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Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200464416423Original ArticleFatty acid biosynthesis and solvent toleranceA. Segura et al.

Environmental Microbiology (2004) 6(4), 416–423 doi:10.1111/j.1462-2920.2004.00578.x

Fatty acid biosynthesis is involved in solvent tolerance

in Pseudomonas putida DOT-T1E

Ana Segura,1* Estrella Duque,1 Antonia Rojas,1 Introduction

Patricia Godoy,1 Antonio Delgado,2 Ana Hurtado,1
Aromatic hydrocarbons are extremely toxic to microorgan-
John E. Cronan Jr,3 and Juan-Luis Ramos1
1 isms because they dissolve in the cytoplasmic membrane,
Department of Biochemistry and Molecular and Cellular
causing loss of ions, metabolites, lipids and proteins; dis-
Biology of Plants, CSIC-Estación Experimental del Zaidín,
sipation of the pH gradient and electrical potential; and
E-18008 Granada, Spain.
2 inhibition of membrane protein functions (Sikkema et al.,
Department of Earth Sciences and Environmental
1995; Ramos et al., 1997; Segura et al., 1999). The dam-
Chemistry, CSIC-Estación Experimental del Zaidín,
ages lead to cell death. However, independent laborato-
E-18008 Granada, Spain.
3 ries have isolated Pseudomonas sp. strains able to thrive
Department of Microbiology, University of Illinois Urbana,
on liquid medium in the presence of very high concentra-
Illinois 61801, USA.
tions of toluene, styrene and p-xylene (Inoue and Horiko-
shi, 1989; Cruden et al., 1992; Weber et al., 1994; Ramos
Summary et al., 1995; Kim et al., 1998).
The mechanisms underlying solvent tolerance are not
The unusual tolerance of Pseudomonas putida DOT-
yet fully understood. Several factors have been found to
T1E to toluene is based on the extrusion of this sol-
influence solvent-tolerance, e.g. efflux pumps and alter-
vent by constitutive and inducible efflux pumps and
ations in phospholipids (Heipieper et al., 1992; 1995;
rigidification of its membranes via phospholipid alter-
Weber et al., 1994; Aono and Kobayashi, 1997; Pinkart
ations. Pseudomonas putida DOT-T1E-109 is a sol-
and White, 1997; Kieboom et al., 1998; Junker and
vent-sensitive mutant. Mutant cells were less efficient
Ramos, 1999; Kobayashi et al., 2000; Ramos et al.,
in solvent extrusion than the wild-type cells, as shown
2002). Efflux pumps that expel organic solvents from the
by the limited efflux of 14C-1,2,4-trichlorobenzene from
cell membranes have been identified in several solvent-
the cell membranes, despite the fact that the efflux
tolerant Pseudomonas sp. strains. The SrpABC pump is
pumps are overexpressed as a result of increased
found in Pseudomonas putida S12 and Pseudomonas
expression of the ttgDEF and ttgGHI efflux pump
putida GM73 (Kieboom et al., 1998; Kim et al., 1998), and
operons. This limitation could be the result of alter-
the TtgABC, TtgDEF and TtgGHI pumps are found in P.
ations in the outer membrane because the mutant
putida DOT-T1E (Ramos et al., 1998; Mosqueda and
cells released more b-lactamase to the external
Ramos, 2000; Rojas et al., 2001). These efflux pumps
medium than the wild-type cells. The mutant P. putida
belong to the Resistance-Nodulation-Cell Division (RND)
DOT-T1E-109 showed negligible synthesis of fatty
family and are critical for solvent tolerance, because their
acids in the presence of sublethal concentrations of
inactivation leads to an increase in solvent sensitivity
toluene as revealed by analysis of 13CH3-13COOH
(Kieboom et al., 1998; Kim et al., 1998; Ramos et al.,
incorporation into fatty acids. In contrast, the mutant
1998; 2002; Mosqueda and Ramos, 2000; Rojas et al.,
strain in the absence of solvents, and the wild-type
2001).
strain, both in the presence and in the absence of
Phospholipid alterations mainly involve short-term
toluene, incorporated 13CH3-13COOH at a high rate into
responses that take place within 1 min after solvent expo-
de novo synthesized lipids. The mutation in P. putida
sure, and consists of the rapid transformation of the cis
DOT-T1E-109 increases sensitivity to the solvent
fatty acids C16 : 1,9 and C18 : 1,9 into their trans isomers
because of a limited efflux of the solvent from the cell
(Heipieper et al., 1992; Weber et al., 1994; Sikkema et al.,
membranes with the concomitant inhibition of fatty
1995; Junker and Ramos, 1999). The cti gene that
acid biosynthesis.
encodes the P. putida cis/trans-isomerase has been
cloned (Holwick et al., 1997; Junker and Ramos, 1999),
and a knock-out cti mutant of P. putida DOT-T1E was
isolated and characterized (Junker and Ramos, 1999).
Received 10 September, 2003; accepted 9 December, 2003.
*For correspondence. E-mail ansegura@eez.csic.es; Tel. Growth of this mutant was delayed in the presence of
(+34) 958 181600; Fax (+34) 958 129600. solvents.
© 2004 Blackwell Publishing Ltd
emi_578.fm Page 417 Thursday, March 4, 2004 4:25 PM

Fatty acid biosynthesis and solvent tolerance 417

In this study we describe a P. putida DOT-T1E mutant, et al., 1998; Junker and Ramos, 1999; Ramos et al.,
generated by random mini-Tn5 mutagenesis, that showed 2002), we decided to determine which of these functions
increased sensitivity to toluene. This was a consequence was affected in the P. putida DOT-T1E-109 mutant strain.
of the limited extrusion of toluene, in spite of the hyperex-
pression of two of the efflux pumps involved in the
Expression of the toluene efflux pumps in P. putida DOT-
expulsion of this aromatic hydrocarbon. Membrane disor-
T1E-109 was enhanced
ganization could be the reason for a decreased in toluene
export. Toluene accumulation inside the cells resulted in We tested expression of the ttg efflux operons in P. putida
inhibition of fatty acid biosynthesis which, in turn, seems DOT-T1E and in its P. putida DOT-T1E-109 mutant using
to magnify the negative effects of toluene. a fusion of each of the ttg operon promoter regions to a
promoterless lacZ gene. We found that the expression
level from the ttgA promoter was similar in both strains
Results
regardless of the presence or the absence of toluene
P. putida DOT-T1E-109 exhibits solvent sensitivity (Table 1). In contrast, it was found that the expression
from the ttgD promoter, that was very low in the wild-type
Tolerance to toluene in the wild-type P. putida DOT-T1E is
strain, was 20- and 80-fold higher in the mutant back-
an inducible process. In fact, about 1 in 104 cells tolerated
ground in the absence and in the presence of toluene
a sudden shock when not pre-exposed to low toluene
concentrations (e.g. as supplied via the gas phase),
whereas almost 50–100% of the cells tolerated a sudden Table 1. Expression of solvent-tolerant efflux pumps in the wild-type
shock when pregrown on toluene supplied via the gas and the P. putida DOT-T1E-109 mutant background.
phase (Ramos et al., 1997; and Fig. 1A). Pseudomonas
b-galactosidase (units)
putida DOT-T1E-109 was isolated in a general screening
for toluene-sensitive mutants after mini-Tn5-‘phoA-Km Strain Fusion – Toluene + Toluene
mutagenesis of the wild-type strain. The mutant cells were
DOT-T1E PttgA:lacZ 53 74
unable to tolerate sudden exposure to 0.3% (v/v) toluene PttgDlacZ 9 19
when pregrown on LB (Fig. 1B), and only about 0.001% PttgG:lacZ 402 998
of the DOT-T1E-109 cells tolerated the toluene shock DOT-T1E-109 PttgA:lacZ 72 91
when pregrown on LB plus toluene supplied in the gas PttgD:lacZ 210 1645
PttgG:lacZ 1108 3756
phase (Fig. 1B).
Given that efflux pumps and alterations in phospholipids Pseudomonas putida DOT-T1E and DOT-T1E-109 cells bearing the
are involved in solvent-tolerance in P. putida (Heipieper indicated fusion were grown on LB medium supplemented with Tc
without or with toluene supplied through the gas phase. Data are the
et al., 1992; Weber et al., 1994; Aono and Kobayashi, average of a least three independent assays with standard deviations
1997; Pinkart and White, 1997; Kieboom et al., 1998; Kim below 15% of the given values.

Fig. 1. Tolerance to toluene of the wild-type P. putida DOT-T1E strain (A) and DOT-T1E-109 mutant strain (B). Overnight LB cultures of the wild
type and mutant strains plus (circles) and minus (triangles) toluene in the vapour phase were diluted 100-fold in the same medium and incubated
until they reached the exponential growth phase. Then two aliquots of these cultures were prepared; one of them was kept as a control (open
symbols), and to the other we added 0.3% (v/v) toluene (closed symbols). Survival was monitored by counting CFU per ml at the indicated times.

© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 416–423

emi_578.fm Page 418 Thursday, March 4, 2004 4:25 PM

418 A. Segura et al.

respectively (Table 1). The expression from the ttgG pro-
moter in the mutant strain was enhanced three- to fourfold
with respect to the level of expression in the wild-type
strain both in the absence and in the presence of toluene
(Table 1). This suggests that in P. putida DOT-T1E-109,
solvent sensitivity could not be the result of the limited
synthesis of the efflux pumps involved in toluene extrusion
as the expression of the ttgGHI and ttgDEF operon efflux
pumps is upregulated in this mutant.

Functionality of the solvent extrusion pumps is

compromised in the mutant cells growing in the presence
of sublethal concentrations of toluene

To ascertain the functionality of the efflux pumps we mea-

sured the accumulation of the non-metabolizable toluene
analogue 14C-1,2,4-trichlorobenzene in the cell mem-
branes of the wild-type DOT-T1E strain and the DOT-T1E-
109 mutant strain as described before (Ramos et al.,
1998). The data shown in Table 2 revealed low accumu-
lation of this aromatic hydrocarbon in the cell membranes
of both strains when pregrown in the absence of toluene.
However, we unexpectedly found that mutant cells pre-
exposed to toluene in the gas phase were less efficient
than the wild-type cells in aromatic hydrocarbon extrusion
and indeed accumulated fivefold more 1,2,4-[14C] trichlo-
robenzene than the wild-type strain. These results support
that the operation of the efflux pumps was compromised
in cells pre-exposed to toluene. This situation is similar to
that described before in a P. putida DOT-T1E mutant that
lacked the OprL protein and exhibited diminished mem-
brane integrity. The OprL mutant exhibited a very dimin-
ished ability to extrude the toluene analogue and was also
hypersensitive to toluene (Ramos et al., 1997).
P. putida DOT-T1E-109 released periplasmic b-lactamase
to the external medium
To test whether the P. putida DOT-T1E-109 mutant
showed membrane damage, we studied the release of
Table 2. Incorporation of 1,2,4-[14C]trichlorobenze into membranes
of P. putida DOT-T1E and DOT-T1E-109 cells grown in the absence
and in the presence of sublethal concentrations of toluene.
14
C incorporated/mg of cell protein
Fig. 2. Liberation of proteins to the external medium.
A. SDS-PAGE of supernatant proteins stained with Coomassie blue.
B. Immunodetection of b-lactamase in the supernatant fractions. 1.
P. putida DOT-T1E; 2. P. putida DOT-T1E-109; 3. P. putida KT2440
tolQ mutant QX.
proteins into the extracellular medium. Analysis of pro-
teins culture supernatants by SDS-PAGE and Coomassie
staining showed the presence of numerous proteins in
supernatant of strain P. putida KT2440 tolQ mutant (QX)
(Llamas et al., 2000) that was used as a positive control
in our assay but neither the wild-type nor the P. putida
DOT-T1E-109 supernatants showed massive liberation of
proteins (Fig. 2A). To further investigate the release of
periplasmic proteins into the extracellular medium, we
transformed the wild-type and the mutant strain with
plasmid pJB3Tc19, which encodes a periplasmic b-
lactamase and then its presence in the supernatants of
the different P. putida strains was analysed by Western
blot. As shown in Fig. 2B, b-lactamase release was higher
in P. putida DOT-T1E-109 than in the wild-type meaning
that membrane integrity was compromised in the mutant
strain, although the phenotype was not as strong as that
of the control strain P. putida KT2440 tolQ mutant.
Therefore, membrane alteration(s) in P. putida DOT-T1E-
109 can contribute to the malfunctioning of the efflux
pumps.

Strain – Toluene + Toluene

Cis/trans isomerization and alteration of phospholipid
DOT-T1E 21 953 16 513
DOT-T1E-109 28 110 86 117 headgroup are not affected in P. putida DOT-T1E-109

Pseudomonas putida DOT-T1E and DOT-T1E-109 were grown in LB The results obtained with the DOT-T1E-109 mutant and
in the absence of toluene or with toluene supplied via the gas phase. the wild-type strain were similar to those described previ-
Then, cells were treated as described in Experimental procedures to ously regarding cis/trans isomeration and alteration of
assay functionality of the solvent efflux pumps. Data are the average
of three independent assays with standard deviation values in the phospholipid headgroup composition. For example, in the
range of 10–20% of the given values. mutant strain cis/trans isomerization of unsaturated fatty
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 416–423
emi_578.fm Page 419 Thursday, March 4, 2004 4:25 PM
acids took place in less than 1 min after sudden exposure
to sublethal concentrations of toluene (i.e. 0.05% v/v).
Phospholipid headgroup composition of P. putida DOT-
T1E-109 growing on LB medium with toluene supplied via
the gas phase revealed that almost 20% of total was
cardiolipin, a result similar to those reported for the wild-
type strain (Ramos et al., 1997).
Phospholipid biosynthesis in P. putida DOT-T1E-109 is
significantly diminished in the presence of solvents
We measured fatty acid biosynthesis in the presence of
sublethal concentrations (i.e. 0.075% v/v) of toluene. To
determine lipid biosynthesis we analysed the incorpora-
tion of 13CH3-13COOH into fatty acids as described in the
Experimental procedures. We found that the wild-type
strain incorporated 13CH3-13COOH at a high rate both in
the absence and in the presence of toluene (d13C
minute-1 of 51.04 and 29.35 in the absence and presence
of toluene, respectively) (Fig. 3A). In contrast, the mutant
strain incorporated 13CH3-13COOH at a high rate in the
absence of toluene (d13C minute-1 = 42.25), but the incor-
poration of 13CH3-13COOH in the presence of toluene was
severely compromised (d13C minute-1 = 0.44) (Fig. 3A).
The lack of incorporation of the labelled substrate in the
later case was not caused by the loss of viability in DOT-
T1E-109 in the presence of toluene given that survival
throughout the assay was not affected (not shown). Fur-
thermore, the incorporation of 13C into proteins was mea-
sured under the same conditions. We observed that both
the mutant and the wild-type strain were able to incorpo-
rate 13C at a similar rate in cells grown in the absence of
toluene (d13C minute-1 = 37.14 and 39.52, respectively);
however, in P. putida DOT-T1E-109 the incorporation was
less efficient than in the wild type in the presence of
Fig. 3. Time-course incorporation of 13CH3–13COOH by P. putida DOT-T1E and DOT-T1E-109. Wild-type (circles) and DOT-T1E-109 (triangles)
cells growing exponentially on LB were supplemented (closed symbols) with 0.075% (v/v) toluene or kept without addition (open symbols). Then
3 mM 3CH3-13COOH were added and samples removed at the indicated times for determination of the incorporation of 13C in (A) lipids and (B)
proteins, as described in Experimental procedures.
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 416–423
Fatty acid biosynthesis and solvent tolerance 419
toluene, namely d13C minute-1 was 20.72 for the mutant
strain versus d13C minute-1 of 46.205 for the wild-type
strain (Fig. 3B). These results indicated that although
there is a significant decreased in the 13C incorporation
into proteins in the mutant strain in the presence of tolu-
ene, the incorporation of 13C-acetate into lipids was neg-
ligible. Then, it follows that in vivo inhibition of fatty acid
biosynthesis may be caused by the accumulation of tolu-
ene in the cell because of the limited extrusion of the
solvent as shown in Table 2.
To further confirmed that toluene accumulation was
inhibiting fatty acid biosynthesis, we tested the incor-
poration of 13CH3-13COOH in membrane lipids of P. putida
DOT-T1E-18 (ttgB::¢phoA:Km) in which one of the three
efflux pumps involved in toluene tolerance was not
functional. In this mutant strain accumulation of 1,2,4-
[14C]trichlorobenzene in the cellular membranes was pre-
viously demonstrated (Ramos et al., 1998). Pseudomo-
nas putida DOT-T1E-18 was also unable to incorporate
significant amounts of 13CH3-13COOH in membrane lipids
in the presence of sublethal concentrations of toluene (not
shown).
This series of results suggest that P. putida DOT-T1E-
109 was more sensitive towards toluene than the wild-
type strain as a result of the limited solvent extrusion,
which lead to the inhibition of fatty acid biosynthesis.
Identification of the P. putida DOT-T1E-109 phoA::Km
insertion.
To reveal the nature of the mutation in P. putida DOT-T1E-
109 we decided to clone the mutation as described in
Experimental procedures. DNA sequence analysis of the
wild-type gene revealed that the mini-Tn5-phoA-Km trans-
poson had interrupted an orf (the sequence has been
emi_578.fm Page 420 Thursday, March 4, 2004 4:25 PM
420 A. Segura et al.
submitted to GenBank under accession number
AFO31419) that encodes for a 565-residue polypeptide.
The exact insertion site was located within codon 333 of
the ORF polypeptide. Comparison of the deduced
polypeptide sequence against several Database banks
revealed that the translated sequence of the interrupted
orf exhibits 38–45% similarity with the three FadD proteins
(FadD1, Fad2 and FadX) of P. putida strain U (Olivera
et al., 2001) and 99% identity with one of the four FadD-
like proteins of P. putida KT2440 (Nelson et al., 2002). In
consonance with this homology finding is that the orf gene
product has a fatty acyl-CoA synthetase (FAC) signature
motif (Black, 1991; Black et al., 1997) between residues
434 and 458, and highly conserved ATP/AMP binding
motives between residues 213 and 222, and 352 and 357
(Fig. 4).
Because in P. putida DOT-T1E-109 the fadD-like gene
is fused in frame to ‘phoA, we also determined alkaline
phosphatase activity in exponentially growing cells in the
absence of toluene or with toluene in the gas phase.
Alkaline phosphatase levels were similar regardless
growth conditions. It then follows that the fadD-like gene
expression in P. putida DOT-T1E was not influenced by
the presence of organic solvents in the culture medium.
To confirm that mutation in a fadD-like gene lead to
solvent-sensitive phenotype, we mutated the fadD-like
gene by introducing an W:KmR cassette as described in
Experimental procedures. However, the resulting strain (P.
putida DOT-T1E-PS85) was as resistant as the wild type
to the toluene shock (not shown).
Discussion
Solvent tolerance in several P. putida strains is a complex
process that is still not well understood, and which
involves a fine interplay between efflux pumps and the
rigidification of cell membranes as a result of cis to trans
isomerization of unsaturated fatty acids, and an increased
biosynthesis of cardiolipin (Ramos et al., 1998). Our
results show that a P. putida mutant exhibited reduced
toluene tolerance upon a sudden shock with this aromatic
hydrocarbon. This reduced tolerance seems to be the
result of the altered membrane integrity and limited oper-
ation of the efflux pumps that leads to the accumulation
of toluene in the cell. Solvent accumulation inhibits fatty
acid biosynthesis which in turn increases membrane fra-
gility and foster toluene effects. Although previous studies
seem to point out that toxicity of the organic solvents is
caused by general physico-chemical effects and neither
metabolic, nor chemical reactions were associated with
the toxic effects (Heipieper et al., 1991; 1992), our results
indicate that the decrease in lipid biosynthesis could be a
major factor contributing to loss of integrity of the cellular
membrane. Pinkart and White (1997) reported that the
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 416–423
solvent-tolerant Pseudomonas sp. Idaho synthesized lip-
ids at higher rate in the presence of solvents than in the
absence of the toxic compounds, whereas synthesis was
not enhanced in a solvent-sensitive Pseudomonas sp.
strain. The impairment in synthesis of fatty acids observed
in this strain will boost the toxic general effects of toluene
accumulation. In a recent report, Zhang et al. (2003)
showed the downregulation of lipid biosynthesis genes in
response to the organic solvent dimethyl sulphoxide in
Saccharomyces cerevisiae. The effect seems to be quite
specific as none of the genes were responsive to other
environment stresses (Zhang et al., 2003).
Although the exact nature of the mutation of P. putida
DOT-T1E-109 remains elusive, our results support the link
between rigidification of the cell membrane through fatty
acid biosynthesis, efflux pumps functionality, and survival
of P. putida in environments with extreme solvent
concentrations.
Another interesting result of our study is that the
TtgABC efflux pump forms part of a regulatory network
different from that of TtgDEF and TtgGHI: the level of the
former was not altered in this mutant background, in con-
trast with the other two efflux pumps (Table 1). Given that
the level of expression of ttgDEF is almost negligible in
the wild-type and very high in the mutant background, it
is likely that cells use this efflux pump as an emergency
element to overcome extreme conditions when the cell
membranes are weakened. In connection with this obser-
vation it is worth mentioning that an increase in induced
levels of ttgDEF expression have also been shown in a
TtgGHI knock-out mutant (W. Terán, M.T. Gallegos and
J.L. Ramos, unpublished results).
Experimental procedures
Bacterial strains, plasmids and culture media.
Pseudomonas putida DOT-T1E is a rifampin (Rif)-resistant
derivative of the solvent-tolerant strain P. putida DOT-T1
(Ramos et al., 1997). Pseudomonas putida DOT-T1E-109 is
a solvent-sensitive derivative of DOT-T1E isolated, after mini-
Tn5-¢phoA mutagenesis, in a general screening for solvent-
sensitive mutants (Ramos et al., 1998). Competent Escheri-
chia coli JM109 and E. coli DH5a cells were used in cloning
experiments (Ausubel et al., 1991).
Bacterial strains were routinely grown on Luria–Bertani
(LB) liquid medium. Pseudomonas putida strains were also
grown on M9 minimal medium with 10 mM citrate or oleate
(Rock and Jackowski, 1985) as the sole carbon source. Cul-
tures were incubated at 30∞C and shaken on an orbital plat-
form operating at 200 strokes per min. Growth was usually
determined as colony forming units (CFU) per millilitre.
Antibiotics were used at the following concentrations
(mg ml-1): Ampicillin (Ap), 100; chloramphenicol (Cm), 30–90;
kanamycin (Km), 25; rifampin (Rif), 10; tetracycline (Tc), 10;
and piperacillin (Pi), 50.
emi_578.fm Page 421 Thursday, March 4, 2004 4:25 PM
DNA manipulations
Preparation of plasmids and total DNA of P. putida, digestion
with restriction enzymes, separation of DNA and Southern
blots were done according to standard protocols (Ausubel
et al., 1991). Plasmid DNA was sequenced in both strands
with universal, reverse or specifically designed primers in an
automatic DNA sequencer (Applied Biosystems ABI-PRISM
3100). DNA amplification reactions were done in a Perkin-
Elmer GeneAmp 2400 thermal cycler with the appropriate
primers.
Cloning the mutation in P. putida DOT-T1E-109
To locate the gene inactivated by the mini-transposon, the
mutation in DOT-T1E-109 was cloned as follows: chromo-
somal DNA was digested with SphI. This enzyme cuts the
mini-transposon once upstream from the Km gene, so that
the Km-resistant marker plus adjacent DNA can be rescued
upon ligation on appropriate vectors and selection of Km-
resistance colonies (Ausubel et al., 1991). SphI-digested
DNA of DOT-T1E-109 was ligated to pUC18 cut with the
same enzyme, and one Km-resistant colony carrying plasmid
pANA3 was isolated. Restriction mapping allowed us to iden-
tify a 900-bp SphI-NotI fragment in pANA3. The NotI site lies
at the mini-Tn5 border and the SphI site corresponds to the
SphI closest to the mini-Tn5 insertion in the chromosome of
DOT-T1E-109. The 900-bp fragment was amplified by poly-
merase chain reaction (PCR) and labelled with digoxigenin.
The labelled PCR product was used for colony screening
hybridization against a DOT-T1E gene bank generated before
(Ramos et al., 1998). A clone called pANA19 was selected
for sequencing of the insert on both strands.
Generation of a P. putida fadD-like::WKm mutant
Plasmid pANA19 was cut with BamHI and the insert was
cloned into the BamHI site of plasmid pUC18Not to generate
pANA130. Plasmid pANA130 was cut with ApaI, an enzyme
that cuts only once within the fadD-like gene (position 1457
of sequence AF031419). The Klenow fragment and the four
deoxynucleoside triphosphates (dNTPs) were used to fill in
the ends and make them blunt (Ausubel et al., 1991). The
2kb W-Km cassette of plasmid pHP45W-Km (Fellay et al.,
1987) was obtained after digestion with EcoRI, and these
ends made blunt as above and ligated to the linearized
pANA130 plasmid. The ligation was transformed into E. coli
DH5a and cells were selected on LB plates supplemented
with ampicillin and kanamycin. After digestion analysis, plas-
mid pANA131 was selected. Electroporation was used to
transfer pANA131 to P. putida DOT-T1E. pANA131 behaves
as a suicide vector, and because of identical sequences,
pANA131 integration into the host chromosome via homolo-
gous recombination can be selected on LB solid medium with
Rif, Km and Pi. A merodiploid clone was grown overnight on
LB to allow a second recombination event in which the wild-
type gene was replaced by the mutant allele. For this selec-
tion, colonies were plated again on LB supplemented with Rif
and Km. Among these colonies, those that did not grow in
the presence of piperacillin were selected as putative
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 416–423
Fatty acid biosynthesis and solvent tolerance 421
resolved merodiploids. These mutants were checked by
Southern blot hybridization (not shown). One clone that
exhibited the correct mutation was called P. putida DOT-T1E-
PS85 and kept for further studies.
Incorporation of [14C]trichlorobenzene into cell
membranes
Exponentially growing cells were harvested by centrifugation,
washed twice with LB, and suspended in 2.5 ml of LB to
reach a cellular density of about 150–200 mg of cell
protein ml-1. Then the cells were incubated for 10 min at 30∞C
and exposed to 5 mCi of 1,2,4-[14C]trichlorobenzene. After
10 min, 250 ml of the cell suspension was filtered through a
0.45-mm Millipore filter and washed with 2 ml of LB medium.
The filters were dried, and the 14C associated with cell pellet
(disintegrations min-1) was determined in a Packard Radio-
chemical detector.
Leakage of proteins into the extracellular medium
Plasmid pJB3Tc19 (Blatny et al., 1997) was electroporated
into P. putida DOT-T1E and P. putida DOT-T1E-109 as
described before (Enderle and Farwell, 1998). Strain P. putida
KT2440 tolQ mutant (QX) was described before (Llamas
et al., 2000). Cells were grown on LB medium overnight and
the following day, cultures were centrifuged (15000 g, 7 min,
4∞C). Fifteen millilitres of the supernatant fraction were fil-
tered through cellulose acetate filters (0,22 mm pore diame-
ter) and precipitated by incubation for 30 min at 20∞C with
75 ml of absolute ethanol. After 15 min centrifugation
(15 000 g) at 4∞C the dry pellet was suspended in Laemmli
sample buffer. Proteins were separated on SDS-polyacrila-
mide (10% wt/vol) gel electrophoresis (Laemmli, 1970) and
stained with Coomasie blue or transferred onto PVDF mem-
branes (Towbin et al., 1979) and immunodetected with an
anti-b-lactamase polyclonal antibody raised against the b-
lactamase encoded by pBR322. The equivalent of the super-
natant of 3 ¥ 108 cells were loaded on the gel. The Western
blots were performed as described previously (Ausubel et al.,
1991) and developed with the Kit Super SignalâWest Dura
Extended Duration (Pierce) following the manufacturers
instructions.
Analysis of phospholipids
Phospholipids were extracted according to Bligh and Dyer
(1959) and trans-esterified as described before (Bannon
et al., 1982). After gas chromatographic separation, the fatty
acids were identified by mass spectrometry.
Incorporation of 13CH3–13COOH into cell components
Cells were grown on LB medium overnight at 30∞C. On the
following day, the cultures were diluted 1 : 100 and grown
under the same conditions until they reached a turbidity of
about 0.8 at 660 nm. Then, the cultures were divided into two
halves, to one of the samples of each strain we added
0.075% (v/v) toluene, and the other half was kept as control.
emi_578.fm Page 422 Thursday, March 4, 2004 4:25 PM
422 A. Segura et al.
Cultures were incubated for 5 min at 30∞C with shaking and
then 1 mM of 13CH3-13COOH was added to all of them. Phos-
pholipids were extracted as described above, and resus-
pended in 100 ml n-hexane. For protein extraction cells were
treated for 10 min with 0.1% (wt/vol) SDS at room tempera-
ture and then for another 10 min at 96∞C, then trichloroacetic
acid was added to reach 25% (v/v) and upon 10 min on ice
protein was collected by centrifugation at 12 000 g. Proteins
were finally suspended in 100 ml water.
To determine incorporation of 13CH3-13COOH in the above
fractioned cell components 25 ml of each sample were evap-
orated during 1 h in tin capsules at 50∞C, and then analysed
in an Elemental Analyzer-Isotope Ratio Mass Spectrometer
on line with a Delta Plus XL mass spectrometer. The overall
precision of analyses was ±0.1 per thousand for 13C. The
stable isotope composition is reported as D-values per thou-
sand D(Rsample/Rraudad-1) ¥ 1000 where R = 13C/12C for d13C val-
ues. The international standard used for 13C/12C was Pee Des
Belemnites, a fossil marine carbonate of biogenic origin.
Regression coefficients and slopes of the incorporation
lines were calculated using the Microsoft Excel program. For
this analysis we used only the linear part of the graphic (from
time 10–60 min for the incorporation into lipids and from 30
to 120 for the protein incorporation).
Alkaline phosphatase assay
For alkaline phosphatase (Apase) measurements (Sambrook
et al., 1989), cells of DOT-T1E-109 grown on LB medium
were harvested by centrifugation, washed once with APase
buffer [0.1 M Tris-HCl (pH 7.6), 1 mM ZnCl2, 1 mM MgCl2],
and resuspended in a 1 ml of APase buffer containing 2% (v/
v) Triton X-100. The reaction was started by adding 10 mM
4-nitrophenyl phosphate at 37∞C and was stopped after 5 min
of incubation at 37∞C with 200 ml of 1 M NaOH. The sample
was centrifuged for 10 min in a microcentrifuge at 12 000 g.
The absorbance of the supernatant was measured at 405 nm
and compared with a standard curve for 4-nitrophenol.
b-Galactosidase assay
Fusions of the promoter of ttgA, ttgD and ttgG genes to a
promoterless lacZ gene in the low copy number vector
pMP220 (Spaink et al., 1987) are available in our laboratory
(W. Terán, M.T. Gallegos and J.L. Ramos, unpublished;
Duque et al., 2001; Rojas et al., 2003). These plasmids were
electroporated into the wild-type P. putida DOT-T1E and its
mutant derivative DOT-T1E-109. Transformants were grown
overnight in LB medium with Tc. Next day, cultures were
diluted 100-fold in the same medium. After 1 h incubation,
cultures were split in two halves. One half was used as a
control and the other was supplemented with toluene sup-
plied via the gas phase. After 5 h incubation, b-galactosidase
activity was assayed in permeabilized whole cells according
to Miller (1972).
Acknowledgements
This work was supported by a grant from the European
Commission (QLRT-CT2001-00435). We kindly thank R.
Lloubes (C.N.R.S. Marseille, France) for anti-b-lactamase
© 2004 Blackwell Publishing Ltd, Environmental Microbiology, 6, 416–423
antibody. We thank M.M. Fandila and C. Lorente for their help
in the preparation of this manuscript, and K. Shashok for
improving the use of English.
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