Molecular Microbiology (2001) 39(4), 11001106

Global and cognate regulators control the expression of

the organic solvent efflux pumps TtgABC and TtgDEF of
Pseudomonas putida
Estrella Duque, Ana Segura, Gilberto Mosqueda and
Juan L. Ramos*
Consejo Superior de Investigaciones Cientficas, Estacion
Experimental del Zaidn, C/Profesor Albareda 1, E-18008
Granada, Spain.
Summary
Pseudomonas putida DOT-T1E grows on a water
toluene double liquid phase. Toluene tolerance in this
microorganism is mainly achieved by at least two
efflux pumps that belong to the RND family. The
TtgDEF efflux pump is induced by toluene, whereas
the other efflux pump, called TtgABC, is expressed at
a high level in cells not exposed to toluene and at a
lower level in cells grown with toluene. The ttgR gene
is adjacent to the ttgABC operon and is transcribed
divergently from ttgA. The expression level of ttgR
was fourfold higher in cells growing in the presence
of toluene than in its absence. In a TtgR-deficient
background, expression from the ttgA promoter
increased about 20-fold, suggesting that TtgR
represses expression from the ttgA promoter. In
this mutant, background expression of the ttgR
gene was also much higher than in the wild-type
background; however, its level of expression increased
in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed
the same pattern of expression as in the wild type.
Analysis of a P. putida pTn5cat mutant that exhibited
increased sensitivity to a sudden toluene shock,
regardless of whether or not it was previously exposed
to low toluene concentrations, revealed that pTn5cat
had interrupted an lrp-like gene. The ttgR gene was
expressed at very high levels in this mutant, with
concomitant repression of expression of the ttgABC
operon. The second ttgDEF efflux pump was expressed
at low levels in this mutant strain, suggesting that the
Lrp-like protein is a global regulatory protein involved
in the solvent-tolerant response of this strain.

Accepted 6 December, 2000. *For correspondence. E-mail jlramos@

eez.csic.es; Tel. (134) 958 121 011; Fax (134) 958 129 600.
Q 2001 Blackwell Science Ltd

Introduction
Organic solvents with a logPow between 1.5 and 3.5 are
extremely toxic to microorganisms (Sikkema et al., 1995)
because they dissolve in the cytoplasmic membrane,
disorganize it and collapse the cell membrane potential.
This, together with the induced loss of lipids and proteins,
leads to irreversible damage, resulting in the death of the
cell (de Smet et al., 1978; Sikkema et al., 1995).
However, a number of strains belonging to the genus
Pseudomonas grow in the presence of supersaturating
concentrations of toluene (Inoue and Horikoshi, 1989;
Cruden et al., 1992; Weber et al., 1994; Ramos et al.,
1995; Li et al., 1998). Tolerance to organic solvents in
these Pseudomonas putida strains is achieved mainly by
a series of efflux pumps that remove the organic solvents
from cell membranes (Isken and de Bont, 1996; Ramos
et al., 1997; 1998; Kieboom et al., 1998a, b; Mosqueda
and Ramos, 2000).
In P. putida DOT-T1E cells, two efflux pumps, TtgABC
and TtgDEF (Ttg stands for toluene tolerance
genes), prevent the accumulation of toluene and other
related aromatic hydrocarbons in the cell membranes
(Ramos et al., 1998; Mosqueda and Ramos, 2000). The
pumps belong to the resistancenodulationdivision
(RND) efflux pump family (Zgurskaya and Nikaido,
1999; Koronakis et al., 2000). The TtgABC pump is
expressed at high basal levels (Ramos et al., 1998),
whereas the TtgDEF is induced by aromatic hydrocarbons
(Mosqueda and Ramos, 2000). The importance of the
TtgABC pump for toluene tolerance was revealed by the
fact that a ttgB knock-out mutant was hypersensitive to a
sudden toluene shock (Ramos et al., 1998). However,
toluene exclusion functioned efficiently in this mutant
when cells were pregrown on LB medium with toluene in
the gas phase, which revealed the importance of the
inducible pump.
In this study, we show that the expression of the ttgABC
operon is regulated by the ttgR gene product. The ttgABC
genes are, in fact, overexpressed in a P. putida mutant
lacking the TtgR protein. Expression of the ttgR gene is
controlled, in turn, by another repressor belonging to the
Lrp family of global regulators. The Lrp-like protein is
also involved in the control of expression from the ttgD
promoter.

Regulation of drug exclusion 1101

Results and discussion
Identification of a gene upstream from the ttgABC operon
that is divergently transcribed with respect to the efflux
pump genes
Evidence that the P. putida ttgABC genes are transcribed
at relatively high basal levels comes from studies of
alkaline phosphatase (AP) activity in the P. putida DOTT1E-18 strain, in which a 0 phoA gene was inserted in
frame within the ttgB gene. In P. putida DOT-T1E-18 cells
growing on LB, AP activity was < 4 ^ 1 mmol of 4nitrophenol min21  OD660 (n 4) regardless of the
growth phase. Because the TtgABC pump is involved in
the efflux of toluene, we also determined AP activity in
cells exposed to toluene supplied via the gas phase.
We found that AP activity was lower, in the range
2.5 ^ 0.2 mmol of 4-nitrophenol min21  OD660 (n 6).
These results were surprising, because they suggested
that the TtgABC pump could be downregulated in
response to toluene.
Regulators of drug efflux pumps in Pseudomonas
aeruginosa, Escherichia coli, Neisseria gonorrhoeae and
other microorganisms are often adjacent to the genes
coding for the efflux pumps (Klein et al., 1990; Ma et al.,
1993; Pan and Spratt, 1994; Moore et al., 1999; Srikumar
et al., 2000). We therefore decided to sequence the DNA
region surrounding the ttgABC genes. To this end, we
screened a P. putida library in pUC18 with a ttgA gene
probe and found a plasmid, called pED2, that carried
about 2 kb of DNA upstream from ttgA and 600 bp of DNA
downstream from ttgA. The sequenced DNA contained a
single open reading frame (ORF; 633 bp) upstream from
the ttgABC operon and transcribed in the opposite
direction.
Analysis of the deduced 211-amino-acid polypeptide
revealed a potential helixturnhelix DNA-binding
domain between residues 33 and 55. Comparison of
this deduced polypeptide with sequences deposited in
several databases showed about 5060% similarity to a
number of transcriptional repressors, such as AcrR (Ma
et al., 1993) and EnvR (Klein et al., 1990), from E. coli, the
MtrR repressor from N. gonorrhoeae (Pan and Spratt,
1994), the AmrR regulator from Burkholderia pseudomallei and P. aeruginosa (Moore et al., 1999; WestbrockWadman et al., 1999) and the MexZ regulator from P.
aeruginosa (Ramos-Aires and Plesiat, 1999; Tan et al.,
1999), all of which control efflux pumps. We called the
protein TtgR because it is involved in the control of the
ttgABC operon (see below).
Identification of the transcription initiation point of the
divergent ttgA and ttgR promoters
The transcription initiation points of ttgA and ttgR were
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 11001106

mapped as described in Experimental procedures. Each

of the two genes was transcribed from a single promoter
(Fig. 1A and B). The 5 0 end of the mRNA ttgA starts at
T99 in the sequence shown in Fig. 1C, whereas ttgR
starts at G128 in the same sequence. The location of the
start sites indicates that the promoters of the divergently
transcribed ttgR and ttgA fully overlap. The 210 region of
the ttgA and ttgR promoters exhibits some degree of
similarity to promoters recognized by RNA polymerase
with sigma 70 (Fig. 1D) and lower similarities in their 235
regions.
The intensity of the cDNA bands in Fig. 1A and B was
used to estimate the relative strength of the ttgA and ttgR
promoters under different conditions. Densitometric analysis suggested that the level of ttgA mRNA in cells
growing in the presence of toluene was 1.5-fold (n 3)
lower than that in the absence of the aromatic compound
(Fig. 1A), which is in agreement with the results obtained
in the AP assay. The level of ttgR mRNA was four- to

Fig. 1. Transcription initiation point of the ttgA and ttgR genes in

the P. putida wild-type strain and the ttgR mutant under different
growth conditions.
A. Transcription initiation mapping of the ttgA gene.
B. Transcription mapping of the ttgR gene.
In (A) and (B), lanes 3 and 4 correspond to the wild-type strain and
lanes 5 and 6 to the ttgR mutant DOT-T1E-13 strain. Cells were
pre-exposed to toluene in the gas phase (lanes 4 and 6) or grown
in the absence of the solvent (lanes 3 and 5). Lanes 1 and 2
correspond to a ladder.
C. DNA sequence at the divergent PttgA and PttgR promoter regions.
The transcription initiation point is in bold, and the arrow indicates
the direction of gene transcription. The ATG start codon of each
gene is shown in bold.
D. The ttgA and ttgR promoter sequences. The proposed 210 and
235 boxes are shown.

1102 E. Duque, A. Segura, G. Mosqueda and J. L. Ramos

eightfold higher in cells growing on toluene than in its
absence (Fig. 1B).
We have shown before that P. putida DOT-T1E
cells pre-exposed to toluene are more tolerant to
sudden toluene shocks than uninduced cells. Therefore,
the decreased expression of the TtgABC pump
described above should be efficiently compensated
for by the induction of the TtgDEF pump in cells preexposed to toluene, so that toluene tolerance is not
compromised.

Expression from the ttgD promoter is not compromised in

a ttgR null background
As in the wild-type strain, the ttgDEF operon was
expressed in the ttgR mutant only in response to toluene,
and the induction level in the wild-type and the ttgR
mutant strains was similar (not shown). These results
indicate that TtgR controls expression from the ttgA
promoter but not from the ttgDEF operon.

Phenotypic analysis of the ttgR null mutant of P. putida

DOT-T1E-13
Analysis of the expression of ttgA and ttgR genes in the
ttgR null mutant
Analysis of the primary sequence of TtgR predicted it to
be a repressor belonging to the TetR family (Klein et al.,
1990; Ma et al., 1993; Pan and Spratt, 1994; Deckert et al.,
1998; Palumbo et al., 1998; Moore et al., 1999; RamosAires and Plesiat, 1999; Tan et al., 1999; WestbrockWadman et al., 1999; Orth et al., 2000). To determine
whether the TtgR protein was involved in the control of
ttgABC expression, we generated a null ttgR mutant
called P. putida DOT-T1E-13 (see Experimental procedures). We tested the level of expression from the ttgA
promoter and from the overlapping and divergent ttgR
promoter in the DOT-T1E-13 background. The assays
were performed with mRNA prepared from DOT-T1E-13
cells grown in the absence and in the presence of toluene
(Fig. 1A and B). Expression of the ttgA gene was about
20-fold higher than in the wild-type background, regardless of the growth conditions. Therefore, these results
indicate that TtgR represses expression from the ttgA
promoter. In P. putida DOT-T1E-13 cells growing in the
presence of toluene, expression from ttgR was found to
be about eightfold higher than that in cells growing in the
absence of the aromatic hydrocarbon. The basal and
induced level of expression from ttgR in the TtgR-deficient
background was at least twofold higher than in the wildtype background. These results indicated that TtgR might
be involved in its own control and that another regulator
must be responsible for the increased expression from the
ttgR promoter in the presence of toluene (see below).
Although in vitro assays are needed to define the site of
action of the TtgR protein at the ttgA and ttgR promoters,
it is worth noting that an inverted repeat overlaps the 210
box of the ttgA promoter, whereas in ttgR, it is located
between the 210 and 235 regions (Fig. 1D). The TtgR
repressor might hinder access of RNA polymerase to the
promoters in the same way that TetR inhibits expression
from its cognate promoters (Rojo, 1999; Orth et al., 2000).
However, the different location of the inverted repeat in
the two promoters might account for some of the
differences observed in their pattern of expression.

Our previous results showed that the TtgABC pump is

involved in the efflux of toluene, isopropylbenzene and pxylene. It is probably also involved in the efflux of
chloramphenicol (Cm), carbenicillin (Cb), nalidixic acid
(Nal) and tetracycline (Tc) in P. putida DOT-T1E because
resistance to these antibiotics was compromised in a ttgB
knock-out strain gene (Ramos et al., 1998). This indicates
that the TtgABC pump might remove both aromatic
hydrocarbons and antibiotics of very different chemical
structure. Therefore, we analysed tolerance to aromatic
hydrocarbons and resistance to antibiotics in P. putida
DOT-T1E-13 and the ttgB mutant P. putida DOT-T1E-18.
The higher levels of TtgABC proteins in the TtgR
mutant led us to expect that DOT-T1E and DOT-T1E-13
would be able to grow on LB medium with 0.3% (v/v)
isopropylbenzene, p-xylene and toluene, as was the case
(not shown). As toluene tolerance in P. putida DOT-T1E
cultures is influenced by growth conditions, we determined the survival of P. putida DOT-T1E and DOT-T1E13 cells in liquid culture medium when cells were
pregrown on LB liquid medium without toluene or with
toluene supplied via the gas phase. We also included in
this series of assays the ttgB:: 0 phoA DOT-T1E-18 mutant
grown under the same conditions. These cells were then
challenged with 0.3% (v/v) toluene, and their short-term
survival was determined (Fig. 2). The wild-type and DOTT1E-13 strains behaved similarly, and growth conditions
influenced the response obtained. Most of the wild-type or
DOT-T1E-13 cells that were pre-exposed to low toluene
concentrations survived the solvent shock (Fig. 2),
whereas the number of cfu ml21 cells not pre-exposed
to toluene decreased to 0.01%-0.001% of the initial
number after 30 min (Fig. 2). After prolonged incubation,
the cells that had survived the shock multiplied (not
shown). In agreement with our previous findings, DOTT1E-18 was more sensitive to a sudden toluene shock
than the wild-type strain regardless of the growth
conditions (Fig. 2).
Our earlier studies showed that the TtgABC pump
expelled trichlorobenzene and that the efflux of 14C-labelled
1,2,4-trichlorobenzene was competitively inhibited by
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 11001106

Regulation of drug exclusion 1103

Fig. 2. Survival of wild-type DOT-T1E, DOTT1E-13, DOT-T1E-18 and DOT-T1E-15 from
a sudden toluene shock. Cells were grown in
30 ml of LB medium (circles) or LB medium
with toluene in the gas phase (triangles) until
the culture reached a turbidity of about 1 at
660 nm. These cultures contained about 109
cfu ml21. The cultures were divided in two
halves; to one, we added 0.3% (v/v) toluene
(closed symbols), and the other was kept as a
control (open symbols). The number of viable
cells was determined before the addition of
toluene and at the indicated times.

toluene, isopropylbenzene and xylenes, but not by

benzene. P. putida DOT-T1E is able to grow on LB
medium supplemented with 15 mM benzene, but 25 mM
of this aromatic hydrocarbon prevented cell growth.
However, DOT-T1E-13 was as sensitive to benzene as
DOT-T1E, suggesting that the TtgABC pump does not
remove benzene (not shown).
The sensitivity of the wild type and the ttgR and ttgB
mutants to several antibiotics was tested by determining
the minimal inhibitory concentration (MIC). Table 1 shows
that the null ttgR mutant, which overproduces the efflux
pump proteins, was more resistant than the wild type to
Cb, Cm, Nal and Tc, whereas the TtgB mutant was more
sensitive than the wild type to these antibiotics. This
provides further evidence that the TtgABC pump is
involved in the efflux of these antibiotics, and that an
increase in the expression of the efflux pump proteins as a
consequence of the lack of repression of the ttgABC
genes leads to increased tolerance to antibiotics. The
TtgABC pump system is therefore similar to the MexAB
OprM pump of P. aeruginosa (Li et al., 1998) and to the
AcrABTolC pump of E. coli (Aono et al., 1998), which
expel antibiotics and hydrocarbons. However, these
findings contrast with the results of Mosqueda and
Ramos (2000) and Isken and de Bont (1998), who
showed that the TtgDEF pump of DOT-T1E and the
SrpABC pump of P. putida S-12 remove aromatic
hydrocarbons but not antibiotics.
Involvement of an lrp-like gene product in control of the
expression of the ttgR gene
One of the questions left open by the results described
above was how the expression of the ttgR gene is
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 11001106

controlled. To identify new genes involved in toluene

tolerance, we generated a battery of mutants of P. putida
DOT-T1E with a Tn5-derived plasmosome, pTn5cat, and
isolated mutants unable to tolerate a sudden toluene
shock, as described before (Ramos et al., 1998). Some of
the mutants had insertions in the ttgABC operon (our
unpublished results), and we also found an insertion in a
chromosomal locus different from the ttgABC genes. This
mutant was called P. putida DOT-T1E-15. The location of
this pTn5cat insertion, the rescue of the wild-type gene
and sequence analysis of this new gene are described in
Experimental procedures. This set of analyses revealed
that the plasmosome had knocked out an lrp-like gene
that we called ttgX.
Tolerance to a sudden toluene shock was assayed in
strain DOT-T1E-15 in cells pre-exposed or not to toluene
through the gas phase (Fig. 2). Regardless of the growth
conditions, the strain was much more sensitive to
toluene than the wild type. These results prompted us to
determine expression from the ttgA, ttgR and ttgD
promoters in cells grown in the absence and in the
presence of toluene supplied through the gas phase. In
the ttgX mutant, ttgR was expressed at higher levels than
in the wild-type background (Fig. 3), with concomitant
inhibition of the expression of the ttgABC operon (Fig. 3).
Table 1. Susceptibility of P. putida DOT-T1E and its ttgB and ttgR
mutants to antibiotics.
MIC (mg ml21)
Strain

Genotype

Cb

Cm

Nal

Tc

DOT-T1E
DOT-T1E-13
DOT-T1E-18

Wild type
ttgR::VKm
ttgB::Tn5

512
1024
16

128
256
32

64
256
8

4
16
0.24

1104 E. Duque, A. Segura, G. Mosqueda and J. L. Ramos

Fig. 3. Expression from the ttgA, ttgR and
ttgD promoters in the wild-type P. putida
DOT-T1E and its mutant DOT-T1E-15 bearing
an insertion at the ttgX gene. Wild-type or
DOT-T1E-15 (TtgX-deficient) cells were grown
in the absence () and in the presence (1) of
toluene, and transcription from the ttgA, ttgR
and ttgD promoters was determined as
described in Experimental procedures.

In the wild-type strain, the ttgDEF operon was not

expressed in cells that had not been pre-exposed to
toluene, whereas expression was strongly induced in cells
pre-exposed to toluene. Surprisingly, in contrast to the
wild-type strain, low levels of expression from the ttgDEF
operon were observed in DOT-T1E-15, regardless of
whether or not it was exposed to toluene (Fig. 3).
The expression pattern of the efflux pumps is consistent
with the finding that DOT-T1E-15 does not withstand a
sudden toluene shock, in contrast to the wild type. In
addition, these results indicate that the TtgX protein is a
global regulator of efflux pumps in P. putida DOT-T1E,
although at present we do not know whether TtgX controls
expression from ttgABC, ttgR and ttgDEF directly or
through a specific regulator.
In short, our results indicate that toluene tolerance in
P. putida DOT-T1E involves at least two efflux pumps
controlled by cognate regulators and global ones, so that
cells respond rapidly to solvent exposure.
Experimental procedures
Bacterial strains, culture medium and growth conditions
P. putida DOT-T1E is a rifampicin-resistant derivative of the
solvent-tolerant strain P. putida DOT-T1 (Ramos et al.,
1995); P. putida DOT-T1E-18 is a miniTn5- 0 phoA mutant of
P. putida DOT-T1E that shows sensitivity to toluene, Nal,
Cm, Cb and Tc (Ramos et al., 1998). P. putida DOT-T1E-13
is a ttgR::Km mutant generated in this study by site-directed
mutagenesis (see below). P. putida DOT-T1E-15 has a
pTn5cat plasmosome inserted in the ttgX gene (as identified
in this study) and is sensitive to toluene. E. coli S17-1lpir was
used to replicate plasmid pUIRM504 (Marsch-Moreno et al.,
1998), and E. coli DH5a was used in cloning experiments
(Sambrook et al., 1989).
Cells were grown routinely on liquid LB medium at 308C
and shaken on an orbital platform operating at 150200
rotations min21 (Abril et al., 1989). Growth was measured by
the increase in cfu ml21 on LB solid medium supplemented
with appropriate antibiotics.
Antibiotics were used at the following concentrations:
ampicillin, 50 mg ml21; kanamycin 50 mg ml21; rifampicin
20 mg ml21, tetracycline 0.2510 mg ml21; or at the concentrations indicated in Table 1.

Recombinant DNA techniques

Preparation of P. putida DOT-T1E total DNA, colony screening hybridization isolation of plasmid DNA, digestion of DNA
with restriction enzymes and agarose gel electrophoresis
were performed using standard methods (Sambrook et al.,
1989). DNA was sequenced by the dideoxy sequencing
termination method using T7 phage DNA polymerase as
well as universal or specific 20-mer fluorescently labelled
oligonucleotides to prime synthesis. Amplification of DNA
by polymerase chain reaction (PCR) was performed as
described previously (Ramos et al., 1998; Mosqueda and
Ramos, 2000).

Construction of a ttgR null mutant of P. putida DOT-T1E

by gene replacement
We first knocked out the ttgR gene in vitro. Plasmid pED2 is a
pUC18 derivative that carries the ttgR gene and flanking
DNA. This plasmid has a single BsiWI site in the middle of the
ttgR gene. Upon digestion with BsiWI, the ends were filled in,
and the plasmid was ligated to a 2 kb blunt-ended EcoRI
SacI fragment of plasmid pHP45VKm that contains the VKm
interposon (Fellay et al., 1987) to yield plasmid pED2-Km,
which was used for in vivo gene replacement.
The suicide plasmid pED2-Km encodes resistance to
piperacillin (Pi) and kanamycin (Km) and was electroporated
into P. putida DOT-T1E. Transformants that had acquired the
inactivated gene (KmR) were selected. Among the KmR
clones, we searched for Pi-sensitive ones, which were
expected to be the result of a double recombination event.
One of the KmR, PiS clones, called DOT-T1E-13, was chosen
and confirmed by Southern blot to have the wild-type ttgR
gene replaced by the mutant allele ttgR::Km (not shown).

Isolation of toluene-sensitive pTn5cat mutants of P. putida

DOT-T1E.
About 500 KmR transconjugants of P. putida DOT-T1E were
obtained after mating with E. coli S17lpir (pUIRM504). Each
mutant was tested for its ability to grow on LB medium
supplemented with 0.3% (v/v) toluene. Four mutants failed to
grow in the presence of toluene. By Southern blot analysis,
we determined that pTn5cat had inserted in the ttgABC
operon in three of the mutants. In one of the transconjugants,
called DOT-T1E-15, the plasmosome was inserted somewhere
else.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 11001106

Regulation of drug exclusion 1105

Cloning of the mutation in P. putida DOT-T1E-15 and
analysis of the surrounding DNA sequence
To investigate where pTn5cat had been inserted in the
mutant DOT-T1E-15, we digested about 4 mg of chromosomal DNA from this mutant with EcoRI. This enzyme does not
cut within the plasmosome. After heat inactivation of the
restriction enzyme, the mixture was incubated with 1 unit of
T4 DNA ligase. The ligation mixture was transformed in E.
coli DH5a and plated on LB medium with Km. One of the
clones was chosen randomly, and the plasmid was called
pED3. A 551 bp EcoRISmaI fragment of pED3 was
subcloned in pUC18.
The 551 bp fragment was PCR amplified, marked with
digoxigenin and used to rescue the corresponding wild-type
gene by colony screening hybridization from a library of the
wild-type strain. One clone carrying 4 kb of the chromosomal
DNA was found and sequenced, and the sequence was
compared with the 551 bp fragment adjacent to the pTn5cat
insertion. The DNA sequence revealed that the knocked-out
gene in DOT-T1E-15 corresponded to an ORF that extended
over 462 bp and encoded a putative polypeptide of about
19 kDa. Comparison of the amino acid sequence of the
deduced polypeptide with sequences in several databases
revealed that the polypeptide showed about 31% identity with
the Lrp protein of Salmonella typhimurium (Friedberg et al.,
1995). Because the knocked-out gene in P. putida DOT-T1E
seems to be involved in the global response of toluene efflux
in the wild-type strain, we called the DNA sequence encoding
this global regulator ttgX.

Primer extension analysis

The 5 0 mRNA start of the transcript originating from the ttgR,
ttgA and ttgD promoters was determined according to
Marques et al. (1993). The oligonucleotides used, 5 0 -CCCG
CGCTTGTAGAAGGCCC-3 0 , 5 0 -CTTACAGCCACTGAGCA
GGG-3 0 and 5 0 -CTCTACGAACATGCGTTTCTGCAG-3 0 ,
were complementary to the ttgR, ttgA and ttgD mRNAs.
They were 5 0 end-labelled with [g-32P]-ATP and polynucleotide kinase and annealed to 20 mg of total RNA prepared
from P. putida DOT-T1E, DOT-T1E-13 and DOT-T1E-15
cells growing exponentially in LB medium without toluene or
with toluene in the gas phase. cDNA was synthesized using
avian myeloblastosis virus reverse transcriptase as described
previously (Marques et al., 1993). The cDNAs were separated in ureapolyacrylamide sequencing gels, which were
exposed for the required time to Amersham RPN-8 films for
autoradiography.

Minimal inhibitory concentration (MIC) assays

These assays were performed in LB medium by the microtitre
broth dilution method (Amsterdam, 1991). Microtitre plates
were inoculated with < 1  105 cfu ml21 and incubated for
20 h at 308C.

Alkaline phosphatase activity

Alkaline phosphatase activity was assayed in Triton X-100Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 11001106

permeabilized whole cells as described previously (Wagner

et al., 1995; Junker and Ramos, 1999).

Nucleotide sequence accession number

The P. putida ttgR and ttgX gene sequences were deposited
in the EMBL data bank under accession nos AF238479 and
AF355622 respectively.

Acknowledgements
We thank M. M. Fandila, K. Shashok and C. Lorente for their
assistance in the preparation of this manuscript. This work
was supported by a grant from the European Commission
(BIO4-CT97-2270) and the Comision Interministerial de
Ciencia y Tecnologa (FEDER IFD97-1437).

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