Microbiology (2003), 149, 795–805
DOI 10.1099/mic.0.26046-0
Expansion of growth substrate range in
Pseudomonas putida F1 by mutations in both
cymR and todS, which recruit a ring-fission
hydrolase CmtE and induce the tod catabolic
operon, respectively
Eun Na Choi,1 Min Chul Cho,13 Youngsoo Kim,2 Chi-Kyung Kim3 and
Kyoung Lee1
1
Department of Microbiology, Changwon National University, Kyongnam 641-773, Korea
Correspondence
Kyoung Lee
2,3
Department of Pharmacy and Research Center for Bioresource and Health2 and Department
of Microbiology3, Chungbuk National University, Cheongju 361-736, Korea
Klee@sarim.changwon.ac.kr
Received 9 October 2002
Revised
27 November 2002
Accepted 27 November 2002
Pseudomonas putida F1 can assimilate benzene, toluene and ethylbenzene using the toluene
degradation pathway, and can also utilize p-cymene via p-cumate using the p-cymene and
p-cumate catabolic pathways. In the present study, P. putida F1 strains were isolated that were
adapted to assimilate new substrates such as n-propylbenzene, n-butylbenzene, cumene and
biphenyl, and the molecular mechanisms of genetic adaptation to an expanded range of aromatic
hydrocarbons were determined. Nucleotide sequence analyses showed that the selected strains
have mutations in the cymR gene but not in todF gene. The impairment of the repressor CymR by
mutation led to the constitutive expression of CmtE, a meta-cleavage product hydrolase from the
cmt operon. This study also showed that CmtE has a broad range of substrates and can hydrolyse
meta-cleavage products formed from biphenyl and other new growth substrates via the toluene
degradation pathway. However, the artificially constructed strain P. putida F1(cymR : : Tcr) and a
recombinant P. putida F1, which expressed CmtE constitutively, could not grow on the new
substrates. The adapted strains possess the tod operon, which is induced by new growth
substrates that are poor inducers of wild-type P. putida F1. When the todS gene from the adapted
strains was introduced in a trans manner to P. putida F1(cymR : : Tcr), the resulting recombinant
strains were able to grow on biphenyl and other new substrates. This finding indicates that the TodS
sensor was altered to recognize these substrates and this conclusion was confirmed by nucleotide
sequence analyses. Amino acid substitutions were found in the regions corresponding to the
receiver domain and the second PAS domain and their boundaries in the TodS protein. These
results showed that P. putida F1 adapted strains capable of growth on n-propylbenzene,
n-butylbenzene, cumene and biphenyl possess mutations to employ CmtE and to induce the tod
catabolic operon by the new growth substrates.
INTRODUCTION
Pseudomonas putida F1 can assimilate benzene, toluene and
ethylbenzene (Gibson et al., 1968). The enzymic pathway
responsible for converting these aromatic hydrocarbons to
TCA cycle intermediates is called the toluene degradation
(tod) pathway (Finette et al., 1984; Gibson et al., 1990). This
tod pathway is one of the cis-dihydrodiol pathways and has
3Present address: Korea Research Institute of Bioscience and
Biotechnology (KRIBB), Yusong, Taejon 305-600, Korea.
Abbreviations: GFP, green fluorescent protein; HOHD, 2-hydroxy-6oxohexa-2,4-dienoate; Tc, tetracycline; tod, toluene degradation.
0002-6046 G 2003 SGM
been well characterized biochemically and genetically. The
catabolic genes of this pathway are clustered in a configuration of todXFC1C2BADEGIH (Fig. 1) (Lau et al., 1997;
Menn et al., 1991; Wang et al., 1995; Zylstra & Gibson, 1989;
Zylstra et al., 1988). The tod pathway consists of seven
enzymic reactions and is initiated by a multicomponent
toluene dioxygenase (encoded by todC1C2BA) (Zylstra &
Gibson, 1989) that catalyses, for instance, the formation of
cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene from
toluene with the consumption of NADH and O2 (Gibson
et al., 1970). The cis-dihydrodiol compound then undergoes
dehydrogenation and dioxygenation reactions due to the
action of the TodD and TodE enzymes, respectively, to form
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795
E. N. Choi and others
Fig. 1. Genetic organization of the p-cymene (cym), p-cumate (cmt) and toluene (tod) degradation operons in P. putida F1.
The arrows indicate the direction of transcription. TodR is a nonfunctional regulator. Chemical structures relevant to this study
are shown: I, p-cymene; II, p-cumate; III, 6-isopropyl-HOHD; IV, 2-hydroxypenta-2,4-dienoate. In the tod pathway, while
aromatic hydrocarbons with R can serve as growth substrates, those with the R9 substituent are blocked, in terms of reaction
with TodF.
a ring-fission product, namely 2-hydroxy-6-oxo-6-methylhexa2,4-dienoate (6-methyl-HOHD), which is hydrolysed by
TodF to acetate and 2-hydroxypenta-2,4-dienoate. This
latter product is further degraded to pyruvate and acetylCoA by three consecutive enzyme steps (TodGHI). The todX
gene is known to encode an outer-membrane protein
and may be involved in the uptake of aromatic hydrocarbon
substrates (Wang et al., 1995). Studies on the substrate
specificity of the tod pathway have revealed that npropylbenzene, n-butylbenzene, cumene and biphenyl are
degraded to only ring-fission dead-end products (Cho et al.,
2000; Gibson et al., 1968), which demonstrates that TodF is
limited in terms of channeling substrates into TCA cycle
intermediates (Furukawa et al., 1993).
The expressions of the tod catabolic genes are regulated by
the gene products of todST, which are located downstream
of the tod structural genes as a separate transcriptional unit
(Lau et al., 1997). The relatively large TodS protein (978 aa
with a calculated mass of 108 kDa) possesses multiple
protein domains: a basic leucine zipper motif at the N
terminus for dimerization and DNA binding, and a receiver
domain of response regulator located at the centre of the
protein flanked by one set of a PAS domain, followed by a
canonical histidine kinase (see Fig. 2). PAS domains are
known as signal sensors and are found in various redox, light
and hydrocarbon sensor proteins in archaea, bacteria and
eukarya (Taylor & Zhulin, 1999; Zhulin et al., 1997). The
796
todT gene encodes a protein that shows significant sequence
similarity with many response regulators of two-component
signal transduction systems (Hoch & Silhavy, 1995; Reizer &
Saier, 1997). Thus, TodS is regarded as a sensory hybrid
kinase that uses ATP to autophosphorylate at a specific
Fig. 2. Mutations found in the CymR (a) and TodS (b) proteins
of the adapted F1 strains (strain numbers are in parentheses).
The numbers of amino acids in each domain were deduced from
a web-based SMART program provided by http://smart.emblheidelberg.de. HTH, helix–turn–helix motif; ZIP, basic leucine
zipper motif; PAS, PAS domain; HisKA, histidine kinase domain;
RR, receiver domain of response regulator.
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Microbiology 149
F1 adapted strains with versatile catabolism
histidine residue in response to an effector. TodT as a
response regulator receives the phosphate at a conserved
aspartate (e.g. Asp-56) and then mediates changed gene
expression. This two-component signal transduction system
controls the positive regulation of the tod operon (Lau et al.,
1997). Although TodT has been reported to bind to a
nucleotide sequence (termed the tod box) in the promoter
region in front of the todX gene (Lau et al., 1997), the role
of TodS as a sensor for detecting inducers has not been
clearly demonstrated. The organic solvent-tolerant toluenedegrading P. putida strains CE2010 (Ohta et al., 2001) and
DOT-T1 (Mosqueda et al., 1999) have been reported to
possess tod operons almost identical in nucleotide sequence
to those of P. putida F1. In particular, P. putida CE2010 also
achieved growth on biphenyl.
Eaton showed that P. putida F1 can assimilate p-cymene by
using two cym and cmt operons. These operons encode
enzymes that catalyse p-cymene to p-cumate, and p-cumate
to TCA cycle intermediates, respectively (Fig. 1) (Eaton,
1996, 1997). The cmt degradation pathway is similar to the
tod pathway, except that the former incorporates one more
reaction step of decarboxylation after ring cleavage.
However, the mechanisms for the control of gene expression
exploited are completely different in the two pathways. In
the cym and cmt degradation pathways, CymR of a TetR
protein family (Hillen & Berens, 1994) acts as a repressor in
both catabolic operons with p-cumate as an effector (Eaton,
1997). The locations of the cymR, cym and cmt gene clusters
with respect to the tod operons are shown in Fig. 1.
In the present study, we isolated P. putida F1 cells adapted to
grow on n-propylbenzene, n-butylbenzene, cumene and
biphenyl as carbon and energy sources, and examined the
biochemical and genetic backgrounds of the adapted F1
strains to better understand the mechanisms underlying the
control of the expressions of the cym, cmt and tod catabolic
genes. In addition, we compared the genetic backgrounds
obtained from the strains adapted to grow on biphenyl and
other aromatics at our laboratory with those from the
natural isolate P. putida CE2010.
METHODS
Bacterial strains, plasmids and culture conditions. The bac-
terial strains and plasmids used in this study are listed in Table 1.
Escherichia coli DH5a (Ausubel et al., 1990) was used as the host
strain for the maintenance of plasmids. E. coli cells were grown in
Luria–Bertani (LB) liquid medium (Sambrook & Russell, 2001) or
on plates with 1?5 % agar. P. putida F1 and its adapted strains were
grown in LB or minimal salts (MSB) medium (Stanier et al., 1966)
with carbon source(s) as indicated. Succinate was used at 5 mM
for growth of P. putida. Alkylbenzenes were supplied in the vapour
phase and biphenyl was supplied as a solid at 1 g l21 to support
the growth of P. putida. To maintain the plasmid in the transformed E. coli or P. putida cells during growth, ampicillin
(50 mg ml21), tetracycline (12?5 mg ml21), gentamicin (10 mg ml21),
kanamycin (20 mg ml21) or chloramphenicol (30 mg ml21) was
incorporated into the culture media. Cells were grown in a 50 ml
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culture volume in liquid culture in 250 ml Erlenmeyer flasks in a
shaking incubator at 180 r.p.m. E. coli and P. putida cells were
grown at 37 and 28 ˚C, respectively.
Enzymes and chemicals. The enzymes necessary for nucleic acid
manipulation were purchased from KOSCO, Promega and GibcoBRL. Most of the chemicals used in this study were obtained from
Aldrich Chemicals. The exceptions were IPTG (Duchefa) and 2,3dihydroxybiphenyl (Wako Pure Chemicals). All chemicals were of
analytical grade.
Molecular cloning and Southern blot analysis. The standard
techniques for molecular cloning were performed as described by
Sambrook & Russell, (2001). Plasmids were isolated with a Bioneer
plasmid extraction kit (Taejeon). DNA fragments from gels were
purified using GFX PCR DNA and Gel Band Purification kit
(Amersham Pharmacia Biotech). Southern blot analysis was performed with a Photogene Nucleic Acid Detection System version 2.0.
(Gibco-BRL), using a biotin-labelled probe made using a BioPrime
DNA labelling kit according to the manufacturer’s protocol.
Mutational analysis of the todF, cymR and todS genes from
adapted strains. The todF and cymR genes from adapted strains
were retrieved by PCR amplification. PCR fragments were cloned
into a pGEM-T easy vector and the resulting plasmids were amplified in E. coli DH5a. The plasmids purified from the E. coli recombinant strains were subjected to nucleotide sequence analysis. The
mutation site(s) found was additionally confirmed by nucleotide
sequence analysis from a second clone. For the mutational analysis
of the todS gene from adapted strains, pTodS-B1, -B4, -C1, -C4 and
-C6 (Table 1) were used for nucleotide sequencing. Mutation(s) was
also confirmed from the second clone. Nucleotide sequence analysis
was carried out by Genotech using an ABI Prism 3700 DNA
Analyser (Applied Biosystems).
Construction of P. putida F1(cymR : : Tcr). The plasmid pEN-15
harboured in E. coli DH5a was transferred to a P. putida F1 recipient by conjugation with an E. coli HB101(pRK2013) helper strain as
follows. Cells of E. coli DH5a(pEN-15), E. coli HB101(pRK2013)
and P. putida F1, grown freshly overnight in LB, were suspended
individually in saline to OD600 of 2. The suspensions were mixed in
equal volumes and the mixture was placed on a nitrocellulose filter
deposited on the surface of LB agar medium. The plate was then
incubated at 28 ˚C for 12 h. The mated cells were spread, after serial
dilution with saline, on MSB agar containing 0?5 % ethanol and
tetracycline to select P. putida exoconjugants. pEN-15 could not
replicate in P. putida F1 and thus the Tcr gene had to be incorporated into the chromosome for P. putida F1 strain to be resistant
against tetracycline. The double cross-over recombination event was
expected to yield a cymR : : Tcr mutation. The expected exconjugants
were screened by PCR using the primers used for amplification of
cymR. The mutation was also confirmed by Southern analysis using
the Tcr gene as a probe. The right mutants were obtained at the
probability of one in forty.
Measurements of the oxygen uptake and substrate preference of CmtE. The rate of oxygen consumption of P. putida F1 or
of its adapted strains grown in MSB on indicated carbon source(s)
was determined with a Clark-type oxygen electrode (Rank Brothers)
as described previously (Cho et al., 2000). The methods for IPTG
induction of the cloned gene from recombinant E. coli cells, preparation of cell extracts from bacterial cells and the production of
meta-cleavage products were described in a previous report (Cho
et al., 2000). The activities of CmtE were determined at 25 ˚C by
measuring the absorbance decrease of each meta-cleavage product.
Assay mixtures (1 ml total volume) contained 0?1 M potassium
phosphate buffer (pH 7?5), meta-cleavage product (final 50 mM)
and crude extract. The wavelengths (extinction coefficients in
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E. N. Choi and others
Table 1. Plasmids used in this study
Ap, ampicillin; Cm, chloramphenicol; Gm, gentamicin; Km, kanamycin.
Plasmid
pGEM-T easy
pUC19
pRK2013
p34S-Tc
pHRP309
pJFF350
pBBR1MCS-2
pBBR1-GFP
pPROBE-GT
pAG408
pRE611
pJHE-W1
pEN-3
pEN-9
pEN-10
pEN-12
pEN-13
pEN-14
pEN-15
pEN-19
pTodS-F1
pTodS-B1
pTodS-B4
pTodS-C1
pTodS-C4
pTodS-C6
Description
Reference or source
PCR cloning vector, Ap
Cloning vector, Apr
ColE1 ori, RP4 mobilization function, Kmr
Tetracycline cassette vector, Tcr
Broad-host-range lacZ transcriptional fusion vector, Gmr
Mini-Tn5, Kmr
Broad-host-range cloning vector, IncQ, lacZa, Kmr
Broad-host-range promoter-probe vector, Gmr, promoterless gfp–cat
Broad-host-range promoter-probe vector, Gmr, promoterless gfp
Mini-Tn5–gfp, tnp, RP4 oriT, R6K oriV, Kmr Gmr Apr
7?4 kb HindIII fragment containing 39 terminus cym and
59 terminus cmt operons from F1 inserted into pLV59, Cmr
XhoI–EcoRI digested 1?3 kb PCR product, made using primers
59-GCA TTC CCG GAT CCA CGG AGG AGA CGG C-39 and
59-GTT CGA ATT CAC GTT GGC TTC ATT CAT G-39 with F1 chromosomal
DNA as template, inserted into pBBR1MCS-2, carries Plac–cmtE, Kmr
KpnI–HindIII digested gfp fragment from pBBR1-GFP inserted into
pHRP309 removing lacZ, Gmr
BamHI–KpnI digested 960 bp PCR product (todR–PtodX), made using primers
59-TCA GGA TCC CGG CGA GCC TCA CGC CAT-39 and
59-GTG GTA CCT AGG GGG CTC GAT TAT T-39 with
F1 chromosomal DNA as template, inserted into pEN-3, Gmr
1?8 kb KpnI fragment of gfp–Kmr from pAG408 inserted into pEN-9, Gmr Kmr
pEN-10 derivative lacking a 1?8 kb EcoRI fragment, carries todR–PtodX–gfp, Gmr
BamHI digested 0?7 kb fragment from pJFF350 inserted into pUC19,
carries RP-4 oriT, Apr
780 bp PCR product (cymR gene), made using primers
59-GAT CGG TAC CCT CAT TAT CAA CGA GCA G-39 and
59-TAT CGA ATT CCT CGC GCT GCC ACA CTA G-39 with F1 chromosomal
DNA as template, inserted into pGEM-T easy where Tc cassette from p34S-Tc
had been inserted at HindIII site, Tcr Apr
KpnI–EcoRI digested 2?2 kb fragment from pEN-14 inserted into pEN-13, Tcr Apr
HindIII–BglII digested 1?9 kb DNA fragment from pRE611 inserted into pPROBE-GT;
carries PcmtAa–gfp, Gmr
HindIII–XbaI digested 3530 bp PCR product, made using primers
59-CCG AAG CTT CTC CTC ATC AAA AAA GTA TTC T-39 and
59-AGA GGA TCC ACT CTG GCA TGG TAT TGG-39 with
chromosomal DNA from F1 as template, inserted into pBBR1MCS-2,
TodS expression vector, Kmr
As pTodS-F1 but with PCR product from strain B1
As pTodS-F1 but with PCR product from strain B4
As pTodS-F1 but with PCR product from strain C1
As pTodS-F1 but with PCR product from strain C4
As pTodS-F1 but with PCR product from strain C6
Promega
New England Biolabs
Figurski & Helinski (1979)
Dennis & Zylstra (1998)
Parales & Harwood (1993)
Fellay et al. (1989)
Kovach et al. (1995)
Ouahrani-Bettache et al. (1999)
Miller et al. (2000)
Suarez et al. (1997)
Eaton (1996)
r
mM21 cm21) (Duggleby & Williams, 1986; Seah et al., 1998) used
to monitor the change in concentration of the meta-cleavage
products of catechol, 3-methylcatechol, 4-methylcatechol, 3propylcatechol, 3-isopropylcatechol and 2,3-dihydroxybiphenyl were
376 (40), 389 (11?9), 382 (24?5), 383 (20), 395 (10?7) and 434
(19?8) nm, respectively. Protein was determined using the BCA protein assay (Pierce) with BSA as the standard. Specific enzyme activities are reported as mmol substrate utilized min21 (mg protein)21.
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UV/visible absorbance spectra were measured on a spectrophotometer (Scinco, model 2130). Oxygen uptake and CmtE activity
assays were conducted in triplicate, and the initial rates of the assays
were determined and used for calculation of means and standard
deviations.
PCR analysis. Reaction mixtures (50 ml) contained chromosomal
DNA (20 ng), ExTaq DNA polymerase (1 U), dNTP (0?2 mM each)
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Microbiology 149
F1 adapted strains with versatile catabolism
and the primer set (0?5 mM each) in buffer supplied by the manufacturer (TaKaRa). PCR was carried out with a Bioneer thermal cycler
(Taejeon) under the following conditions: 2 min at 94 ˚C, and then
30 cycles of 30 s at 94 ˚C, 30 s at 55 ˚C, variable times at 72 ˚C and a
final 5 min at 72 ˚C. The times used for the DNA polymerization
step at 72 ˚C were 1?5, 1?5, 1 and 4 min for the PCR products
designed for pJHE-W1, pEN-9, pEN-14 and TodST-F1, respectively.
Primers were synthesized by Bioneer. When required, the sequences
were confirmed by nucleotide sequence analysis.
Fluorescence measurements. Cells harvested by centrifugation
at a specific incubation time were washed twice with saline and
resuspended to OD600 ~0?2. The intensity of the fluorescence was
measured using a spectrofluorophotometer (model RF-5391PC,
Shimadza). Samples were prepared from three independent cultures.
The excitation and emission wavelengths used were 393 and
509 nm, respectively, with each 3?0 nm of wavelength split. The specific fluorescence intensity of each sample was defined as the measured fluorescence intensity divided by the OD600.
Measurement of meta-cleavage product accumulation with
a resting cell system. P. putida F1 and its recombinant cells were
cultured in a shaking incubator for 24 h with toluene as a carbon
and energy source. Cells were harvested by centrifugation and suspended in 0?1 M potassium phosphate buffer (pH 7?5) to OD600
0?5. Twenty microlitres of 0?1 M chemical stock dissolved in methanol was added to 10 ml the cell suspension in 250 ml Erlenmeyer
flasks. The reaction mixtures were incubated at 28 ˚C with shaking
at 180 r.p.m. and the supernatants from 1 ml aliquots were obtained
every hour by centrifugation. The accumulation of meta-cleavage
product from n-propylbenzene, cumene and biphenyl was monitored at the wavelengths described above; that from n-butylbenzene
was monitored at 388 nm.
RESULTS
Isolation of the adapted F1 strains which grew
on a broad range of aromatic hydrocarbons
To study the natural adaptation of the tod pathway to new
aromatic hydrocarbons, we isolated spontaneous P. putida
F1 mutants capable of growing on biphenyl. P. putida F1
cells were spread on MSB agar and exposed to biphenylloaded vapour. The mutants (called adapted strains)
developed to yield colonies of 1–3 mm after a 1 month
incubation. The frequency of adapted strain generation was
about 1029. Five colonies (C1, C4, B1, B4 and C6) with
enhanced growth were selected for further studies. When the
colonies were restreaked on MSB agar with biphenyl, new
colonies appeared in 3 days. Growth of the adapted strains
was also observed within a week on MSB agar in the presence of benzene, toluene, ethylbenzene n-propylbenzene,
n-butylbenzene and cumene as sole carbon and energy
sources, although the strains grew at different levels.
However, the adapted strains could not grow on nalkylbenzenes with a side chain length greater than C5.
The similarities between the ranges of the aromatic
hydrocarbons compatible with the adapted strains implied
that these strains had similar genetic background(s).
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The adapted strains had a mutation in cymR
that led to the constitutive expression of the
cym and cmt operons
Early studies showed that the meta-cleavage products formed
from n-propylbenzene, n-butylbenzene, cumene and biphenyl
through consecutive actions of toluene dioxygenase, TodD
and TodE enzymes could be hydrolysed inefficiently by
TodF in the tod pathway (Cho et al., 2000; Gibson et al.,
1968). Therefore we believed that the todF gene might be
altered in the adapted strains to act on a broad range of
substrates. However, nucleotide sequence analysis showed
no mutations in any of the PCR-cloned todF genes from the
five adapted strains.
The second possibility for the hydrolysis of the metacleavage products was that the adapted strains employed
CmtE, as the cmt pathway substrate (6-isopropyl-HOHD)
of the enzyme is the same as that formed by cumene from the
tod pathway (Fig. 1). The cmt operon is induced in the
presence of p-cumate and regulated by a repressor CymR
(Eaton, 1997). For the adapted strains to express cmtE in the
absence of an effector, the CymR protein has to be incapable
of functioning. At first, the level of CmtE expression in
P. putida F1, and in its adapted strains, was determined by
measuring the promoter activity of the cmt operon based on
the green fluorescent protein (GFP)-reporter vector (pEN19). The results are shown in Table 2. The cmt operon in
P. putida F1 was not induced by succinate, n-propylbenzene,
n-butylbenzene, cumene, biphenyl and toluene, but was
induced by p-cymene and p-cumate. In contrast, the cmt
operon was constitutively expressed in all five adapted
strains at levels similar to that in P. putida F1(cymR : : Tcr).
The constitutive expression of the catabolic cym operon in
the adapted strains and P. putida F1(cymR : : Tcr) was also
confirmed by oxygen consumption assays using p-cymene as
a substrate (data not shown). These results indicate that the
cym and cmt catabolic operons are constitutively expressed
in all five adapted strains, and that this is probably achieved
by the impairment of the repressor, CymR, resulting in weak
or the non-binding of CymR to operator regions in both the
cym and cmt promoters. Sequencing of the cymR gene from
the adapted strains confirmed the presence of different types
of mutations, which included point mutation in strains C1
(T284C), C4 (C35A), C6 (G528A) and B4 (G541A), an
insertion in strain B1 (342TGATT343) and a deletion in
strain B4 (C537). Numbers indicate nucleotide position of
the DNA sequence GenBank accession no. U24215. Number
1 corresponds to 841 of the sequence accession number.
Changes in nucleotides of the cymR gene yield mutations in
the repressor as shown in Fig. 2.
Substrate preference of CmtE
If the expression of the cmtE gene is responsible for the
adaptation of P. putida F1 to various aromatic hydrocarbons, then CmtE is able to recognize HOHD derivatives
formed from the aromatic hydrocarbons. To determine the
substrate preference of CmtE, a cmtE expression vector
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E. N. Choi and others
Table 2. Expression of cmt operon in P. putida F1 and its adapted strains
Cells were grown on the carbon source(s) indicated for 48 h in MSB medium with shaking as described in Methods.
Strain
F1
F1(pEN-19)
F1(pEN-19)
F1(pEN-19)
F1(pEN-19)
F1(pEN-19)
F1(pEN-19)
F1(pEN-19)
F1(pEN-19)
B1(pEN-19)
B4(pEN-19)
C1(pEN-19)
C4(pEN-19)
C6(pEN-19)
F1(cymR : : Tcr)(pEN-19)
Carbon source
Specific GFP expression*
Succinate
Succinate
Succinate+n-propylbenzene
Succinate+n-butylbenzene
Succinate+cumene
Succinate+biphenyl
Toluene
p-Cymene
p-Cumate
Succinate
Succinate
Succinate
Succinate
Succinate
Succinate
9±2?5
12±1?5
13±0
13±0
12±0
10±0?1
3±0?6
56±9?3
41±0?6
154±16?8
162±8?2
154±9?0
154±5?8
203±4?2
142±14?0
*Units are arbitrary.
(pJHE-W1) was constructed and the gene was expressed in
E. coli. A crude extract obtained from recombinant E. coli
DH5a(pJHE-W1) showed the preferred order with metacleavage products of 3-isopropylcatechol>3-propylcatechol>3-phenylcatechol¢3-methylcatechol (Table 3). The
extract showed no measurable activity towards HOHD
and 5-methyl-HOHD, which are meta-cleavage products
formed from catechol and 4-methylcatechol, respectively.
Although the crude extract obtained from P. putida
F1(cymR : : Tcr) grown on succinate showed the overall
specific activities of about 5–10-fold less than shown by
recombinant E. coli cells, it showed a similar substrate
preference towards HOHD derivatives. However, P. putida
F1 cells grown on succinate did not exhibit the hydrolysis
activities. This result indicates that P. putida F1(cymR : : Tcr)
cells constitutively express CmtE, which is responsible for
the hydrolysis of a broad range of HOHD derivatives by
the adapted strains.
New substrates could not induce the tod
catabolic operon enough to support the growth
of the adapted strains
In a previous study, it was concluded that the utilization of
biphenyl as a carbon and energy source by strain P. putida
CE2010 proceeds using the tod pathway enzymes to 6phenyl-HOHD (Ohta et al., 2001). In addition, the adapted
F1 strains were not generated from a P. putida F1 mutant,
which could not grow on toluene. Furthermore, P. putida
F1(cymR : : Tcr) grown on succinate failed to take up oxygen
dependent on biphenyl and other new growth substrates,
indicating that the enzymes encoded by cym and cmt
operons could not catalyse the initial oxidation of npropylbenzene, n-butylbenzene, cumene and biphenyl. In
this context, it appears that the adapted F1 strains also utilize
the tod pathway enzymes to yield ring fission products from
n-propylbenzene, n-butylbenzene, cumene and biphenyl, as
Table 3. Activities of the meta-cleavage product hydrolase in cell extracts of strains E. coli DH5a(pJHE-W1) and P. putida
F1(cymR : : Tcr)
–, No detectable activity.
Strain
Hydrolase activity (mU) towards:
HOHD
DH5a(pJHE-W1)*
F1(cymR : : Tcr)3
–
–
6-Methyl-HOHD
5-Methyl-HOHD
6-Propyl-HOHD
6-Isopropyl-HOHD
6-Phenyl-HOHD
72?4±7?2
12?0±1?4
–
–
92?4±14?5
17?6±1?2
262±8?8
57?2±2?0
90?8±4?5
9?6±2?1
*Cell extract made from cells grown in LB with IPTG induction.
3Cell extract made from cells grown in MSB with succinate.
800
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F1 adapted strains with versatile catabolism
was previously reported for the F1 strain (Cho et al., 2000).
The expression of CmtE in P. putida F1 may direct
conversion of biphenyl and other new growth substrates
to TCA cycle intermediates, which in turn indicates that
P. putida F1 expressing CmtE may grow on the new
substrates (Fig. 1). The biotransformation experiment,
described in Methods, showed that the toluene-grown
P. putida F1(cymR : : Tcr) and P. putida F1(pJHE-W1) did
not accumulate the meta-cleavage products from npropylbenzene, n-butylbenzene, cumene and biphenyl,
whereas toluene-grown P. putida F1 cells did accumulate
these products (data not shown). Despite rationalizations
of the degradation pathway of new substrates, CmtEexpressing strains, such as P. putida F1(cymR : : Tcr) and
P. putida F1(pJHE-W1), failed to grow on the aromatic
hydrocarbons, indicating that the adapted strains have
additional mutation(s).
The growth of adapted F1 strains on biphenyl and other new
aromatic hydrocarbon substrates should be accompanied by
the induction of the tod catabolic operon. Therefore, the
expression of the tod operon was assessed by monitoring the
expression of GFP from a transcriptional fusion todX : : gfp
construct (pEN-12) and by determining the toluenedependent oxygen uptake of P. putida F1 cells exposed to
various aromatic hydrocarbons during growth in the
presence of succinate. The results are shown in Fig. 3. As
expected, the induction of GFP and toluene-dependent
oxygen uptake were observed in the presence of benzene,
toluene and ethylbenzene. In contrast, the expression of the
tod catabolic operon was not detected in either analyses in
response to other aromatic hydrocarbons such as npropylbenzene, n-butylbenzene, cumene and biphenyl. In
fact, GFP induction was not observed in the presence of
Fig. 4. Biphenyl-dependent induction of the tod catabolic
operon in the adapted F1 strains. F1 or its adapted strains
containing pEN-12 were grown on MSB in the presence of
succinate and biphenyl. (a), growth pattern; (b), expression
levels of GFP at given culture times. #, F1(pEN-12); &,
B1(pEN-12); m, B4(pEN-12); %, C1(pEN-12); $, C4(pEN12); and n, C6(pEN-12).
the latter aromatic hydrocarbons during 5 day culture by
24 h testing.
Mutation is required to induce the tod operon
to allow growth of the adapted strains on
biphenyl and other aromatic hydrocarbons
Fig. 3. Induction of the tod catabolic operon by various chemicals. P. putida F1(pEN-12) cells were grown on MSB plus
succinate in the absence (control) or in the presence of each
chemical. The culture conditions used are described in
Methods. The black and white bars represent specific GFP
expression and toluene-dependent oxygen uptake rates, respectively. The levels of specific GFP expression and toluenedependent oxygen uptake were determined from 4 day cultures.
http://mic.sgmjournals.org
With the preceding result, it might be believed that the
adapted strains should contain a tod catabolic operon
mutated to be inducible in the presence of n-propylbenzene,
n-butylbenzene, cumene and biphenyl. To investigate this
possibility, the growth of the strains and the induction of
a gfp-based reporter of the tod catabolic operon (pEN-12)
were determined in the adapted strains using succinate and
biphenyl as carbon and energy sources (Fig. 4). The adapted
strains produced a diauxic growth pattern. Because a second
growth started following onset of the induction of the tod
catabolic operon, we concluded that the adapted cells use
succinate initially and then biphenyl. In a separate experiment, we found that GFP was not expressed in the presence
of succinate alone. This result also indicated that the adapted
strains did not express GFP constitutively. Moreover, the
strains differed in terms of the level and lag period of
the biphenyl-dependent induction of GFP. In addition, the
levels of growth of the adapted strains on biphenyl were not
proportional to the degree of GFP induction. In the presence
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801
E. N. Choi and others
of n-propylbenzene, n-butylbenzene and cumene on MSB
agar plates, the GFP induction was also observed in the
adapted strains but not in P. putida F1. In the presence of
toluene as a sole carbon and energy source, P. putida F1 and
its adapted strains expressed GFP and this expression
reached a maximum after 2 or 3 days of incubation. The
specific activity of GFP expression in the steady state of
P. putida F1 was a mean of 480, and those of C1, C4, C6, B1
and B4 were found to be 130, 400, 100, 550 and 720,
respectively. This result showed that the mutation in the
adapted strains also influences the induction of the tod
catabolic operon by toluene. The relative induction level of
GFP in each adapted strain by toluene was similar to that
induced by biphenyl.
This result indicates that the mutated todS gene gives the
adapted strains the ability to sense the aromatic hydrocarbons as effectors. To identify the amino acid(s) changed
in TodS, we sequenced the nucleotides of todS in the adapted
strains. Strains C4, C1, C6, B1 and B4 contained only
the nucleotide substitutions A1410G, C1956G, C2318A,
G1976C and C1736T, respectively. The numbers indicate
nucleotide position in the DNA sequence, GenBank accession no. U72354 (Lau et al., 1997). Strain B1 had an
additional nonsense mutation, A3613G. Changes in nucleotides of the adapted strains gave rise to the amino acid
changes indicated in Fig. 2.
DISCUSSION
todS mutation in the adapted strains
Because the TodS protein is regarded as an effector sensor, it
is likely that in the adapted strains, the acquisition of tod
operon induction by biphenyl and other new growth
substrates took place by the mutation of the protein. Prior
to the complementation experiments with the mutated
genes, we constructed the expression vector of the todS gene
(pTodS-F1) from P. putida F1, as described in Methods.
The vector was introduced into P. putida F1(cymR : : Tcr),
wherein the induction of the tod operon is the limiting factor
for strain growth on biphenyl and other new aromatics. The
recombinant strain P. putida F1(cymR : : Tcr)(pTodS-F1)
was unable to grow on new substrates. In contrast, when the
todS gene in an expression vector from the adapted strains
was introduced into P. putida F1(cymR : : Tcr), the recombinant strains were able to utilize these substrates (Table 4).
The relative growth of P. putida F1(cymR : : Tcr) containing
various todS alleles on different carbon sources was similar
to the corresponding adapted strains although P. putida
F1(cymR : : Tcr)(pTodS-C1) grows slower than strain C1.
Micro-organisms are readily adapted to degrade many
xenobiotic aromatic compounds through various molecular
adaptation mechanisms (de Lorenzo & Perez-Martin, 1996;
van der Meer et al., 1992). Sometimes, novel metabolic
capabilities are acquired by modifications of pre-existing
metabolic pathways within one strain as shown in this study.
Here, we demonstrated that P. putida F1 adapted strains,
which are generated spontaneously to allow growth additionally on n-propylbenzene, n-butylbenzene, cumene and
biphenyl, basically contained mutations in the cymR and
todS genes. When cells were exposed to biphenyl and other
new substrates, mutated TodS proteins were able to recognize the chemicals as effectors of tod catabolic operon
expression, which is induced insufficiently by the same
chemicals in the wild-type F1 strain to undertake chemical
degradation. However, the tod pathway enzymes are able to
oxidize these chemicals to meta-cleavage products only,
which become dead-end metabolites because of the narrow
substrate preference of TodF. On the other hand, the cymR
mutation leads to constitutive expression of CmtE, which
Table 4. Growth characteristics of adapted and corresponding recombinant strains carrying todS variant
Growth was measured by streaking the recombinant strains on MSB agar with the chemical supplied in the vapour phase. The growth level
was determined after 10 days at 28 ˚C and is indicated by the number of + symbols. No growth is indicated by –.
Strain
Growth
n-Propylbenzene
F1
F1(cymR : : Tcr)(pTodS-F1)
B1
F1(cymR : : Tcr)(pTodS-B1)
B4
F1(cymR : : Tcr)(pTodS-B4)
C1
F1(cymR : : Tcr)(pTodS-C1)
C4
F1(cymR : : Tcr)(pTodS-C4)
C6
F1(cymR : : Tcr)(pTodS-C6)
802
–
–
++++
++++
++
+
+++
++
++
++
+++
++++
n-Butylbenzene
Cumene
Biphenyl
Toluene
–
–
+++
+++
++
+++
++
+
++
+++
+++
+++
–
–
++
++
++
+++
+++
+
+++
+++++
+++
+++
–
–
++
+++
++
++
+++
++
+
+
+++
++++
+++
+
+++
+++
++
+
+++
++
+++
++++
+++
+++
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F1 adapted strains with versatile catabolism
has a broad range of substrates. Therefore, these intermediates are hydrolysed by CmtE to 2-hydroxypenta-2,4dienoate, which is a metabolite common to both the tod and
cmt pathways (Fig. 1), and which could be degraded to
pyruvate and acetyl-CoA to enter the TCA cycle. This could
be the best explanation as to why the adapted strains can
grow on biphenyl and other aromatics.
It has been previously demonstrated that a natural isolate of
P. putida CE2010 assimilates biphenyl in a CmtE and tod
catabolic operon manner, via a mosaic of pathways (Ohta
et al., 2001). Although the biochemical backgrounds of the
growth of P. putida CE2010 and of the adapted F1 strains on
biphenyl appear to be similar, the ways in which the
expressions of the degradation enzymes are controlled
appear to differ as follows. P. putida CE2010 differs by a
single base in the cmt promoter-operator region from
P. putida F1 in the DNA region from the 39 terminus of cymE
to the 59 terminus of todF (GenBank accession number
AB042508) (Fig. 1). It was concluded that P. putida CE2010
first degrades biphenyl to 6-phenyl-HOHD using the tod
pathway enzymes, and degraded further by CmtE (Fig. 1),
which is constitutively expressed by the less effective binding
of the repressor CymR to the cmt operator region (Eaton,
1997) due to the changed nucleotide. No base difference was
found in cymR in P. putida strains F1 and CE2010. Although
the involvement of CmtE in the hydrolysis of the ring-fission
products in both P. putida CE2010 and the adapted F1
strains is the same, the expression levels of CmtE could
differ. While in P. putida CE2010 CmtE was constitutively
expressed at a low level, though inducible by biphenyl, in the
adapted F1 strains CmtE was constitutively expressed at a
high level, and was not inducible by biphenyl. Furthermore,
it was interesting to find in the same study that when a
P. putida CE2010-type cmt promoter region was artificially
introduced into P. putida F1, the recombinant P. putida F1
could not grow on biphenyl as the sole carbon source (Ohta
et al., 2001). This result indicates that the constitutive
expression of CmtE is not enough for P. putida F1 to achieve
the biphenyl-dependent growth, and thereby suggests that
the induction of the tod operon by biphenyl differs in two
strains. Because P. putida CE2010 can grow on biphenyl, it is
expected that the tod operon is inducible by biphenyl, and
that this probably reflects the amino acid differences of
TodSCE2010 and TodSF1, the latter of which is unaffected by
biphenyl. The nucleotide sequence of TodSCE2010 is not
available in the databases.
The level of induction of the tod catabolic operon in P. putida
F1 by various aromatic hydrocarbons has been determined
previously by examining the level of TodE expression and
the presence of its mRNA (Cho et al., 2000). While npropylbenzene and cumene induced the TodE activity at
a level of one third that induced by toluene and also yielded
mRNA signals, n-butylbenzene and biphenyl induced a basal
level of TodE activity and gave negative mRNA signals. In the
present study, measurements of the activity of a gfp-based
reporter of the promoter of todX, and of toluene-dependent
http://mic.sgmjournals.org
oxygen uptake, indicated that the expression of the tod catabolic operon is negligible in the presence of n-propylbenzene,
n-butylbenzene, cumene or biphenyl (Fig. 3). The discrepancy
between the previous and the present work in induction of
the tod catabolic operon by the chemicals may be due to
the methods of cell culture applied. The cells used for
the previous experiments were from plates whereas the
cells used in this study were from liquid culture. In many
aspects including availabilities of oxygen, water and other
nutrients, the conditions imposed on the cells would be
not the same in the two culture systems. Nevertheless,
the induction of the tod catabolic operon even by
n-propylbenzene and n-butylbenzene may be insufficient
to support the growths of the recombinant strains P. putida
F1(cymR : : Tcr) or P. putida F1(pJHE-W1), in which CmtE
is expressed constitutively.
CmtE hydrolyses 6-phenyl-HOHD (Ohta et al., 2001) as
well as 6-isopropyl-HOHD (Eaton, 1996). In the present
study, we further characterized the substrate preference of
CmtE. As pointed out by Ohta et al. (2001), the level of
amino acid sequence identity between CmtE and the known
hydrolases identified from aromatic degradation pathways
is relatively low. In the web databases, CmtE was found to
have a maximum sequence identity of 34 % with Grampositive dibenzofuran-degraders Terrabacter sp. strain
DBF63 (Habe et al., 2002) and Rhodococcus sp. strain YK2
(Iida et al., 2002), which indicated that CmtE is probably
separated from the groups of known hydrolases. In this
respect, it may be that CmtE has different substrate
preference compared to the well-characterized BphDLB400,
which is also known to hydrolyse 6-isopropyl- and 6phenyl-HOHD. The latter enzyme has been identified in the
biphenyl degradation pathway of Burkholderia sp. LB400
(Hofer et al., 1993; Mondello, 1989). CmtE showed higher
activity toward 6-isopropyl-HOHD than 6-phenyl-HOHD
and showed relatively high activity toward 6-methyl-HOHD
(Table 3), whereas BphDLB400 showed higher activity
toward 6-phenyl-HOHD than 6-isopropyl-HOHD, and
little activity to 6-methyl-HOHD (Seah et al., 1998). This
substrate preference of CmtE seems to lie between that of
BpbDLB400 and TodF, which showed highest specificity for
HOHDs with small substituents at C-6 (Seah et al., 1998).
An early study showed that the introduction of the bphD
gene obtained from the biphenyl degrader Pseudomonas
pseudoalcaligenes KF707 into P. putida F1, enabled its
growth on biphenyl (Furukawa et al., 1993). However, as
biphenyl could not induce the tod catabolic operon in
P. putida F1, the recombinant strain could not grow on
biphenyl. In the study, however, it is likely that the selected
recombinant strains might contain additional mutation(s)
to allow biphenyl recognition as an effector. During this
study, we found that P. putida F1(pJHE-W1), which was
isolated initially from LB with kanamycin and contained the
expected plasmid, could not grow on biphenyl as a sole
carbon and energy source. However, a few colonies were
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803
E. N. Choi and others
formed with increased incubation time (2–3 weeks) due to
additional mutation(s).
The involvement of a two-component signal transduction
system as a transcriptional regulatory unit in the tod
pathway is intriguing, because many other degradation
pathways of biphenyl and other aromatics employing
similar ‘cis-dihydrodiol’ pathways use single component
transcriptional regulators (Diaz & Prieto, 2000). The
situation is complicated by the fact that TodS is composed
of multiple protein domains as shown in Fig. 2. The
presence of a receiver domain and two PAS domains in TodS
implies that the sensor may recognize different environmental and/or intracellular signals. In this regard, both
TodS and TodT proteins are known to be required for the
chemotaxis to aromatic hydrocarbons in P. putida F1
(Parales et al., 2000), indicating that the catabolism and
microbial behaviour are co-ordinately regulated, and the
chemotaxis is probably controlled at the transcriptional
level. Similar two-component signal transduction systems
have been identified in the pathways of aerobic styrene
degradation (O’Leary et al., 2002; Panke et al., 1999; Santos
et al., 2000; Velasco et al., 1998), in other forms of aerobic
toluene degradation (Mosqueda et al., 1999), and in anaerobic toluene degradation (Coschigano & Young, 1997;
Leuthner & Heider, 1998). The sensor proteins found in
anaerobic toluene degradation are shorter than TodS and
contain two PAS domains and one histidine kinase domain.
However, the functions of each domain from the aromatic
hydrocarbon sensors have not been identified with the
exception of that of the leucine zipper dimerization motif
from TodS in P. putida F1 (Lau et al., 1997). In the present
study, we were able to identify amino acids in TodS
responsible for the recognition of new and the original
effectors. The mutations were found in the receiver domain,
the second PAS domain and in their boundaries (Fig. 2).
Mutations found in strains C1 and B1 increased the hydrophobicities of the amino acid residues, and those in strains
C6, B4 and C4 introduced charge changes. The exact
functions of those amino acids used for effector binding or
for the activation of TodS require further investigation.
Finally, this study provides a means to determine the
important amino acid residues of CymR in terms of its
structural stability or DNA binding, and the residues
required by TodS for effector binding. These modified
proteins could be obtained from F1 strains adapted to
assimilate n-propylbenzene, n-butylbenzene, cumene and
biphenyl.
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ACKNOWLEDGEMENTS
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