Previous Article | Next Article 
Journal of Bacteriology, June 1999, p. 3578-3581, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of Colicin S4 and Its Receptor,
OmpW, a Minor Protein of the Escherichia coli Outer
Membrane
Holger
Pilsl,*
David
majs,
and
Volkmar
Braun
Mikrobiologie/Membranphysiologie,
Universität Tübingen, Tübingen, Germany
Received 10 February 1999/Accepted 5 April 1999
 |
ABSTRACT |
Analysis of the nucleotide sequence of an Escherichia
coli colicin S4 determinant revealed 76% identity to the
pore-forming domain of the colicin A protein, 77% identity to the
colicin A immunity protein, and 82% identity to the colicin A lysis
protein. The N-terminal region, which is responsible for the
Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal
region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked
the OmpW protein were resistant to colicin S4.
| |
TEXT |
Colicins are plasmid-encoded
proteins that are synthesized by Escherichia coli and kill
sensitive strains of E. coli and closely related species
(3, 5, 10, 11). The narrow host range is determined by a
highly specific uptake into sensitive cells. Colicins use proteins of
the outer membrane to bind to the E. coli cell. These
proteins include porins and receptors for vitamin B12,
siderophores, and nucleosides (3, 5, 10, 11). Genetic studies have shown that, in addition to the receptor proteins, two
different translocation systems are required for colicin import (6, 7). Group B colicins use the Ton system, which consists of the proteins TonB, ExbB, and ExbD (3), and group A
colicins utilize the Tol system, which consists of the proteins TolA,
TolB, TolQ, and TolR (21). Colicin S4 belongs to the group A
colicins (7). However, Ferber et al. (8)
described E. coli 
mutants supposedly mutated in the
tonB gene that were insensitive to colicin S4. A dependence
on both import systems, Tol and Ton, has only been described, by us,
for a colicin U mutant (17). For this reason and since the
receptor protein of colicin S4 is unknown, we characterized the colicin
S4 genes and the colicin S4 import proteins.
Sequencing of the colicin S4 genes.
The colicin S4 plasmid
pColS4 (Fig. 1) coding for the colicin S4
genes was isolated from strain E. coli K-12, which was
originally obtained from a patient with an uncharacterized infection.
E. coli K-12 was identified as a colicin S4-producing strain
by showing cross-immunity to the colicin S4-producing reference strain
Shigella dispar P15. The colicin S4 determinant was excised
from plasmid pColS4 (Fig. 1) with EcoRI, and the 5.2-kb
restriction fragment was cloned into the EcoRI site of
pBCSK+. E. coli 5K transformed with the resulting plasmid
pHP189 released colicin S4 and was immune to crude cell extracts
obtained from E. coli K-12 (pColS4). Both strands of a
2,809-bp fragment were completely sequenced and revealed three open
reading frames, which we designated csa (colicin S4
activity), csi (colicin S4 immunity), and csl
(colicin S4 lysis). csa and csl have the same
transcription polarity, and csi has the opposite polarity,
an arrangement typical for genes of pore-forming colicins.
csa, csi, and csl encode open reading frames of 499 (Mr = 54,085), 179 (Mr = 20,527), and 51 (Mr = 4,911) amino acid residues,
respectively. The electrophoretic mobility of colicin S4 corresponded
to an Mr of 52,000.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 1.
Arrangement of the csa, csi, and
csl genes on the natural plasmid pColS4. The
EcoRI restriction sites were used for cloning of the colicin
S4 genes.
|
|
The nucleotide sequences flanking the genes of the colicin S4 operon
exhibited high similarity to the sequences of other colicin
determinants. The promoter region of
csa is 97% identical
to the
corresponding region of colicin 10 (Fig.
2). The nucleotide sequences
upstream of
the colicin 5 and K genes display the same high degree
of identity
(Fig.
2). The sequence of the colicin S4 genes shows
a mosaic-like
structure (Fig.
2) like that of other colicins (
15,
16,
20).
Colicin S4 consists of three domains: an N-terminal
domain, which is
responsible for translocation through the outer
membrane; a central
domain, which binds to the receptor in the
outer membrane; and a
C-terminal domain, which contains the lethal
activity.
csa
and the colicin K gene
cka display 97% sequence
identity in
the 5' region; the N-terminal 48 amino acids of colicin
S4 and colicin
K are 94% identical (Fig.
2). Since the N-terminal
domain of the
colicins is responsible for interaction with the
proteins of the
translocation systems (
2), the N-terminal domains
of
colicins S4 and K could be responsible for interaction of these
colicins with the Tol system proteins.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 2.
Comparison (percent sequence identity) of the activity,
immunity, and lysis genes and the encoded proteins to the corresponding
genes (within the bars) and proteins (above the bars) of colicin S4.
|
|
The region of the hydrophobic pore-forming domain of colicin S4 is
nearly identical to that of colicin A (Fig.
3); in colicin
A, it consists of a
central hydrophobic hairpin (helices 8 and
9) surrounded by eight
amphipathic helices (
14). Our previous
assignment of the
immunity-specifying region to the hydrophobic
hairpin (
18)
is supported by the complete cross-resistance to
colicin S4 and colicin
A of cells producing one of the two colicins
(data not shown). The
cross-immunity is also reflected by the
high degree of sequence
identity (77%) between the Csi and Cai
immunity proteins, which stands
in contrast to the low sequence
similarities between the immunity
proteins of other colicins of
the colicin A family (
18).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 3.
Sequence comparison of the C-terminal 200 amino acids of
colicins S4 and A. Asterisks denote identical residues; dashes indicate
similar residues. The hydrophobic segment is boxed, and the amino acids
of the tip of the hydrophobic hairpin are indicated in boldface type.
|
|
The central part of colicin S4 shows no sequence similarities to any
known proteins. This may reflect the unique receptor
specificity of
colicin S4, since in all colicins the receptor
binding domain has been
assigned to the central domain. Colicin
S4 contains a pair of identical
sequences in this central domain
that might have evolved by duplication
of a gene fragment (Fig.
4). The
duplicated sequence may imply that colicin S4 binds to
two copies of
the receptor protein to enter cells. Colicin S4
represents another
example that supports our previous proposal
that colicins evolved by
mixing DNA fragments that encode functional
domains (
15,
16,
20).
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 4.
Sequence duplication in the putative receptor binding
domain of colicin S4. Asterisks denote identical residues; dashes
indicate similar residues.
|
|
Identification of the receptor protein of colicin S4.
To
identify the colicin S4 receptor, colicin S4-resistant mutants of
E. coli 5K were isolated from colonies in zones of growth inhibition on plates onto which a crude extract of colicin S4 had been
applied. Mutations in the tol genes were excluded by testing
the sensitivity of the colicin S4-insensitive mutants to colicins A and
K. Ten colicin S4-insensitive mutants (HP151 to HP160) were highly
sensitive to colicins A and K. To identify the receptor protein of
colicin S4 in the outer membrane, mutant HP151 was transformed with an
E. coli gene bank (2- to 6-kb fragments of the E. coli chromosome ligated into pACYC184; kindly provided by Silke
Patzer of this institute). One out of 800 transformants contained a
plasmid (pHP205) that restored colicin S4 sensitivity to E. coli HP151. The 3.6-kb fragment of pHP205 comprises
yciB to trpB at 28.5 min of the E. coli chromosome. No functions have been assigned to the open
reading frames of this region, except to trpA. By
differential solubilization, two-dimensional gel electrophoresis, tryptic digestion, mass spectrometry of the isolated peptides, and
comparison of the nucleotide sequence with the E. coli
genome sequence, a protein was assigned to the gene product of the
yciD gene and named OmpW (13) on the basis of its
sequence similarity to the OmpW protein of Vibrio cholerae
(9). The subcloned yciD gene (located on a 1-kb
SspI fragment; pHP206) conferred colicin S4 sensitivity to
all resistant mutants (HP151 to HP160), indicating that OmpW is the
receptor or an essential part of the receptor. All E. coli
strains tested with defined mutations in genes coding for outer
membrane proteins were fully sensitive to colicin S4, including the
BL21 omp8 mutant, whose genes (ompF,
ompC, lamB, and ompA) encoding major
outer membrane proteins are deleted (19).
The previously observed TonB dependence of colicin S4 uptake
(
8) probably resulted from a deletion that included
tonB and
yciD. Larger deletions in this region of
the chromosome are frequently
observed (
4).
E. coli H2300, with a deletion extending from
trpB to
tonB (
8a), was insensitive to colicin S4.
E. coli H2300
transformed with pHP206 was sensitive to
colicin S4, which indicates
that
yciD alone complements the
colicin S4-resistant phenotype,
in contrast to a cloned
tonB
gene, which did not restore the colicin
S4 sensitivity of
E. coli H2300. These data demonstrate that uptake
of colicin S4 into
sensitive cells is not
tonB dependent but requires
the
yciD gene
product.
Comparison of outer membranes of the colicin S4-resistant
E. coli HP151 with that of the colicin S4-sensitive
E. coli 5K revealed
that only a weak band present in the outer
membrane fraction of
E. coli 5K was missing in the outer
membrane fraction of
E. coli HP151 (Fig.
5, compare lanes 1 and 2). To increase
the amounts
of the OmpW protein,
yciD was cloned downstream
of the phage T7
promoter and transcribed by the T7 RNA polymerase
(
22). OmpW
from the induced recombinant strain formed two
strong bands; the
band with the lower mobility was probably the OmpW
precursor with
the signal sequence (Fig.
5, lane 4). After induction of
the RNA
polymerase, more OmpW was synthesized, and synthesis of the
OmpF
and OmpC porins and the OmpA protein was strongly suppressed (Fig.
5, compare lanes 3 and 4). Like other outer membrane proteins,
OmpW was
heat modifiable, as revealed by the electrophoretic mobility
of samples
prepared at 37 and 95°C (data not shown).
View larger version (154K):
[in this window]
[in a new window]
|
FIG. 5.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of outer membranes of E. coli 5K (lane 1),
HP151 (lane 2), BL21(pHP211) uninduced (lane 3), and BL21(pHP211)
induced (lane 4). The arrows indicate the 21-kDa OmpW protein and its
presumed 24-kDa precursor.
|
|
Colicin S4 binding to OmpW was assayed by mixing 1 ml of cell
suspension (2 × 10
9 cells per ml) with 1 ml of
colicin S4 solution (10
3 dilution titer). The mixture was
incubated for 20 min at 37°C
and centrifuged, and the amount of
unbound colicin in the supernatant
was determined. OmpW had to be
overproduced to bind 90% of colicin
S4 to cells of
E. coli BL21(pHP211).
Sequence similarities of E. coli OmpW to other
proteins.
OmpW of E. coli shows significant sequence
similarities to OmpW of V. cholerae, a protein that is
highly immunogenic (12). The biological function of the OmpW
protein of V. cholerae is unknown. Like E. coli
OmpW, V. cholerae OmpW is produced in minor amounts under
laboratory conditions (12). Recently it has been shown that
synthesis of Omp21 from Comamonas acidovorans, which is 30%
identical to E. coli OmpW, is enhanced by oxygen depletion (1).
Thorne and Corwin (
23) localized a gene locus between the
trp genes and
tonB of
E. coli that is
involved in the uptake of
aromatic amino acids. Using indole acrylic
acid, they isolated
an
E. coli mutant with a 60 to 80%
decrease in tryptophan uptake.
Therefore, we tested whether OmpW serves
as a pore for tryptophan
uptake across the outer membrane. The colicin
S4-resistant
E. coli mutant HP151 showed no reduction in the
uptake of
3H-labeled tryptophan (data not shown), which
suggests that the
gene locus identified by Thorne and Corwin
(
23) is not
yciD.
Since OmpW belongs to a new
family of outer membrane proteins
with unknown functions
(
1), it will be interesting to investigate
the biological
function of OmpW for the
E. coli cell, apart from
its being
the colicin S4 receptor
protein.
Nucleotide sequence accession number.
The EMBL GenBank
accession number for the colicin S4 sequence is Y18684.
| |
ACKNOWLEDGMENTS |
We thank Karen A. Brune for critical reading of the manuscript.
We thank the Deutsche Forschungsgemeinschaft for financial support (SFB
323, project B1).
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Mikrobiologie/Membranphysiologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany. Phone: (49) 7071 2974620. Fax: (49) 7071 294634. E-mail:
Holger.Pilsl{at}mikrobio.uni-tuebingen.de.
Present address: Microbiology and Molecular Genetics, University of
Texas Medical School, Houston, Texas.
| |
REFERENCES |
| 1.
|
Baldermann, C.,
A. Lupas,
J. Lubieniecki, and H. Engelhardt.
1998.
The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded  -sheet proteins by its sequence and properties.
J. Bacteriol.
180:3741-3749[Abstract/Free Full Text].
|
| 2.
|
Bouveret, E.,
A. Rigal,
C. Lazdunski, and H. Benedetti.
1998.
Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli.
Mol. Microbiol.
27:143-157[Medline].
|
| 3.
|
Braun, V.,
H. Pilsl, and P. Gross.
1994.
Colicins: structures, modes of action, transfer through membranes, and evolution.
Arch. Microbiol.
161:199-206[Medline].
|
| 4.
|
Conkell, M. B., and C. Yanofsky.
1971.
Influence of chromosome structure on the frequency of tonB trp deletions in Escherichia coli.
J. Bacteriol.
105:864-872[Abstract/Free Full Text].
|
| 5.
|
Cramer, W. A.,
J. B. Heymann,
S. L. Schendel,
B. N. Deriy,
F. S. Cohen,
P. A. Elkins, and C. V. Stauffacher.
1995.
Structure-function of the channel-forming colicins.
Annu. Rev. Biophys. Biomol. Struct.
24:611-641[Medline].
|
| 6.
|
Davies, J. K., and P. Reeves.
1975.
Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group B.
J. Bacteriol.
123:96-101[Abstract/Free Full Text].
|
| 7.
|
Davies, J. K., and P. Reeves.
1975.
Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group A.
J. Bacteriol.
123:102-117[Abstract/Free Full Text].
|
| 8.
|
Ferber, D. M.,
J. M. Fowler, and R. R. Brubaker.
1981.
Mutations to tolerance and resistance to pesticin and colicins in Escherichia coli  .
J. Bacteriol.
146:506-511[Abstract/Free Full Text].
|
| 8a.
| Günther, K. Unpublished results.
|
| 9.
|
Jalajakumari, M. B., and P. A. Manning.
1990.
Nucleotide sequence of the gene, ompW, encoding a 22 kDa immunogenic outer membrane protein of Vibrio cholerae.
Nucleic Acids Res.
18:25.
|
| 10.
|
James, R.,
C. Kleanthous, and G. R. Moore.
1996.
The biology of E colicins: paradigms and paradoxes.
Microbiology
142:1569-1580[Free Full Text].
|
| 11.
|
Lazdunski, C. J.,
E. Bouveret,
A. Rigal,
L. Journet,
R. Lloubes, and H. Benedetti.
1998.
Colicin import into Escherichia coli cells.
J. Bacteriol.
180:4993-5002[Free Full Text].
|
| 12.
|
Manning, P. A.,
E. J. Bartowsky,
D. I. Leavesly,
J. A. Hackett, and M. W. Heuzenroeder.
1985.
Molecular cloning using immune sera of a 22-kDal outer membrane protein of Vibrio cholerae.
Gene
34:95-103[Medline].
|
| 13.
|
Molloy, M. P.,
B. R. Herbert,
B. J. Walsh,
M. I. Tyler,
M. Traini,
J.-C. Sanchez,
D. F. Hochstrasser,
K. L. Williams, and A. A. Gooley.
1998.
Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.
Electrophoresis
19:837-844[Medline].
|
| 14.
|
Parker, M. W.,
J. P. M. Postma,
F. Pattus,
A. D. Tucker, and D. Tsernoglou.
1992.
Refined structure of the pore-forming domain of colicin A at 2.4Å resolution.
J. Mol. Biol.
224:639-657[Medline].
|
| 15.
|
Pilsl, H., and V. Braun.
1995.
Novel colicin 10: assignment of four domains to the TonB- and TolC-dependent uptake via the Tsx receptor and to pore formation.
Mol. Microbiol.
16:57-67[Medline].
|
| 16.
|
Pilsl, H., and V. Braun.
1995.
Strong function-related homology between the pore-forming colicins K and 5.
J. Bacteriol.
177:6973-6977[Abstract/Free Full Text].
|
| 17.
|
Pilsl, H., and V. Braun.
1998.
The Ton system can functionally replace the TolB protein in the uptake of a mutated colicin U.
FEMS Microbiol. Lett.
164:363-367[Medline].
|
| 18.
|
Pilsl, H.,
D. majs, and V. Braun.
1998.
The tip of the hydrophobic hairpin of colicin U is dispensable for colicin U activity but is important for interaction with the immunity protein.
J. Bacteriol.
180:4111-4115[Abstract/Free Full Text].
|
| 19.
|
Prilipov, A.,
P. S. Phale,
P. Van Gelder,
J. P. Rosenbusch, and R. Koebnik.
1998.
Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli.
FEMS Microbiol. Lett.
163:65-72[Medline].
|
| 20.
|
Roos, U.,
R. E. Harkness, and V. Braun.
1989.
Assembly of colicin genes from a few DNA fragments. Nucleotide sequence of colicin D.
Mol. Microbiol.
3:891-902[Medline].
|
| 21.
|
Sun, T. P., and R. E. Webster.
1987.
Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.
J. Bacteriol.
169:2667-2674[Abstract/Free Full Text].
|
| 22.
|
Tabor, S., and C. C. Richardson.
1985.
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.
Proc. Natl. Acad. Sci. USA
82:1074-1078[Abstract/Free Full Text].
|
| 23.
|
Thorne, G. M., and L. M. Corwin.
1975.
Mutations affecting aromatic amino acid transport in Escherichia coli and Salmonella typhimurium.
J. Gen. Microbiol.
90:203-216[Abstract/Free Full Text].
|
Journal of Bacteriology, June 1999, p. 3578-3581, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Arnold, T., Zeth, K., Linke, D.
(2009). Structure and Function of Colicin S4, a Colicin with a Duplicated Receptor-binding Domain. J. Biol. Chem.
284: 6403-6413
[Abstract]
[Full Text]
-
Burgess, N. K., Dao, T. P., Stanley, A. M., Fleming, K. G.
(2008). {beta}-Barrel Proteins That Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro. J. Biol. Chem.
283: 26748-26758
[Abstract]
[Full Text]
-
Gualdi, L., Tagliabue, L., Landini, P.
(2007). Biofilm Formation-Gene Expression Relay System in Escherichia coli: Modulation of {sigma}S-Dependent Gene Expression by the CsgD Regulatory Protein via {sigma}S Protein Stabilization. J. Bacteriol.
189: 8034-8043
[Abstract]
[Full Text]
-
White-Ziegler, C. A., Malhowski, A. J., Young, S.
(2007). Human Body Temperature (37{degrees}C) Increases the Expression of Iron, Carbohydrate, and Amino Acid Utilization Genes in Escherichia coli K-12. J. Bacteriol.
189: 5429-5440
[Abstract]
[Full Text]
-
Partridge, J. D., Sanguinetti, G., Dibden, D. P., Roberts, R. E., Poole, R. K., Green, J.
(2007). Transition of Escherichia coli from Aerobic to Micro-aerobic Conditions Involves Fast and Slow Reacting Regulatory Components. J. Biol. Chem.
282: 11230-11237
[Abstract]
[Full Text]
-
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]
-
Hong, H., Patel, D. R., Tamm, L. K., van den Berg, B.
(2006). The Outer Membrane Protein OmpW Forms an Eight-stranded beta-Barrel with a Hydrophobic Channel. J. Biol. Chem.
281: 7568-7577
[Abstract]
[Full Text]
-
Hu, W. S., Li, P.-C., Cheng, C.-Y.
(2005). Correlation between Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium and Expression of Outer Membrane Proteins OmpW and Ail/OmpX-Like Protein, Which Are Regulated by BaeR of a Two-Component System. Antimicrob. Agents Chemother.
49: 3955-3958
[Abstract]
[Full Text]
-
Nandi, B., Nandy, R. K., Sarkar, A., Ghose, A. C.
(2005). Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae. Microbiology
151: 2975-2986
[Abstract]
[Full Text]
-
Nikaido, H.
(2003). Molecular Basis of Bacterial Outer Membrane Permeability Revisited. Microbiol. Mol. Biol. Rev.
67: 593-656
[Abstract]
[Full Text]
-
Weinstock, G. M.
(2001). Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes. J. Bacteriol.
183: 3949-3957
[Abstract]
[Full Text]
-
Mammarappallil, J. G., Elsinghorst, E. A.
(2000). Epithelial Cell Adherence Mediated by the Enterotoxigenic Escherichia coli Tia Protein. Infect. Immun.
68: 6595-6601
[Abstract]
[Full Text]
-
Nandi, B., Nandy, R. K., Mukhopadhyay, S., Nair, G. B., Shimada, T., Ghose, A. C.
(2000). Rapid Method for Species-Specific Identification of Vibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW. J. Clin. Microbiol.
38: 4145-4151
[Abstract]
[Full Text]
Top
Abstract
Text
