Previous Article | Next Article 
Journal of Bacteriology, May 1999, p. 3281-3283, Vol. 181, No. 10
Department of Enzymology and Protein
Engineering,
Received 13 January 1999/Accepted 15 March 1999
Bacteriocin release protein is known to activate outer membrane
phospholipase A (OMPLA), which results in the release of colicin from
Escherichia coli. In vivo chemical cross-linking
experiments revealed that the activation coincides with dimerization of
OMPLA. Permeabilization of the cell envelope and dimerization were
characterized by a lag time of 2 h.
Colicins are plasmid-encoded
proteins produced by Escherichia coli and capable of killing
E. coli and closely related species (18).
Bacteriocin release protein (BRP) (also referred to as lysis protein)
is a small lipoprotein that is essential for the release of colicin
into the medium. BRP activates the endogenous outer membrane (OM)
phospholipase (OMPLA) (6, 14, 20) which is important for the
permeabilization of the cell envelope, since strains defective in the
structural gene for OMPLA, pldA, do not release colicin.
OMPLA is constitutively expressed, but enzymatic activity is detected
in vivo only under adverse conditions (2, 7, 15, 17).
Chemical cross-linking with formaldehyde suggested that OMPLA is
present in whole cells in a monomeric state, whereas the dimeric form
of the enzyme could be detected after sonication of the cells
(10). We have shown in vitro that the monomeric state of
OMPLA is inactive and that activation requires dimerization (10). In this study, we have investigated whether under
physiologically relevant conditions activation of OMPLA involves dimerization.
The pACYC184-based plasmid pJL4 encodes chloramphenicol
acetyltransferase (CAT) and contains the BRP gene from pCloDF13 of Enterobacter cloacae under control of the lpp/lac
tandem promoter/operator (13). Plasmid pRB1 (4)
contains the wild-type pldA gene under its own, constitutive
promoter; pRB1-S144A (5) contains a pldA allele,
which encodes an inactive S144A substitution mutant OMPLA. A 3,300-bp
XmnI/NruI fragment of plasmid pMF20
(22), carrying the phoA gene encoding alkaline
phosphatase (AP) under control of a constitutive, mutant promoter was
subcloned into the unique HincII site of pRB1 and of
pRB1-S144A, yielding plasmids pND19 and pND20, respectively. E. coli K-12 strain CE1303 (pldA recA56 [9]) transformed with pJL4 and either pND19 or pND20
was grown under agitation at 37°C in Luria broth supplemented with
chloramphenicol (37 µg/ml) and ampicillin (50 µg/ml) and with 10 mM
MgCl2 to prevent BRP-induced lysis (14, 20).
When the optical density at 600 nm reached 0.3, 150 µM
isopropyl- The permeabilization of the cell envelope was monitored by measuring
the release of cytosolic CAT and of periplasmic AP into the medium.
Measurement of CAT activity (23) revealed the presence of 1 to 2% of the total CAT activity in the medium before induction of BRP
synthesis (Fig. 1). After induction, CAT
activity in the medium increased to 10% of the total amount after a
lag period of approximately 2 h (Fig. 1). This lag time is
consistent with previous observations for the release of colicin E2
(20), cloacin DF13 (14) and marker enzymes
(19). The release of CAT was dependent on the action of
OMPLA, since it was much lower when an inactive mutant of OMPLA, in
which the active-site serine was replaced by an alanine, was used (Fig.
1). However, also in the latter case, the levels of CAT in the
supernatant were significantly higher than when BRP synthesis was not
induced (Fig. 1), indicating that BRP alone is capable of a partial
permeabilization of the cell envelope. Measurement of AP activity
(26) revealed the presence of 5% of the total amount of AP
produced in the medium when BRP synthesis was not induced. After
induction of BRP synthesis, the release of AP increased to 27% of the
total activity (data not shown). The release of AP was dependent on the
activity of wild-type OMPLA and was again characterized by a lag time
of 2 h (data not shown). These data show that periplasmic AP can
enter the secretion pathway, suggestive of a sequential secretion
process.
Dimerization of OMPLA was analyzed in vivo by chemical cross-linking
with formaldehyde as described previously (10). Without induction of BRP synthesis, OMPLA was detected mostly as a monomeric species (Fig. 2, odd-numbered lanes).
However, when BRP synthesis was induced, considerable amounts of dimer
could be trapped (Fig. 2, lanes 4, 6, 8 and 10). The absence of dimeric
cross-linking products before activation could be explained by the
existence of OMPLA in a dimeric but cross-linking-incompetent state
under normal conditions. However, when gluteraldehyde was used as a cross-linker with a longer spacer, dimers were also not observed (results not shown). Therefore, it is unlikely that OMPLA preexists in
a dimeric state in the OM, although this possibility cannot be totally
excluded. The dimerization of OMPLA was characterized by a considerable
lag time, which correlates with the lag time observed for the release
of CAT and AP into the medium. Moreover, it has been reported that the
formation of lysophosphatidylethanolamine, the major hydrolysis product
of the OMPLA-catalyzed reaction, is characterized by a similar lag time
(6, 14, 20). BRP-induced dimerization was also detected for
the inactive mutant OMPLA (data not shown).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Bacteriocin Release Protein Triggers Dimerization
of Outer Membrane Phospholipase A In Vivo
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
-D-1-thiogalactopyranoside (IPTG) was added to
induce BRP synthesis.
View larger version (18K):
[in a new window]
FIG. 1.
Release of CAT after induction of BRP synthesis. CE1303
cells containing pJL4 and either pND19 (wild-type OMPLA; filled
circles) or pND20 (inactive mutant OMPLA; open circles) were induced
with 150 µM IPTG at time zero. The release of CAT from the cells into
the culture medium was monitored over time. The release is expressed as
a percentage of enzyme activity present in the culture supernatant of
the total enzyme activity determined after complete lysis of the
culture by sonication. In control experiments, no IPTG was added
(filled and open squares for wild-type and mutant OMPLA, respectively).
The results are from three independent cultures, and standard
deviations are indicated by the error bars.
View larger version (61K):
[in a new window]
FIG. 2.
In vivo cross-linking of OMPLA. CE1303 cells containing
pJL4 and pND19 were grown either with (+) or without ( 
) IPTG to
induce BRP synthesis. At several time points after the addition of IPTG
(indicated in hours over the lanes), samples were collected,
cross-linked, and analyzed on a gel. OMPLA was visualized by Western
blotting with a monoclonal antibody and subsequent chemiluminescence
detection (Renaissance Western blot ECL kit; DuPont). The positions of
the monomer (M) and dimer (D) forms of OMPLA are indicated to the right
of the gel.
What can we propose now for the molecular mechanism of BRP-mediated activation of OMPLA? The lag time of 2 h after induction of BRP expression before the onset of permeabilization of the cell envelope and dimerization of OMPLA is intriguing. Although the posttranslational processing of BRP is a slow process and takes minutes for completion (11), it is still rapid compared to the observed lag time. BRP is produced in large amounts up to 105 copies per cell (12), which are much larger than the small amounts of OMPLA present. Therefore, a specific, direct interaction between BRP and OMPLA is not very likely the direct trigger for activation. Diffusion rates within the OM are very low (25) due to the interactions between the negatively charged lipopolysaccharide molecules, which are interconnected via Mg2+- or Ca2+-mediated salt bridges and via hydrophobic interactions between the lipid A parts (16). The low diffusion rate together with the low abundance of the protein (380 copies per cell [21]) might normally prevent the dimerization of OMPLA. The incorporation of BRP in the OM could destabilize the membrane, especially if the localization is restricted to one leaflet (24). Such action correlates with our finding that, although with reduced efficiency, BRP alone is capable of permeating the OM. Morphological changes induced by BRP have been observed as blebs in the OM (27). Furthermore, low-level expression of the pColE1 BRP causes partial exfoliation of the OM (1). One can envisage that the BRP-mediated membrane perturbation relieves the lipid asymmetry of the OM and accelerates diffusion in the OM, thereby triggering OMPLA activation. In our model, we assume a uniform distribution of monomeric OMPLA within the OM. Recently, a method for the specific in vivo labeling of cysteine residues in the OM protein FhuA has been reported (3). The labeling of single-cysteine mutants of OMPLA with fluorescent probes could provide insight in the distribution of OMPLA over the cell surface, and we are currently investigating this possibility. Once activated, the enzyme will generate fatty acid and, more importantly, lysophospholipid, a compound known to destabilize membranes (8). The water solubility of lysophospholipid is likely sufficient to permit transport to the cytoplasmic membrane. The presence of large amounts of BRP and lysophospholipid in both membranes could allow for the direct transport of colicins and reporter enzymes through these membranes in a sequential manner.
| |
ACKNOWLEDGMENTS |
|---|
We thank B. Oudega for the generous gift of plasmid pJL4 and for useful discussions.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Enzymology and Protein Engineering, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Phone: 31-30-2532458. Fax: 31-30-2522478. E-mail: n.dekker{at}chem.uu.nl.
Hubertus M. Verheij passed away in a tragic accident on 1 August 1998.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Aono, R. 1991. Envelope alteration of Escherichia coli HB101 carrying pEAP31 caused by Kil peptide and its involvement in the extracellular release of periplasmic penicillinase from an alkaliphilic Bacillus. Biochem. J. 275:545-553. |
| 2. | Audet, A., G. Nantel, and P. Proulx. 1974. Phospholipase A activity in growing Escherichia coli cells. Biochim. Biophys. Acta 348:334-343[Medline]. |
| 3. |
Bös, C.,
D. Lorenzen, and V. Braun.
1998.
Specific in vivo labeling of cell surface-exposed protein loops: reactive cysteine in the predicted gating loop mark a ferrichrome binding site and a ligand-induced conformational change of the Escherichia coli FhuA protein.
J. Bacteriol.
180:605-613 |
| 4. |
Brok, R. G. P. M.,
E. Brinkman,
R. van Boxtel,
A. C. A. P. A. Bekkers,
H. M. Verheij, and J. Tommassen.
1994.
Molecular characterization of enterobacterial pldA genes encoding outer membrane phospholipase A.
J. Bacteriol.
176:861-870 |
| 5. | Brok, R. G. P. M., I. Ubarretxena Belandia, N. Dekker, J. Tommassen, and H. M. Verheij. 1996. Escherichia coli outer membrane phospholipase A: role of two serines in enzymatic activity. Biochemistry 35:7787-7793[Medline]. |
| 6. |
Cavard, D.,
D. Baty,
S. P. Howard,
H. M. Verheij, and C. Lazdunski.
1987.
Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing.
J. Bacteriol.
169:2187-2194 |
| 7. | Cronan, J. E. J., and D. L. Wulff. 1969. A role for phospholipid hydrolysis in the lysis of Escherichia coli infected with bacteriophage T4. Virology 38:241-246[Medline]. |
| 8. | Cullis, P. R., and B. de Kruijff. 1979. Lipid polymorphism and the functional role of lipids in biological membranes. Biochim. Biophys. Acta 559:399-420[Medline]. |
| 9. | de Geus, P., I. van Die, H. Bergmans, J. Tommassen, and G. de Haas. 1983. Molecular cloning of pldA, the structural gene for outer membrane phospholipase of E. coli K12. Mol. Gen. Genet. 190:150-155[Medline]. |
| 10. |
Dekker, N.,
J. Tommassen,
A. Lustig,
J. P. Rosenbusch, and H. M. Verheij.
1997.
Dimerization regulates the enzymatic activity of outer membrane phospholipase A.
J. Biol. Chem.
272:3179-3184 |
| 11. |
Howard, S. P., and L. Lindsay.
1998.
In vivo analysis of sequence requirements for processing and degradation of the colicin A lysis protein signal peptide.
J. Bacteriol.
180:3026-3030 |
| 12. |
Luirink, J.,
D. M. Clark,
J. Ras,
E. J. Verschoor,
F. K. de Graaf, and B. Oudega.
1989.
pCloDF13-encoded bacteriocin release proteins with shortened carboxyl-terminal segments are lipid modified and processed and function in release of cloacin DF13 and apparent host cell lysis.
J. Bacteriol.
171:2673-2679 |
| 13. | Luirink, J., F. de Graaf, and B. Oudega. 1987. Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli. Mol. Gen. Genet. 206:126-132[Medline]. |
| 14. |
Luirink, J.,
C. van der Sande,
J. Tommassen,
E. Veltkamp,
F. K. de Graaf, and B. Oudega.
1986.
Effects of divalent cations and of phospholipase A activity on excretion of cloacin DF13 and lysis of host cells.
J. Gen. Microbiol.
132:825-834 |
| 15. | Michel, G. P. F., and J. Stárka. 1979. Phospholipase A activity with integrated phospholipid vesicles in intact cells of an envelope mutant of Escherichia coli. FEBS Lett. 108:261-265[Medline]. |
| 16. | Nikaido, H. 1996. Outer membrane, p. 29-47. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C. |
| 17. | Patriarca, P., S. Beckerdite, and P. Elsbach. 1972. Phospholipases and phospholipid turnover in Escherichia coli spheroplasts. Biochim. Biophys. Acta 260:593-600[Medline]. |
| 18. | Pugsley, A. P. 1984. The ins and outs of colicins. Part I. Production and translocation across membranes. Microbiol. Sci. 1:168-175[Medline]. |
| 19. |
Pugsley, A. P., and J. P. Rosenbusch.
1981.
Release of colicin E2 from Escherichia coli.
J. Bacteriol.
147:186-192 |
| 20. | Pugsley, A. P., and M. Schwartz. 1984. Colicin E2 release: lysis, leakage or secretion? Possible role of a phospholipase. EMBO J. 3:2393-2397[Medline]. |
| 21. | Scandella, C. J., and A. Kornberg. 1971. A membrane-bound phospholipase A1 purified from Escherichia coli. Biochemistry 10:4447-4456[Medline]. |
| 22. | Scholten, M., R. van Boxtel, and J. Tommassen. Unpublished results. |
| 23. | Shaw, W. V. 1975. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43:737-755[Medline]. |
| 24. |
Sheetz, M. P., and S. J. Singer.
1974.
Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions.
Proc. Natl. Acad. Sci. USA
71:4457-4461 |
| 25. |
Smit, J., and H. Nikaido.
1978.
Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium.
J. Bacteriol.
135:687-702 |
| 26. |
Tommassen, J., and B. Lugtenberg.
1980.
Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase.
J. Bacteriol.
143:151-157 |
| 27. | van der Wal, F. J., J. Luirink, and B. Oudega. 1995. Bacteriocin release proteins: mode of action, structure, and biotechnological application. FEMS Microbiol. Rev. 17:381-399[Medline]. |
Journal of Bacteriology, May 1999, p. 3281-3283, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Cascales, E., Buchanan, S. K., Duche, D., Kleanthous, C., Lloubes, R., Postle, K., Riley, M., Slatin, S., Cavard, D.
(2007). Colicin Biology. Microbiol. Mol. Biol. Rev.
71: 158-229
[Abstract] [Full Text] -
Bos, M. P., Tefsen, B., Voet, P., Weynants, V., van Putten, J. P. M., Tommassen, J.
(2005). Function of Neisserial Outer Membrane Phospholipase A in Autolysis and Assessment of Its Vaccine Potential. Infect. Immun.
73: 2222-2231
[Abstract] [Full Text] -
Cavard, D.
(2004). Role of Cal, the colicin A lysis protein, in two steps of colicin A release and in the interaction with colicin A-porin complexes. Microbiology
150: 3867-3875
[Abstract] [Full Text] -
Cavard, D.
(2002). Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion. J. Bacteriol.
184: 3723-3733
[Abstract] [Full Text] -
Langen, G. R., Harper, J. R., Silhavy, T. J., Howard, S. P.
(2001). Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degP Mutants and Induces the Cpx and {sigma}E Extracytoplasmic Stress Responses. J. Bacteriol.
183: 5230-5238
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»