Previous Article | Next Article ![]()
Applied and Environmental Microbiology, January 1999, p. 294-296, Vol. 65, No. 1
Department of Bioengineering, Faculty of
Bioscience and Biotechnology, Tokyo Institute of Technology,
Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan
Received 17 August 1998/Accepted 9 October 1998
The organic solvent tolerance of Escherichia coli was
measured under conditions in which OmpF levels were controlled by
various means as follows: alteration of NaCl concentration in the
medium, transformation with a stress-responsive gene (marA,
robA, or soxS), or disruption of the
ompF gene. It was shown that solvent tolerance of E. coli did not depend upon OmpF levels in the membrane.
We previously constructed
Escherichia coli mutants displaying improved organic solvent
tolerance (3). E. coli JA300 (19), used as the parent, produced OmpF porin protein in the membrane even
when grown in the presence of 1% NaCl. In contrast, the levels of OmpF
protein were markedly decreased in the mutants because of mutations in
marR (4, 9). It is reported that OprF porin protein is absent in a toluene-tolerant mutant of Pseudomonas aeruginosa (22). Hydrophobic First, we controlled OmpF synthesis in JA300 by growth under different
salinity conditions. OmpF synthesis is repressed under conditions of
high environmental osmolarity (13). This osmoregulation is
mediated mainly by an increase in expression of micF RNA, an antisense RNA that inhibits OmpF translation. The extent of
osmoregulation was evaluated for JA300 grown in the medium that we have
usually used. A membrane fraction was prepared from sonicated lysate of the cells grown in modified Luria broth (LBGMg) (1% [wt/vol] Bacto Tryptone [Difco Laboratories, Detroit, Mich.], 0.5% Bacto Yeast Extract [Difco], 0.1% glucose, and 10 mM MgSO4)
containing or not containing 1% (wt/vol) NaCl. JA300 produced a
considerable amount of OmpF in the presence of NaCl, although the
amount was less than that produced in the absence of NaCl (Fig.
1 and Table 1). The effect of NaCl on the OmpF level
of JA300 carrying a vector plasmid, pBluescript II SK(+) (Toyobo
Biochemical Inc., Osaka, Japan; hereafter pBS) was similar to that
found in the host cell (results not shown).
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Organic Solvent Tolerance of Escherichia
coli Is Independent of OmpF Levels in the Membrane
![]()
ABSTRACT
Top
Abstract
Text
References
![]()
TEXT
Top
Abstract
Text
References
-lactam antibiotics
pass through OmpF channels faster than through OmpC channels
(28). It seemed likely that organic solvent molecules also
could pass through the OmpF porin. Therefore, it was supposed that the
decreased levels of OmpF or loss of OmpF might contribute to
improvement of organic solvent tolerance in E. coli, as
suggested for acquisition of multiple antibiotic resistance (2,
6). In this study, this possibility was explored by measuring the
organic solvent tolerance of E. coli under conditions in
which OmpF synthesis was controlled by various means.

View larger version (52K):
[in a new window]
FIG. 1.
Sodium dodecyl sulfate-urea-polyacrylamide gel
electrophoresis of envelope protein. JA300 cells transformed with the
plasmids were grown in LBGMg medium. The cells were broken by
sonication. Membrane fractions were extracted with 0.5% sodium
N-lauroylsarcosine at room temperature (11).
Insoluble proteins (45 µg) were electrophoresed on 0.1% sodium
dodecyl sulfate-4 M urea-10% (wt/vol) polyacrylamide gels
(1). Protein was stained with Coomassie Brilliant Blue
R-250. Lanes: M, molecular size markers; 1 to 4, growth in the absence
of NaCl; 5 to 8, growth in the presence of 1% (wt/vol) NaCl; 1 and 5, no plasmid; 2 and 6, pMarA; 3 and 7, pRob; 4 and 8, pSoxS. Bands
containing OmpC, OmpF, and OmpA are shown.
TABLE 1.
Organic solvent tolerance levels of E. coli
JA300 and its derivative
LBGMg agar on which JA300 was plated was overlaid with a ca. 2-mm layer of an appropriate organic solvent and incubated at 37°C overnight. When JA300 grew confluently, it was considered that JA300 was tolerant of the organic solvent overlaying the agar (5). The toxicity of the organic solvent is reflected by its log POW value (5, 15), shown in Table 1. This value is inversely correlated with the toxicity of organic solvent. Here, log POW is the common logarithm of POW, the partition coefficient of the organic solvent between n-octanol and water. Growth without NaCl did not greatly lower the organic solvent tolerance level of JA300, although the OmpF level was high, as described above. We found that growth in the absence of NaCl reduced the organic solvent tolerance of JA300(pBS), compared with that grown in the presence of 1% (wt/vol) NaCl. This difference was observed probably because less growth occurred on the agar not containing NaCl.
Second, the OmpF levels were reduced by overexpression of marA, robA, or soxS. Transcriptional activators, MarA, Rob, and SoxS, positively regulate expression of mar-sox regulon genes including micF (8, 10, 17). Overproduction of these proteins triggered low production of OmpF. We intended to control OmpF synthesis through the repression brought about by overexpression of the stress-responsive genes and by growth at high osmolarity (salinity). The plasmids used here, pMarA, pRob, and pSoxS, were referred to previously as pHA105 (9), pOST42BR (26), and pHc3R (27), respectively.
The level of OmpF was reduced upon introduction of one of the plasmids (Fig. 1 and Table 1). The extent of repression differed depending on the plasmids carried and NaCl concentration. The repression caused by pMarA was independent of NaCl concentration. That caused by pRob was high in the absence of NaCl. In contrast, pSoxS severely repressed OmpF production in the presence of NaCl and slightly repressed it in the absence of NaCl. Consequently, various levels of OmpF production were achieved in JA300 cells.
We reported that overexpression of the genes improved the organic solvent tolerance of E. coli, based on the tolerance measured by monitoring growth in the presence of NaCl (9, 26, 27). The overexpression made JA300 grown without NaCl as highly tolerant as that grown with NaCl (Table 1). As far as examined, the organic solvent tolerance of each transformant did not differ between cells grown with NaCl and those grown without NaCl. It is particularly notable that JA300(pSoxS) grown without NaCl produced a high level of OmpF and was tolerant of cyclohexane, indicating that the increased level of OmpF did not reduce tolerance of cyclohexane. These results suggest that organic solvent tolerance levels are not directly related to OmpF levels.
The OmpF levels were controlled via micF expression in the experiments described above. These controls are indirect for OmpF production. In particular, growth of the organisms under different conditions such as salinity might cause unexpected effects on the cell membrane structures other than OmpF production. Finally, we constructed an ompF disruptant from JA300 and examined the solvent tolerance level. From E. coli RK4786 (14), ompF::Tn5 was transduced into JA300 by generalized transduction with P1kc (25). An OmpF-nonproducing transductant (JOF501) was selected from among kanamycin-resistant clones grown on LBGMg agar containing kanamycin (50 µg/ml). This P1 transductant did not display OmpF in the membrane at all, regardless of NaCl concentration (results not shown). The organic solvent tolerance level of the ompF disruptant, measured on LBGMg agar containing NaCl, was identical to that of JA300 (Table 1). The ompF disruption did not result in improvement of the organic solvent tolerance.
The organic solvent tolerance of JA300 was not altered to any detectable extent as a direct result of a decreased or increased level of OmpF in the membrane or its absence, except for JA300(pBS). The extremely low level of organic solvent tolerance of the tolC disruptant derived from JA300 is not improved by transformation with pMarA, pRob, or pSoxS (7), although the OmpF levels decreased in the transformants (results not shown). This fact supports the conclusion described above. It is likely that the solvent tolerance of the mutants or transformants is improved mainly by elevated expression of acrAB and tolC (7), not by repression of OmpF synthesis. Genes acrAB and tolC encode AcrA, AcrB, and TolC, consisting of a proton motive force-dependent efflux pump to extrude multiple antibiotics (12, 24). Recently, energy-dependent efflux systems extruding organic solvents have been reported to contribute to the organic solvent tolerances of E. coli (7, 32), P. aeruginosa (23, 29), and Pseudomonas putida (16, 18, 30, 31). Probably the AcrAB and TolC efflux system plays the most important role in organic solvent tolerance in E. coli.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan. Phone: (81) 45-924-5766. Fax: (81) 45-924-5819. E-mail: raono{at}bio.titech.ac.jp.
| |
REFERENCES |
|---|
|
|
|---|
| 1. |
Achtman, M.,
A. Mercer,
B. Kusecek,
A. Pohl,
M. Heuzenroeder,
W. Aaronson,
A. Sutton, and R. P. Silver.
1983.
Six widespread bacterial clones among Escherichia coli K1 isolates.
Infect. Immun.
39:315-335 |
| 2. | Alekshun, M. N., and S. B. Levy. 1997. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob. Agents Chemother. 41:2067-2075[Medline]. |
| 3. | Aono, R., K. Aibe, A. Inoue, and K. Horikoshi. 1991. Preparation of organic solvent tolerant mutants from Escherichia coli K-12. Agric. Biol. Chem. 55:1935-1938. |
| 4. | Aono, R., and H. Kobayashi. 1997. Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12. Appl. Environ. Microbiol. 63:3637-3642[Abstract]. |
| 5. | Aono, R., H. Kobayashi, K. N. Joblin, and K. Horikoshi. 1994. Effects of organic solvents on growth of Escherichia coli K-12. Biosci. Biotechnol. Biochem. 58:2009-2014. |
| 6. | Aono, R., M. Kobayashi, H. Nakajima, and H. Kobayashi. 1995. A close correlation between organic solvent tolerance and multiple antibiotic resistance systems. Biosci. Biotechnol. Biochem. 59:213-218[Medline]. |
| 7. |
Aono, R.,
N. Tsukagoshi, and M. Yamamoto.
1998.
Involvement of outer membrane protein TolC, a possible member of the mar-sox regulon, in maintenance and improvement of organic solvent tolerance level of Escherichia coli K-12.
J. Bacteriol.
180:938-944 |
| 8. |
Ariza, R. R.,
A. Li,
N. Ringstad, and B. Demple.
1995.
Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein.
J. Bacteriol.
177:1655-1661 |
| 9. | Asako, H., H. Nakajima, K. Kobayashi, M. Kobayashi, and R. Aono. 1997. Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl. Environ. Microbiol. 63:1428-1433[Abstract]. |
| 10. |
Chou, J. H.,
J. Greenberg, and B. Demple.
1993.
Posttranscriptional repression of Escherichia coli OmpF protein in response to redox stress: positive control of the micF antisense RNA by the soxRS locus.
J. Bacteriol.
175:1026-1031 |
| 11. |
Filip, C.,
G. Fletcher,
J. L. Wulff, and C. F. Earhart.
1973.
Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate.
J. Bacteriol.
115:717-722 |
| 12. |
Fralick, J. A.
1996.
Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli.
J. Bacteriol.
178:5803-5805 |
| 13. |
Hasegawa, Y.,
H. Yamada, and S. Mizushima.
1976.
Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12.
J. Biochem.
80:1401-1409 |
| 14. |
Heller, K.,
B. J. Mann, and R. J. Kadner.
1985.
Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.
J. Bacteriol.
161:896-903 |
| 15. | Inoue, A., and K. Horikoshi. 1989. A Pseudomonas thrives in high concentrations of toluene. Nature 338:264-265. |
| 16. |
Isken, S., and J. A. M. de Bont.
1996.
Active efflux of toluene in a solvent-tolerant bacterium.
J. Bacteriol.
178:6056-6058 |
| 17. |
Jair, K.-W.,
R. G. Martin,
J. L. Rosner,
N. Fujita,
A. Ishihama, and R. E. J. Wolf.
1995.
Purification and regulatory properties of MarA protein, a transcriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters.
J. Bacteriol.
177:7100-7104 |
| 18. |
Kieboom, J.,
J. J. Dennis,
J. A. M. de Bont, and G. J. Zylstra.
1998.
Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance.
J. Biol. Chem.
273:85-91 |
| 19. | Kingsman, A. J., L. Clarke, R. K. Mortimer, and J. Carbon. 1979. Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trp1 region. Gene 7:141-152[Medline]. |
| 20. | Laane, C., S. Boeren, K. Vos, and C. Veegar. 1987. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30:81-87. |
| 21. | Leo, A. J. 1993. Calculating log Poct from structures. Chem. Rev. 63:1281-1306. |
| 22. | Li, L., T. Komatsu, A. Inoue, and K. Horikoshi. 1995. A toluene-tolerant mutant of Pseudomonas aeruginosa lacking the outer membrane protein F. Biosci. Biotechnol. Biochem. 59:2358-2359[Medline]. |
| 23. |
Li, X.-Z.,
Z. Li, and K. Poole.
1998.
Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance.
J. Bacteriol.
180:2987-2991 |
| 24. | Ma, D., D. N. Cook, M. Alberti, N. G. Pon, H. Nikaido, and J. E. Hearst. 1995. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol. Microbiol. 16:45-55[Medline]. |
| 25. | Miller, J. H. 1972. Experiments in molecular genetics, p. 201-205. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 26. | Nakajima, H., K. Kobayashi, M. Kobayashi, H. Asako, and R. Aono. 1995. Overexpression of robA gene increases organic solvent tolerance and multiple antibiotic and heavy metal ion resistance in Escherichia coli. Appl. Environ. Microbiol. 61:2302-2307[Abstract]. |
| 27. | Nakajima, H., M. Kobayashi, T. Negishi, and R. Aono. 1995. soxRS gene increases the level of organic solvent tolerance in Escherichia coli. Biosci. Biotechnol. Biochem. 59:1323-1325[Medline]. |
| 28. |
Nikaido, H.,
E. Y. Rosenberg, and J. Foulds.
1983.
Porin channels in Escherichia coli: studies with -lactams in intact cells.
J. Bacteriol.
153:232-240 |
| 29. | Noguchi, K., H. Nakajima, and R. Aono. 1997. Effects of oxygen and nitrate on growth of Escherichia coli and Pseudomonas aeruginosa in the presence of organic solvents. Extremophiles 1:193-198. [Medline] |
| 30. |
Ramos, J. L.,
E. Duque,
P. Godoy, and A. Segura.
1998.
Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E.
J. Bacteriol.
180:3323-3329 |
| 31. |
Ramos, J. L.,
E. Duque,
H. J. J. Rodriguez,
P. Godoy,
A. Haidour,
F. Reyes, and B. A. Fernandez.
1997.
Mechanisms for solvent tolerance in bacteria.
J. Biol. Chem.
272:3887-3890 |
| 32. |
White, D. G.,
J. D. Goldman,
B. Demple, and S. B. Levy.
1997.
Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli.
J. Bacteriol.
179:6122-6126 |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»