Bacteria able to grow in aqueous:organic
two-phase systems have evolved resistance mechanisms to the toxic
effects of solvents. One such mechanism is the active efflux of
solvents from the cell, preserving the integrity of the cell interior.
Pseudomonas putida S12 is resistant to a wide variety of
normally detrimental solvents due to the action of such an efflux pump.
The genes for this solvent efflux pump were cloned from P. putida S12 and their nucleotide sequence determined. The deduced
amino acid sequences encoded by the three genes involved show a
striking resemblance to proteins known to be involved in
proton-dependent multidrug efflux systems. Transfer of the
genes for the solvent efflux pump to solvent-sensitive P. putida strains results in the acquisition of solvent resistance. This opens up the possibilities of using the solvent efflux system to
construct bacterial strains capable of performing biocatalytic transformations of insoluble substrates in two-phase aqueous:organic medium.
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INTRODUCTION |
The microbial transformation of hydrocarbons is important not only
in environmental applications such as soil remediation and waste stream
purification, but also in biocatalytic applications for the production
of specialty chemicals. The metabolic pathways by which many of these
compounds are degraded in various bacteria have been elucidated and in
many cases the genes coding for the enzymes involved have been cloned
and sequenced (1). A major problem in applying hydrocarbon degrading
bacteria to industrial processes is their susceptibility to the toxic
effects of the very substrate that the organism is utilizing as a
carbon source. This is often due to accumulation of the hydrophobic
compound in bacterial membranes which can cause devastating effects on membrane structure (2, 3). A second problem in the application of
catabolic pathways in the synthesis of fine chemicals is that many of
the desired substrates of enzymatic reactions are sparingly soluble in
water and thus may not be fully bioavailable to microorganisms. The use
of solvent tolerant bacteria allows the introduction of a nonpolar
phase to the medium, dissolving the desired substrate, and increasing
the exposure of the cell to the substrate.
Many different mechanisms have been described that contribute to
solvent resistance (Fig. 1) but despite
these efforts no comprehensive overview is available to explain the
physiological response of microorganisms to toxic organic solvents (for
a recent review, see Weber and de Bont (4)). Our laboratories have been investigating the ability of Pseudomonas putida S12 to
withstand toxic concentrations of toluene and other organic solvents
(5). This organism has evolved at least two mechanisms to combat the accumulation of hydrophobic solvents in the membrane or the interior of
the cell. One key observation was the detection of trans-
rather than cis-unsaturated fatty acids in the membrane of
the solvent-tolerant bacterium upon exposure to solvents (6, 7). The
conversion of cis- to trans-unsaturated fatty
acids by a direct isomerization alters the packing of the phospholipids
in the bacterial membrane. This results in a change in membrane
fluidity, making the membrane less likely to allow solvents to
partition into it, decreasing the detrimental effects on the membrane
due to solvent partitioning, and thus increasing the solvent resistance
of the cell (8). Recently it has been shown that a second mechanism of
solvent resistance is possessed by P. putida S12. This is an
energy-dependent active efflux system for solvents such as
toluene (9) that may function in a fashion similar to that for
multidrug efflux pumps found in many antibiotic-resistant
microorganisms. Thus, P. putida S12 employs at least two
mechanisms for active defense against the detrimental effects of
solvents: one functioning to keep solvents out of the interior of the
cell and a second functioning to prevent solvents from partitioning
into the cell membrane.

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Fig. 1.
Schematic representation of essential
features of solvent-resistant bacteria. A, solvents diffuse
to and preferentially partition to the cytoplasmic membrane where they
cause disruptions in membrane functions by increasing membrane fluidity
and affecting bilayer stability (3, 4). B, to compensate for
these effects, solvent-resistant bacteria modify the composition of the
membrane either by isomerizing cis- into
trans-unsaturated fatty acids from the membrane lipids (9,
26) or by changing the headgroup composition (4, 37). This compensation
will only be partial and thus in a dynamic process, solvents have to be
removed continuously from the membrane. C, removal to a
certain extent may be by degradation (32). D, very recently,
on the basis of whole cell experiments, evidence was obtained that an
unprecedented energy-dependent export system for
hydrophobic solvents is in operation (9). E, in combination
with a retarded influx of solvents due to modifications at the outer
membrane (31, 44), the new efflux pump functions as the key factor in
solvent tolerance.
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In the present report we describe the construction of transposon
mutants of P. putida S12 that have lost the solvent tolerant phenotype. This allowed the cloning of the genes responsible for a
solvent efflux pump that is involved in the ability of P. putida S12 to withstand toxic concentrations of organic solvents.
The nucleotide sequence of the genes involved was determined and their relationship to other bacterial efflux systems is discussed.
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EXPERIMENTAL PROCEDURES |
Bacterial Strains, Plasmids, Media, and Growth of
Strains--
P. putida S12 (10) is the wild-type strain
capable of growth at supersaturated solvent concentrations (5) and is
the object of the present investigation. P. putida JK1 is a
solvent-sensitive mutant of P. putida S12 derived in the
present work by transposon mutagenesis with TnMod-KmO.
P. putida JJD1, also derived in the present work, contains a
kanamycin gene/ColE1 origin cassette insertion in the genome of
P. putida S12. The solvent-sensitive strain P. putida PPO200 is P. putida mt-2 cured of the TOL
plasmid (11). The artificial transposable element
TnMod-KmO1
contains a kanamycin resistance gene and the ColE1 origin of replication between Tn5 inverted repeats. The Tn5
transposase gene and an origin of transfer for conjugation are present
outside the inverted repeats. Escherichia coli JM109
(recA1 endA1 gyr A96 thi hsdR17 supE44 relA1
(lac-proAB) (F
traD36 proAB
lacIqZ
M15) (12)) was utilized as the
host strain for all recombinant plasmids. The cloning vector pUC19
(12), the pGEM series of cloning vectors (Promega, Madison, WI), and
the broad host range vector pUCP22 (13) were used for the construction
of subclones.
L broth (14) was used as complete medium. Minimal medium was prepared
as described by Stanier et al. (15). Solid media contained
2% agar. Ampicillin (100 µg/ml), kanamycin (50 µg/ml), or
gentamicin (25 µg/ml) were added to the medium to maintain recombinant plasmids in E. coli. Gentamycin (25 µg/ml) was
used to maintain the pUCP22 subclones in P. putida and
kanamycin (250 µg/ml) was used in the selection of P. putida S12 transposon mutants. E. coli strains were
routinely cultured at 37 °C and P. putida strains were
grown at 30 °C. Growth of bacterial strains in the presence of
various solvents (indicating solvent resistance) was determined
essentially as described by Weber et al. (5). The experiments were conducted twice with duplicate samples each time.
Generation and Screening of TnMod-KmO Insertion Mutants--
The
conjugatable suicide transposon donor TnMod-KmO was
introduced into P. putida S12 by triparental mating using
pRK2013 as the mobilizing plasmid by established procedures (16, 17). Kanamycin-resistant colonies were tested for the ability to grow on L
agar plates in the presence of saturating vapor amounts of toluene.
This was accomplished by placing the agar plates in a sealed glass
dessicator along with a small beaker containing toluene. Growth was
scored after 12 h at 30 °C.
DNA Techniques--
Total genomic DNA from P. putida
strains was prepared by the CTAB procedure (18). Plasmid DNA was
isolated by the alkaline-sodium dodecyl sulfate lysis method of
Birnboim and Doly (19). DNA was digested with restriction enzymes and
ligated with T4 ligase as recommended by the supplier (Life
Technologies, Inc., Gaithersburg, MD). DNA restriction fragment and
PCR2 products were visualized
by 0.7% or 1.0% agarose gel electrophoresis in 40 mM
Tris, 20 mM acetate, 2 mM EDTA buffer. DNA from
agarose gels was isolated using the method of Vogelstein and Gillespie (20). Plasmid DNA was introduced into either E. coli JM109
or P. putida JK1 cells by electroporation (21) using a Gene
Pulser (Bio-Rad Laboratories).
All sequencing and PCR reactions were performed using a Gene Amp PCR
System 9600 (Perkin-Elmer, Foster City, CA). Nucleotide sequencing
reactions were performed with purified double strand plasmid DNA or PCR
products using AmpliTaq FS DNA polymerase fluorescent dye terminator
reactions (Perkin-Elmer) as recommended by the supplier. Sequencing
products were detected using an Applied Biosystems 373A stretch
automated DNA sequencer (Applied Biosystems Inc., Foster City, CA).
Nucleotide sequence analysis was performed either with the Genetics
Computer Group analysis package (22) or with the National Center for
Biotechnology Information BLAST server (23). PCR reactions for
amplifying the region of genomic P. putida S12 DNA
containing the insertion point for the transposon were performed using
Taq DNA polymerase (Perkin-Elmer). The reaction mixture (100 µl) was treated for 1 min at 94 °C followed by 25 cycles of 1 min
at 96 °C, 1 min at 55 °C, and 1 min at 72 °C before finishing
for 10 min at 72 °C. Primers for this reaction were 5
-CGTTTGCAACCGGTGAG-3
and 5
-TATCGGACGCAAACG-3
corresponding to
positions 3735 to 3752 and 4238 to 4253 of the nucleotide sequence, respectively.
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RESULTS |
Isolation of Solvent-sensitive Mutants--
P. putida
S12 was chosen for a molecular study of the basis of solvent resistance
due to the extensive physiological studies that have been performed on
the strain (4-6, 9, 24-26). The organism can grow in the presence of
a wide variety of normally toxic solvents with log POW
values ranging from 2.3 to 3.5 (Table I).
Initially, several solvent-sensitive transposon mutants were constructed using TnMod-KmO. P. putida S12 mutants
which are no longer resistant to solvents were detected by the
inability of kanamycin-resistant exconjugants to grow on L medium in
the presence of supersaturated vapor concentrations of toluene as
described under "Experimental Procedures." Several
toluene-sensitive mutants were obtained and one of these, designated
strain JK1, was chosen for further analysis. Besides toluene, JK1 is
sensitive to a number of other solvents with log POW values
less than or equal to 3.5 (Table I), indicating that a single genetic
trait is responsible for resistance to all of the solvents tested.
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Table I
Growth of P. putida S12 on L/acetate in the presence of various
solvents
Strain JK1 is a transposon mutant of the genes for a solvent efflux
pump. pJD105 and pJD106 are clones containing the three genes for the
solvent efflux pump (srpABC) cloned into the vector pUCP22.
pJD105 has the genes in the same orientation as the lac promoter on the plasmid while pJD106 has the genes in the reverse orientation. Different solvents (1% final concentration) were separately added to identical subcultures in L medium with 60 mM acetate during the early exponential growth phase.
Growth of the culture was measured at various times after solvent
addition with no continued growth indicating solvent sensitivity. A
plus indicates growth with OD >0.7 after 24 h while a minus
indicates no growth with OD <0.1 after 120 h. Log Pow
values were calculated according to the method of Rekker and de Kort
(43) and is defined as the logarithm of the concentration of a solvent
in octanol divided by the concentration of a solvent in water.
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Cloning and Analysis of the Genes for Solvent Resistance--
To
characterize the genes for solvent resistance in P. putida
S12 in more detail, the region of the genome containing the transposon
insertion in mutant JK1 was cloned. This was aided by the fact that the
TnMod-KmO transposable element utilized in the
construction of the mutants contains an origin of replication derived
from plasmid ColE1. Total genomic DNA from JK1 was cleaved with
BamHI, ligated to form circular molecules, and
electroporated into E. coli JM109 with selection for
kanamycin resistance. Since BamHI does not cleave the
transposon, the resulting clone, pJD101 (Fig.
2), must contain DNA from both sides of
the transposon insertion. Approximately 11 kilobases of genomic DNA was
cloned along with the transposon which contains the origin of
replication and the kanamycin resistance gene (2 kb). The point at
which the transposon is inserted is only 1 kb away from one end of the
BamHI fragment cloned from strain JK1. This being the case,
a second, overlapping 4-kb PstI fragment was cloned from
strain JK1 (designated pJD102, Fig. 2).

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Fig. 2.
Restriction maps of the clones derived from
the transposon and insertional mutant strains. A cartoon of the
srpABC nucleotide sequence is shown at the
bottom, in proportion to the restriction maps and showing
the positions of the genes relative to the restriction enzyme cutting
sites. The triangles in the restriction maps of pJD101
and pJD102 and the boxes in the restriction maps of
pJD103 and pJD104 indicate the location of the inserted kanamycin
resistance gene and the ColE1 origin of replication in the cloned
genomic DNA. The arrows next to pJD105 and pJD106 indicate
the direction of transcription of the lac promoter from the
vector pUCP22. The asterisk indicates that not all of the PstI sites were mapped in these plasmids.
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The cloned genomic DNA was initially analyzed by determining the
nucleotide sequence to either side of the transposon insertion point
using primers specific for either end of the transposon. Screening for
similar nucleotide sequences in the GenBank data base revealed a
significant match with genes coding for multidrug resistance export
pumps, consistent with the hypothesis that a solvent efflux pump is a
key solvent resistance mechanism of P. putida S12. The
complete nucleotide sequence of an operon into which the transposon had
inserted was determined using a series of subclones and internal
oligonucleotide primers. Both strands of a 6.5-kb SstI
fragment were sequenced in their entirety. To accurately determine the
nucleotide sequence at the transposon insertion point, a 0.5-kb DNA
fragment was amplified by PCR from the genome of the wild-type strain
P. putida S12 using oligonucleotide primers flanking the
transposon insertion point in strain JK1. A cartoon of the nucleotide
sequence obtained is shown in Fig. 2 to show the relationship of the
open reading frames with the two clones pJD101 and pJD102 and the
complete nucleotide sequence is shown in Fig.
3. The three open reading frames show
significant homology to the three proteins that assemble to form
proton-dependent multidrug resistance efflux pumps (27, 28)
and thus the genes were labeled srp for solvent
resistance pump.

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Fig. 3.
Nucleotide sequence of an SstI
fragment containing the srpABC genes. The deduced
amino acid sequences of the encoded proteins are shown below
the nucleotide sequence. Termination codons are indicated with an
asterisk, putative ribosome-binding sites are
underlined, a putative RNA polymerase-binding site is underlined, and the TnMod-KmO insertion
point is marked with an arrow. The GenBank accession number
for the srpABC sequence is AF029405.
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Complementation of P. putida JK1--
To prove that the three open
reading frames detected in the cloned fragment at the point of
insertion of the TnMod-KmO transposon actually are
responsible for solvent resistance, complementation experiments were
performed. This required reconstruction of the operon since the clones
obtained (pJD101 and pJD102) contain the transposon mutagenized DNA. A
BglII-SstI kanamycin resistance cassette also
containing the ColE1 origin of replication from TnMod-KmO was inserted at the BglII and
SstI sites of a 6.7-kb ClaI fragment derived from
pJD101. The resulting plasmid (pJD103, Fig. 2) was electroporated into
P. putida S12 to construct a new mutation by site-specific
reciprocal recombination. The resulting strain, JJD1, contains a
kanamycin resistance gene adjacent to the srpABC genes in
the genome. A 12-kb EcoRI genomic fragment containing the
kanamycin/ColE1 cassette was cloned from JJD1. This plasmid, designated
pJD104 (Fig. 2), contains the intact srpABC genes. A 6.5-kb
SstI fragment was cloned from pJD104 into the vector pUCP22
in both orientations with respect to the lac promoter. JK1
containing either of these two plasmids, designated pJD105 and pJD106
(Fig. 2), regained solvent resistance. JK1(pJD105), containing the
srpABC genes in the same orientation as the lac promoter, regained resistance to all of the solvents that the original
strain, P. putida S12, was resistant to (Table I). However, JK1(pJD106), containing the srpABC genes in the opposite
orientation to the lac promoter, regained resistance to only
two solvents, hexane and cyclohexane, with log POW values
near the border of the resistance phenotype. These results are
consistent with the solvent resistance phenotype being dependent on the
level of expression of the srpABC genes.
Transfer of the Solvent Resistance Phenotype--
P.
putida S12 displays multiple physiological responses to organic
solvents (see Introduction). Intuitively, a solvent efflux pump would
be the most important mechanism of solvent resistance since it would be
involved in actively removing solvents from the cell. Experiments were
therefore performed to determine whether the solvent efflux pump by
itself is capable of imparting the solvent resistance phenotype on
other P. putida strains. The two plasmids, pJD105 and
pJD106, were electroporated into the normally solvent sensitive
P. putida PPO200. The resulting recombinant strains are able
to grow on rich medium in a toluene-saturated atmosphere, whereas the
parent strain PPO200 with the vector pUCP22 could not (Fig.
4). PPO200(pJD106) grew slightly
slower than PPO200(pJD105), probably due to the fact that the
srpABC genes are expressed from the lac promoter
in pJD105. In liquid culture, PPO200(pUCP22) could withstand
concentrations of toluene up to 2.8 mM while both PPO200(pJD105) and PPO200(pJD106) showed resistance to the toxic effects of toluene up to a concentration of 4.9 mM (Table
II). Neither of the recombinant strains were resistant to a second phase of toluene (5.6 mM).
These experiments indicate that the solvent resistance phenotype can be
transferred to other bacterial strains and that the resistance can be
enhanced by higher levels of gene expression.

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Fig. 4.
Photograph of the solvent-sensitive P. putida PPO200 plated on solid L medium in the presence of
saturating vapor concentrations of toluene with and without the
srpABC genes. Upper: P. putida PPO200(pUCP22), no growth. Lower left: P. putida
PPO200(pJD105), good growth. Lower right: P. putida
PPO200(pJD106), slow growth.
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Table II
Growth of P. putida PPO200 containing the cloned srp genes on L
medium in the presence of toluene
Toluene was added at different concentrations to identical subcultures
in L medium during the early exponential growth phase. A plus indicates
growth with OD >0.7 and a minus indicates no growth with OD <0.1
after 24 h.
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DISCUSSION |
In the past several years solvent-resistant microorganisms have
been isolated directly from the environment (5, 29-33) or through the
process of mutation of a solvent-sensitive strain (34-36). It is
evident that these organisms must therefore have specific adaptation
mechanisms that impart the solvent resistance. Physiological studies on
microorganisms isolated from the environment that are naturally
resistant to high levels of organic solvents have revealed that many
different factors may play a role (4, 7). Naturally solvent-resistant
bacteria have been shown to alter the composition of the cell membrane
by increasing the ratio of trans- to
cis-unsaturated fatty acids (26) or by changing the
headgroup composition (37). This change in the cell membrane produces a
physical barrier, preventing solvents from entering the cell by
decreasing membrane fluidity. This would not entirely prevent solvents
from entering the cell, only slow down their diffusion into the cell
and increase the time needed to reach equilibrium with the external
environment. An intuitively better method of solvent resistance would
be to physically remove the solvent from the cell. One way of doing
this would be to degrade the solvent but this would only be effective
against low concentrations of solvents. Evidence was recently obtained
that an energy-dependent export system for hydrophobic
solvents functions to remove solvents from the interior of whole cells
(9). This solvent efflux pump should be a key element in solvent
resistance by naturally solvent-resistant bacteria. The genes for such
a solvent efflux pump in P. putida S12 were identified via
transposon mutagenesis to construct a solvent-sensitive strain. The
genes were cloned and sequenced and their role in solvent resistance
verified through complementation of the transposon mutation. The three
genes involved were labeled srpABC for solvent
resistance pump.
The deduced amino acid sequences of the proteins encoded by the
srpABC genes have extensive homology with those for
proton-dependent multidrug efflux systems of the
resistance/nodulation/cell division family (27, 28). This "RND"
family of efflux pumps is composed of three protein components that
together span the inner and outer membranes of Gram-negative bacteria:
an inner membrane transporter (SrpB analogues), an outer membrane
channel (SrpC analogues), and a periplasmic linker protein (SrpA
analogues). Members of this family have been shown to be involved in
export of antibiotics, metals, and oligosaccharides involved in
nodulation signaling. Based on the work presented here, this family can
be broadened to include a new class of efflux pump, involved in export
of solvents. Dendrograms showing the phylogenetic relationship of SrpA,
SrpB, and SrpC to other proteins involved in multidrug resistance are shown in Fig. 5. The
srpABC-encoded proteins show the most homology with those
for the mexAB/oprM-encoded multidrug resistance
pump found in Pseudomonas aeruginosa (38, 39). SrpA, SrpB,
and SrpC are 57.8, 64.4, and 58.5% identical to MexA, MexB, and OprM, respectively. The three dendrograms show that the Srp and the Mex
proteins fall into a distinct class, separate from but still closely
related to the other members of the RND family of efflux pumps. The
evolutionary relationship of the solvent resistance pump to multidrug
resistance pumps is not surprising since they both function to export
hydrophobic molecules from the cell. It is logical that a solvent
efflux pump would have evolved since it would enable microorganisms to
survive in close proximity to oil or coal deposits (a rich source of
carbon and energy) in the environment.

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Fig. 5.
Dendrograms showing the levels of homology
between the amino acid sequences of different proteins involved in
proton-dependent efflux systems. A, dendrogram
of periplasmic linker proteins. B, dendrogram of inner
membrane transporter proteins. C, dendrogram of outer
membrane channel proteins. The accession column refers to the GenPep
data base accession number.
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There have been several attempts to clone genes that are involved in
solvent resistance leading in many cases to the identification of a
protein that is somehow involved in making the cell resistant to a
single solvent or to a group of related solvents. Most of these studies
took place using E. coli that was forced under selective pressure to become solvent resistant. Mutations in a number of different genes can result in an increased tolerance to a particular chosen solvent, any one of which can allow the cell to grow in the
presence of the solvent. Genes implicated in increased organic solvent
tolerance of E. coli include the uncharacterized
ostA (for organic solvent
tolerance) (40), ahpC encoding
alkylhydroperoxide reductase (35), robA encoding a global
regulatory protein (41), and soxS encoding regulatory
proteins controlling the superoxide response regulon (42). These genes
enhance the survivability of the organism in the presence of a
particular solvent but are not responsible for solvent resistance
per se since they do not aid in understanding the true
mechanism(s) of solvent resistance found naturally in environmental
isolates. This article, however, represents the first example of
cloning and characterization of genes for a major solvent resistance
mechanism: a proton-dependent solvent efflux pump.
We have shown that the cloned genes can be transferred to another
P. putida strain with the concomitant gain of solvent
resistance by that organism. This has far reaching implications for
industrial applications in the fine chemistry area. Existing and
potential biocatalytic processes for compounds such as catechols,
phenols, medium chain alcohols, and enantiopure epoxides are suboptimal because the products formed are very toxic to normal microorganisms. Product accumulation to a concentration which allows economic downstream processing is inherently prevented by the physical characteristics of these compounds. The ability, as demonstrated here,
to take a normally solvent-sensitive strain and make it solvent
resistant will greatly enhance the ability of a given strain to perform
a desired biocatalytic reaction resulting in otherwise toxic
products.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF029405.