 2006), the Rhodococcus strains able to
degrade ETBE were sensitive to catabolic repression. On the
other hand, this kind of repression was also observed
previously in Rhodococcus sp. DK17 during growth on
phthalate and benzoate (Choi et al., 2007).
Contrary to R. ruber IFP 2001 and R. zopfii IFP 2005,
Gordonia sp. IFP 2009 did not show any induction at the
transcriptional level, whereas we observed an improved
biodegradation of ETBE, MTBE and TAME after growth on
ETBE. To explain this observation, two additional observations should be considered: (1) the growth of Gordonia sp.
IFP 2009 on ETBE was slower than that of R. ruber IFP 2001
and R. zopfii IFP 2005 (data not shown) and (2) the eth
cluster from R. ruber IFP 2001 was transferred to Mycobacterium smegmatis mc2 155 and the transformants obtained
grew very slowly on ETBE (Francois, 2002). Thus, a possible
hypothesis is that there is an increased instability of the
transcripts inside Mycobacterium. Recent results showed
that specific mechanisms (specific polypurine sequence and
specific transcription initiation) contribute to mRNA stability in mycobacteria (Sala et al., 2008). Similar instability of
the transcripts could explain the results obtained with
Gordonia sp. IFP 2009.
Moreover, the induction of the system by ETBE and not
by MTBE could indicate that the O-alkyl branch of the
molecule could interact with the regulator, because the only
FEMS Microbiol Ecol 72 (2010) 289–296
difference between the two molecules is the O-methyl
branch in MTBE instead of an O-alkyl branch in ETBE. This
point is interesting as neither MTBE nor ETBE can be
considered as the natural inducer of the regulation system.
The natural inducer could be a molecule with an O-alkyl
function and should be available in the environment.
In conclusion, we showed that the three different strains
possessing the CYP249 cytochrome P450 and isolated from
different environments were responsible for the degradation
of ETBE and also MTBE and TAME (at a lower level).
Moreover, the ethB gene appears to be specifically induced
by ETBE and not by MTBE or TAME, and based on our
results, the specificity of the Eth cytochrome system is due to
the regulator rather than the cytochrome itself. This point is
important with regard to the extensive use of ETBE in
several European countries and the possibility of finding
ETBE as a groundwater contaminant. The presence and
expression of the eth genes in ether-contaminated aquifers
would have to be studied systematically in order to evaluate
the possibility of natural attenuation.
Acknowledgements
C.M. was partly supported by a CIFRE (Convention Industrielle de Formation par la Recherche) fellowship provided
by ANRT (Association Nationale de la Recherche Technique) and by IFP. We thank Dr Pierre Béguin for providing
the eth gene sequences. We thank Nicolas Lopes-Ferreira for
the numerous and very helpful discussions.
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