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Journal of Bacteriology, December 1998, p. 6769-6772, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Active Efflux of Organic Solvents by
Pseudomonas putida S12 Is Induced by Solvents
Jasper
Kieboom,1,*
Jonathan J.
Dennis,2
Gerben J.
Zylstra,2 and
Jan A. M.
de Bont1
Division of Industrial Microbiology,
Department of Food Technology and Nutritional Sciences, Wageningen
Agricultural University, Wageningen, The
Netherlands,1 and
Biotechnology Center
for Agriculture and the Environment, Rutgers University, New
Brunswick, New Jersey 08901-85202
Received 16 July 1998/Accepted 7 October 1998
 |
ABSTRACT |
Induction of the membrane-associated organic solvent efflux system
SrpABC of Pseudomonas putida S12 was examined by cloning a
312-bp DNA fragment, containing the srp promoter, in the
broad-host-range reporter vector pKRZ-1. Compounds that are capable of
inducing expression of the srpABC genes include aromatic
and aliphatic solvents and alcohols. General stress conditions such as
pH, temperature, NaCl, or the presence of organic acids did not induce
srp transcription. Although the solvent efflux pump in
P. putida S12 is a member of the resistance-nodulation-cell
division family of transporters, the srpABC genes were not
induced by antibiotics or heavy metals.
| |
TEXT |
Several Pseudomonas
putida strains possess an intrinsic resistance to a wide variety
of structurally unrelated hydrophobic solvents (1, 8, 20,
29) that are lethal for most other gram-negative bacteria. The
susceptibility of bacteria to hydrophobic solvents is due to the
accumulation of these compounds in the membrane (25, 26),
causing an adverse effect on its physicochemical properties.
Solvent-resistant bacteria are able to counterbalance these
effects through a variety of mechanisms mostly affecting the lipid
content of the cell membrane: isomerizing cis-unsaturated fatty acids to the more rigid trans-unsaturated fatty acids
(4, 5, 7), changing the head group composition of membrane
fatty acids (28), increasing the phospholipid content
(20), or increasing the basal rate of phospholipid synthesis
(19). These adaptations of the membrane are static, acting
as a physical but still permeable barrier, and cannot explain the
exceptional resistance of some P. putida strains to organic
solvents. Therefore, it was anticipated that a dynamic system for
exporting a broad range of structurally unrelated organic solvents from
the bacterial membrane had to play an essential role in solvent
tolerance (28). Such an efflux system was indeed identified
in P. putida S12 by means of an assay based on radiolabeled
toluene (9). The genes (srpABC) for this solvent
efflux system were subsequently cloned, sequenced, and shown to impart
the solvent-resistant phenotype to solvent-sensitive P. putida strains (12). This efflux system shows strong
homology to those of the resistance-nodulation-cell division family of transporters known to be involved in the extrusion of hydrophobic antibiotics, dyes, detergents, bile salts, heavy metals, and fatty acids from the membrane (17, 18).
Recently, several researchers also reported the involvement of active
organic solvent efflux in solvent-resistant strains of
Pseudomonas (2, 13, 14, 22). Induction in
Pseudomonas species of efflux systems involved in either
multidrug resistance or solvent tolerance has not been studied in detail.
Construction of the lacZ reporter plasmid
pKRZ-srp.
Plasmid isolation, restriction analysis, ligations,
electroporation, sequencing, and PCRs were performed according to
standard methods as described previously (12). The region of
DNA encompassing the putative promoter region of the srp
operon was amplified by PCR from pJD101 (12). Primers for
the PCR were 5'-GGGTCGACGCTGCTCTGGCGATGACC-3' and
5'-GGTCTAGATCTGTCTCACGGTTTGGC-3', which amplify a 312-bp
fragment corresponding to the region immediately upstream of the
srpABC genes (positions 
285 to +8, where 0 is the G of the
GTG start codon of srpA). The primers contain added
recognition sites for SalI and XbaI,
respectively. The PCR fragment was cloned into the lacZ
reporter plasmid pKRZ-1 (23) cut with XbaI and
SalI. The resulting plasmid, containing the sequence
shown in Fig. 1, was designated
pKRZ-srp.
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|
FIG. 1.
Nucleotide sequence of the PCR-amplified
SalI-XbaI fragment containing the
srpABC promoter region. The terminal SalI and
XbaI sites were added by incorporating their cutting sites
into the PCR primers. The amino acid sequences of srpA are
shown extending outward on the right side of the PCR product. This
fragment was inserted into pKRZ-1 so that the promoterless
lacZ gene is downstream of the start codon of
srpA. An asterisk indicates the stop codon inserted into
srpA by the added XbaI site.
|
|
Basal levels of srp promoter activity.
P.
putida S12 strains were grown to late log phase at 30°C in 200 ml of Luria-Bertani broth (24) supplemented with 50 µg of
kanamycin per ml. Cells were harvested by centrifugation at 4°C
(16,000 × g, 10 min) and washed twice with 100 mM
potassium phosphate buffer (pH 7.0). The washed pellet was resuspended
in 2 ml of the same buffer and lysed by sonication for 5 min. Cell debris was removed by centrifuging the crude cell extract at 4°C and
20,000 × g for 20 min. 
-Galactosidase activity in
the extracts was determined in triplicate by the method of Miller
(16). Total protein content in the extracts was determined
in triplicate by the bicinchoninic acid method (27).
P. putida S12 containing either the promoter probe vector or
pKRZ-srp was grown with no added inducers. -Galactosidase activity
for the vector only was 0.9 ± 0.1 nmol min
1
mg1 while the basal activity for the clone containing the
promoter region was 3.8 ± 0.1 nmol min1
mg1. Similar levels of basal activity were observed when
the strains were grown on minimal medium D (3) supplemented
with 50 µg of kanamycin per ml and 20 mM glucose as the sole source
of carbon and energy (0.8 and 3.9 nmol min1
mg1, respectively).
Activation of the srp promoter over time.
In order
to determine the time course for induction of the srp
operon, -galactosidase activity was measured as a function of time
following exposure of P. putida S12 containing
either the promoter probe vector or pKRZ-srp. P. putida
S12(pKRZ-srp) cells were grown in LB broth, and 3 mM toluene was added
when the cells reached an optical density of 0.3 (Fig.
2). Induction of srp-lacZ
expression was observed 30 min after the addition of toluene and
gradually increased over time. The maximum level of induction of the
srp operon was observed when the cells reached stationary
phase. This clearly shows that exposure to one organic solvent results
in a significant increase in transcription of the srp
operon.
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|
FIG. 2.
Activation of P. putida S12 srp
promoter over time. The arrow indicates the addition of 3 mM toluene.
Cell density of P. putida S12(pKRZ-srp) in LB broth ( ,
control;  , induced cells) and -galactosidase activity ( ,
control;  , induced cells) were monitored over time.
-Galactosidase activity in the time scale experiments was determined
in triplicate cultures by the method of Miller (16) by using
chloroform and sodium dodecyl sulfate to permeabilize the cells.
|
|
Activation of the srp promoter by organic
solvents.
The influence of toluene concentration on gene induction
was determined by varying the amounts of toluene added to the cells in
the early exponential phase. -Galactosidase activity was measured in
the late exponential phase of growth (optical density of 1.0 at 600 nm). As shown in Table 1, increasing the
level of toluene added to the growth medium increases the level of
srp-lacZ gene induction, reaching a maximum at 6.0 mM. (The
saturating limit of toluene in aqueous solutions at 30°C is 6.2 mM.)
Induction by toluene thus results in a 15- to 17-fold increase in
induction over basal levels.
Several hydrophobic organic solvents including aromatic compounds,
aliphatic compounds, and aliphatic alcohols were tested for their
ability to induce the srp-lacZ construct. As can be seen by
the data presented in Table 1, all of the compounds tested are able to
induce the srp genes. Certain aromatic compounds and aliphatic alcohols showed the highest levels of induction. The level of
induction seems to correlate with increasing side chain length in the
case of the alkyl-substituted aromatics and with chain length in the
case of the aliphatic alcohols (up to a 15- to 17-fold induction).
Antibiotics and heavy metals.
We previously showed that the
srp operon shows a high level of similarity to
proton-dependent multidrug efflux systems (12), which are
known to be involved in the efflux of a variety of antibiotics and
heavy metals (17, 18).
Firstly, the ability of certain antibiotics to induce the
srp genes was tested. In initial experiments, we determined
the MIC of each antibiotic by twofold serial dilution in LB broth. The
inoculum was 2% of an overnight culture, and growth was determined by
measuring the optical density at 600 nm after 12 h at 30°C. The
level of -galactosidase activity was measured in the late exponential phase of cultures exposed to a level of each antibiotic resulting in approximately 50% decrease in growth rate. Growth of
P. putida S12(pKRZ-srp) in the presence of 128 µg of
chloramphenicol per ml, 128 µg of ampicillin per ml, and 4 µg of
tetracycline per ml resulted in only a twofold increase in induction
over basal levels. No increase in induction over basal levels was
observed in the presence of the other antibiotics tested: 256 µg of
penicillin G per ml, 256 µg of novobiocin per ml, and 4 µg of
rifampin per ml.
Secondly, the ability of heavy metals to induce the srp-lacZ
fusion was determined. Cells of P. putida S12(pKRZ-srp) were grown in LB broth in the presence of six different heavy metals (added
as chloride salts in a final concentration of 1 mM). Zinc, chromium,
cobalt, nickel, and copper had no detectable effect on the
srp promoter, while cadmium resulted in only a 1.6-fold increase in induction.
Environmental factors.
General stress conditions such as NaCl,
ethanol, and stationary phase are known to induce the AcrAB efflux
system in Escherichia coli (15). On this basis,
it was suggested that a general regulatory mechanism exists in
E. coli to prevent hydrophobic compounds from entering
the bacterial cell. Such a general response was also observed in
P. putida S12 in the case of the induction of
cis- to trans-isomerization of the membrane
unsaturated fatty acids by environmental stress such as pH and heavy
metals (6). This change in fatty acid profile coincided with
an increased resistance to organic solvents. In order to investigate
whether the srp promoter activity was induced by these
environmental factors, cells of P. putida S12(pKRZ-srp) were
grown in LB broth under different conditions. Varying the growth
temperature between 15 and 37°C and varying the pH between 6.0 and
8.0 did not result in a change in srp promoter induction.
High levels of inorganic ions (50 g of NaCl per liter) did not affect
srp-lacZ expression, although the growth of P. putida S12(pKRZ-srp) under the conditions tested was
severely inhibited by high levels of NaCl.
Weber et al. (29) previously showed that incubating P. putida S12 with high acetic acid concentrations increased the
survival of the strain after these cells were exposed to organic
solvents. In order to determine if this was due to induction of the
srp operon, the ability of acetate to induce this active
efflux system for organic solvents was tested. P. putida
S12(pKRZ-srp) was grown in LB broth in the presence of 20, 40, and 60 mM acetic acid (pH 6.5). Under each concentration of acetate, only a
twofold induction of the srp-lacZ construct was observed.
These results suggest that the enhanced survival of acetic-acid-adapted
cells is not due to activation of the SrpABC efflux system.
Conclusions.
The data in the present paper clearly shows that
the srpABC operon for the efflux of organic solvents is
induced by lipophilic aromatic and aliphatic solvents and alcohols. The
data suggests that neither aromaticity nor charge is required for
organic solvents to act as an inducer. Unlike other efflux systems, the
srpABC operon is not induced by environmental stress or
heavy metals, demonstrating that the genes are specifically induced and
are not the result of a general regulatory mechanism as described elsewhere for E. coli (15).
The specific induction by solvents is underlined by the
observation that hydrophobic antibiotics do not induce the
SrpABC efflux system. P. putida S12 only becomes more
resistant to hydrophobic antibiotics by preculturing cells in the
presence of toluene, while P. putida S12 pregrown in the
presence of antibiotics does not elicit the solvent-tolerant phenotype
(10). These observations suggest that hydrophobic
antibiotics are removed from the membrane in solvent-induced cells,
while these hydrophobic antibiotics do not induce SrpABC-mediated
efflux of hydrophobic organic solvents.
Interestingly, Ramos et al. very recently reported the existence of at
least two efflux pumps in the solvent-resistant P. putida DOT-T1E. One system apparently was expressed
constitutively, while a second system was inducible (22).
Although the natural function of these resistance-nodulation-cell
division-type efflux systems has not been clarified yet, our data
suggests that the SrpABC system plays a protective role in resistance
to a wide variety of structurally unrelated hydrophobic compounds.
Multidrug efflux systems in P. aeruginosa are not induced by
known substrates of these efflux pumps (21), suggesting that the extrusion by these pumps of hydrophobic compounds is a nonspecific action. At present, the natural substrate(s) of these systems remains
unknown. The SrpABC efflux system apparently does not have a natural
function in excreting hydrophobic antibiotics because (i)
solvent-sensitive mutants of P. putida S12 have normal
levels of antibiotic resistance (11) and (ii) the
srpABC operon is induced solely by solvent stress (this work).
| |
ACKNOWLEDGMENTS |
This work was supported by the Solvay Duphar Corporation (Weesp,
The Netherlands), a National Science Foundation Young Investigator Award, and cooperative agreement CR822634 from the U.S. Environmental Protection Agency Gulf Breeze Environmental Research Laboratory.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Industrial Microbiology, Department of Food Technology and Nutritional Sciences, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31 (0)317 484412. Fax: 31 (0)317 484978. E-mail:
jasper.kieboom{at}imb.ftns.wau.nl.
| |
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Journal of Bacteriology, December 1998, p. 6769-6772, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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