(Received for publication, July 6, 1994; and in revised form, November 14, 1994)
From the
A 25-kDa outer membrane protein, induced following treatment of Vibrio cholerae cells with -lactam antibiotics and
constituting about 8-10% of the total outer membrane proteins of
-lactam-resistant mutants, has been purified to homogeneity. It is
a basic (pI 8.5) protein rich in
-sheet structure and is a
homodimer, the monomers being held together by hydrophobic
interactions. The effective hydrophobicity of the protein is low, and a
large part of the protein is exposed on the surface of the outer
membrane. The protein does not have
-lactamase or autolytic
activity and is not a penicillin-binding protein. The Stoke's
radius of the 25-kDa protein (26 A) is comparable to the pore size of
the V. cholerae OmpF-like porin. Proteoliposome swelling assay
showed that the 25-kDa protein might block the pores of OmpF through
which
-lactam antibiotics normally enter the cells. Twenty-two
amino acid residues from the N-terminal end of the 25-kDa protein have
been sequenced, and a 32-mer oligonucleotide probe was synthesized
using the amino acid residues 2-12. This probe was used to
identify the gene encoding the 25-kDa protein. The
-lactam-resistant cells are insensitive to changes in the
osmolarity of the growth medium in contrast to the wild type cells
which exhibit osmoregulation of OmpF and OmpC synthesis. All
-lactam-resistant mutants examined are resistant to novobiocin.
Vibrio cholerae, a non-invasive Gram-negative bacterium is the etiological agent of cholera, a severe diarrheal disease. The bacterium adheres to the epithelial cells of the proximal small intestine and secretes a potent enterotoxin which can cause acidosis and death. The outer membrane of enteropathogens is one of the several factors involved in the interaction between the bacterium and the epithelial surface and in conferring resistance to bile salts as well as to host defense factors such as lysozyme and leukocyte proteins (Nikaido and Vaara, 1985).
In the course of our studies on the cell
surface of a hypertoxinogenic strain 569B of V. cholerae for a
better understanding of the host-bacterium interactions, several
atypical features of the cell surface emerged. These cells are highly
sensitive to a wide range of chemicals, particularly hydrophobic
compounds and neutral and anionic detergents. This is partly because of
the presence of phospholipids in the outer leaflet of the outer
membrane (Paul et al., 1992). Normally Gram-negative bacteria
with smooth type lipopolysaccharide (LPS) ()in the outer
membrane are resistant to hydrophobic compounds and detergents and do
not have phospholipids exposed on the surface, thereby restricting the
entry of non-polar molecules by diffusion (Sukupolvi and Vaara, 1989).
The LPS of V. cholerae contained O-antigenic sugars
and has less negative charge compared to those in other organisms (Paul et al., 1992).
The interaction of V. cholerae cells with -lactam antibiotics, which inhibit the final step
of murein biosynthesis, has recently been examined primarily for two
reasons. First, although
-lactam antibiotics have, so far, not
been used as a protective drug for cholera, there are a number of
reports describing that these antibiotics can preferentially inhibit
the adherence of enteropathogens by disorganizing the
membrane-associated machinery responsible for fimbrial attachment
(Sugarman and Donta, 1979; Hales and Ames, 1985; Schifferli and
Beachey, 1988). It would be intriguing to examine whether the
-lactam group of antibiotics can be effectively used as a drug
that will exert control over the spread of infection in the intestine.
Second, the murein network of V. cholerae is weak, and the
cells lyse rapidly in hypotonic medium as well as in the presence of
chelating agents such as Tris and EDTA (Lohia et al., 1984,
1985). Purified outer membrane can be directly isolated from whole
cells by treatment with protein denaturants such as urea at room
temperature (Lohia et al., 1984). Unlike other Gram-negative
organisms, V. cholerae cells are equally sensitive to
penicillin and ampicillin and in general more susceptible than Escherichia coli to most of the
-lactam antibiotics
(Sengupta et al., 1992). Prolonged treatment with
-lactam
antibiotics produced cells resistant to these antibiotics (Sengupta et al., 1992), and this represents an example of adaptive
mutation (Cairns et al., 1988). It has been reported that
except in the relative proportion of some low molecular weight
penicillin-binding proteins, the penicillin-binding protein profiles of
-lactam-resistant and wild type cells were identical (Sengupta et al., 1990; 1992). Cells resistant to a
-lactam
antibiotic exhibited broad cross-resistance to other
-lactam
antibiotics. A new 12-kDa outer membrane protein was detected in
SDS-PAGE from both
-lactam-resistant cells and wild type cells
grown in presence of
-lactam antibiotics (Sengupta et
al., 1992). The protein constituted about 8-10% of the total
outer membrane proteins of
-lactam-resistant cells. Since the
-lactam-induced protein was found to be associated with every type
of
-lactam resistant mutant of V. cholerae, studies were
undertaken to investigate its role in conferring
-lactam
resistance. The protein has been purified to homogeneity and
characterized. Results presented here suggest that the
-lactam-induced protein, a homodimer of 12-kDa monomers, might
block the porin OmpF through which
-lactam antibiotics normally
enter the cell.
The outer membrane along with the associated peptidoglycan layer was
isolated from the crude cell envelope by treatment with 1% (w/v)
Sarkosyl NL-97 for 30 min at 25 °C followed by centrifugation at
105,000 g for 1 h. The inner membrane contamination in
the outer membrane preparation was estimated by measuring the
cytochrome b content which is located in the inner membrane.
All the outer membrane preparations used had less than 5% inner
membrane contamination. For extracting the
-lactam-induced protein
from the outer membrane, outer membrane from
-lactam-resistant
cells was suspended in 2.5 M LiCl solution in water. The
suspension was briefly homogenized, centrifuged at 105,000
g for 60 min at 4 °C, and the supernatant was collected.
This was repeated twice, the supernatants were pooled, and then stored
at -20 °C. Prolonged storage in the presence of LiCl was
avoided because this led to extraction of other outer membrane
proteins.
Protein was assayed either by the method of Markwell et al. (1978) or Lowry et al.(1951) with bovine serum albumin as standard. For some experiments, the protein was estimated using Bio-Rad protein assay dye reagent concentrate (Bio-Rad).
The penicillin-binding property of
the protein was examined by suspending 150 µg of cell envelope in
50 µl of 50 mM phosphate buffer (pH 7.0) containing I-PenX as described previously (Sengupta et al.,
1990). The membrane suspension was incubated at 37 °C for 10 min.
The labeling was terminated by adding 1% (w/v) Sarkosyl NL-97 and
centrifuged at 105,000
g for 1 h. The supernatant and
the pellet were analyzed by SDS-PAGE followed by autoradiography.
Figure 1:
Chromatograms of 25-kDa
-lactam-induced protein at different steps of purification. a, elution profile of the LiCl extract of the outer membrane
of V. cholerae penG
mutants from a Sephadex G-75
column (1
90 cm). The column was eluted with 20 mM Tris-Cl (pH 8.0) containing 20 mM EDTA and 0.4 M LiCl, and 0.8 ml fractions were collected at a flow rate of 15
ml/h. The column was calibrated using hemoglobin (64,000), ovalbumin
(45,000), chymotrypsinogen A (25,000), and cytochrome c (12,500). b, elution profile of the 25-kDa protein from a size
exclusion HPLC column. 10 µg of the protein isolated from peak III (a) was loaded onto a TSK G-3000 SW SE-HPLC column (LKB) and
was eluted with 20 mM sodium phosphate buffer (pH 7.2) at a
flow rate of 0.5 ml/min. The major peak (V
= 13 ml) was pooled and was supplemented with 0.4 M LiCl for stabilization. Minor protein peaks of higher
molecular weight (V
= 5-6
ml) are not shown in the figure. Inset A, dependence of
molecular weight on elution volume (V
).
The HPLC column was calibrated with ferritin (450,000), catalase
(240,000), ovalbumin (45,000), chymotrypsinogen A (25,000), and
cytochrome c (12,500). The arrow indicates the
position of the
-lactam-induced protein. Inset B,
dependence of Stoke's radius (
) on V
. The column was calibrated with
catalase (
= 52 Å), hemoglobin
(
= 33.2 Å), ovalbumin (
= 31.2 Å), and cytochrome c (
= 17 Å) (Corbette and Roche, 1984). The arrow indicates the position of the
-lactam induced protein. c, reverse phase HPLC of the 25-kDa protein. 20 µg of the
purified protein was loaded on a C
-µ Bondapak RP-HPLC
column (3.9
300 mm, Waters) and was eluted with a linear
gradient of solvent A as described under ``Experimental
Procedures.''
Figure 2:
SDS-PAGE of the 25-kDa protein at
different stages of purification. Electrophoresis was carried out in a
10-18% gradient of polyacrylamide containing 0.1% SDS. A, protein profile of the outer membrane of V. cholerae
penG mutant (lane c), LiCl extract of the
outer membrane (lane b), and Sephadex G-75 purified fraction
of the LiCl extract (lane a). The numbers in the
margin indicate molecular mass in kDa. B, lane a:
molecular weight markers (in descending order), phosphorylase b (94,000); bovine serum albumin (67,000); ovalbumin (45,000);
carbonic anhydrase (30,000); trypsin inhibitor (20,100); and
-lactalbumin (14,000); Lane b, purified
-lactam-induced protein.
The amino acid composition of the 25-kDa protein was determined (Table 1). No sulfur-containing amino acid could be detected. The distribution of hydrophilic and hydrophobic amino acids is comparable. The number of acidic amino acid residues in the 25-kDa protein is 13 which exceeds the number of basic amino acid residues. Considering that the pI of the protein is 8.5, it is possible that the majority of the aspartic and glutamic acids are present in the amide form.
The CD spectra of the
25-kDa protein was monitored in the far UV region (190-250 nm) in
presence and absence of 0.4 M LiCl. In absence of salt (Fig. 3A), the spectra showed maximum ellipticity at
218 nm characteristic of proteins having significant amounts of
-sheeted structure. The
-helix content of the 25-kDa protein
was estimated to be 6-7%. In the presence of 0.4 M LiCl,
the
-sheeted structure is destroyed, and the protein assumes
largely random coil conformation (Fig. 3B). The
transition is reversible. The
-sheeted structure of the protein is
restored upon removal of the salt.
Figure 3: CD spectra of the 25-kDa protein. A, the protein (50 µg/ml) in 20 mM potassium phosphate buffer (pH 7.0) was scanned in the far UV region (190-250 nm) at 20 °C. B, spectrum under identical conditions except that the buffer contained 0.4 M LiCl. Spectra presented are after base-line correction.
To determine the minimum concentration of LiCl at which the protein remains in soluble form, the protein was kept at 25 °C for 16 h in the presence of different concentrations of LiCl, centrifuged, and the supernatants were analyzed by size exclusion HPLC and SDS-PAGE. At LiCl concentrations below 0.4 M, the protein remained in the soluble form only for a short time after which it formed aggregates. The net amount of the protein was reduced upon prolonged storage. There was, however, no proteolytic degradation during holding indicating that the purified protein is free from proteases.
Size-exclusion HPLC in buffers ranging from pH 3 to 8.8 containing different concentrations of NaCl or KCl (0.5-2.0 M used) failed to dissociate the dimer. Hence, either the solvent protein interaction or the changes in the electrostatic interactions between the subunits failed to monomerize the protein ruling out electrostatic interaction as the major force involved in dimerization.
Using ANS as extrinsic fluorescent probe,
the hydrophobicity of the 25-kDa protein was estimated. Compared to the
weak emission in aqueous solution, about 9-10-fold increase in
fluorescence intensity was observed when the ANS-25-kDa complex was
examined at 520 nm (, 375 nm). A blue shift of 5 nm
of emission maxima (520-515 nm) was detected (Fig. 4a). The addition of low concentrations of ANS
resulted in linear increase in quantum yield leading to a saturation
value exhibiting the typical hyperbolic saturation kinetics (Fig. 4b). The number of hydrophobic sites in the
25-kDa protein accessible to ANS was estimated to be 1.0 from the
Scatchard plot (Fig. 4c) obtained by titrating a fixed
concentration of the 25-kDa protein with increasing concentrations of
ANS. The dissociation constant for the ligand-protein interaction was
0.2 mM as derived from the Hill plot. From the slope of the
linear part of ANS fluorescence with increasing concentration of the
protein, the hydrophobic coefficient of the 25-kDa protein was
estimated and compared with those of several standard proteins as
described under ``Experimental Procedures.'' The hydrophobic
coefficient of the 25-kDa protein was found to be about -0.02
compared to 1.31 for bovine serum albumin. This is in agreement with
the amino acid composition of the protein.
Figure 4: a, enhancement of ANS fluorescence in presence of the 25-kDa protein. The protein (50 µg) in 700 µl of 20 mM sodium phosphate buffer (pH 7.2) was treated with 150, 300, and 500 µM of ANS in water (in ascending order). Excitation wave length was 375 nm. The lowest curve represents emission spectrum of 500 µM free ANS in water. b, determination of dissociation constant of protein-ANS interaction. The enhancement of fluorescence intensity with increase of ANS concentration was recorded at 515 nm. The inset represents the double-reciprocal plot of the hyperbolic kinetics of the fluorescence data. c, Scatchard plot derived from b.
Figure 5:
Radioiodination of the outer membrane of
wild type and penG mutants. Cells were iodinated
as described under ``Experimental Procedures,'' and outer
membrane was isolated and analyzed by 15% SDS-PAGE. Lane a, penG
mutants; lane b, wild type cells.
The numbers in the margin indicate molecular size in
kDa.
It has been reported that penicillin can
damage bacterial cell walls by inducing autolysins (Prestidge and
Pardee, 1957). To examine whether the 25-kDa protein is a
-lactam-induced autolysin, B. megaterium protoplasts were
exposed to crude lysates of
-lactam-resistant V. cholerae cells, and the absorbance was monitored at different times at 670
nm. There was no detectable lysis of B. megaterium protoplasts. B. megaterium protoplasts are stable for at
least 1 h in sucrose-phosphate buffer (pH 7.0).
Penicillin-binding proteins have been reported for both wild type and penicillin-resistant mutants of V. cholerae (Sengupta et al., 1990, 1992). None of the penicillin-binding proteins either of the wild type or the resistant mutants had a molecular size of 12 kDa in SDS-PAGE. It has been shown that all the penicillin-binding proteins of V. cholerae are located in the inner membrane, while the 25-kDa protein is an outer membrane protein.
A comparison of the uptake of crystal violet
in wild type and -lactam-resistant cells suggested reduction in
the permeability of the dye in the mutant cells. However, because of
the nonspecificity of the crystal violet uptake these results could not
be considered conclusive. To examine whether change in permeability is
indeed responsible for
-lactam resistance in the mutant cells, the
more sensitive liposome swelling assay (Nikaido et al., 1991)
was carried out.
Liposomes were prepared as described under
``Experimental Procedures'' and purified OmpF-like protein of V. cholerae, which is a trimer of monomer size 38 kDa, ()was incorporated into these liposomes. The V. cholerae OmpF-like protein allowed arabinose (Fig. 6a) to
enter the liposomes but not stachyose (Fig. 6b) as
detected spectrophotometrically at 400 nm. When the 25-kDa protein was
incorporated into the liposomes and the swelling assay was performed
with different sugars, even arabinose did not enter the liposome (Fig. 6e) showing that the 25-kDa protein itself does
not have any porin-like activity. No swelling of the liposomes occurred
even when arabinose was used for assay (Fig. 6c) when
the 25-kDa protein was added to the liposomes in which the OmpF like
protein of V. cholerae was embedded. However, as soon as water
was added to the liposome-OmpF-25 kDa complex, liposomes swelled almost
instantaneously (Fig. 6g) indicating that there is no
artifact in the experimental conditions. The results of these in
vitro experiments strongly suggest that the 25-kDa protein blocks
the pore of OmpF and does not allow the entry of
-lactam
antibiotics into the cell. Whether the change in permeability is due to
the blocking of the pore as such or through reorganization of the porin
molecules remains to be investigated. Incidentally, the pore size of
OmpF-like proteins and the Stoke's radius of the 25-kDa protein
(26 Å) reported here are comparable.
Figure 6: Liposomes were prepared as described under ``Experimental Procedures'' and the V. cholerae or E. coli OmpF or the 25-kDa protein was incorporated into the liposomes. Permeability of arabinose and stachyose through these proteoliposomes was monitored by measuring the change in absorbance at 400 nm with time. Liposomes with V. cholerae OmpF: a, arabinose (48 mM); b, stachyose (65 mM); c, 25-kDa protein + arabinose; g, 25-kDa protein + arabinose + water. Liposomes with E. coli OmpF: d, arabinose; h, stachyose. Liposome with 25-kDa protein: e, arabinose; f, stachyose. Decrease in absorbance due to the swelling of liposome was taken as a measure of permeability.
Figure 7:
SDS-PAGE of cell lysates of novpenG
mutant (lane a), and nov
mutant (lane b) of V. cholerae 569B. Numbers in the
margin indicate molecular mass in kDa.
Figure 8:
SDS-PAGE of the outer membrane proteins of
569B (lanes a-d) and penG mutants of 569B (lanes e-h) grown in nutrient broth containing 2, 1, 0.5, and
0% NaCl, respectively. The numbers in the margin indicate
molecular mass in kDa.
A 32-mer oligonucleotide was synthesized using the amino acid residues 2-12 of the 25-kDa protein. The oligomer was 5`-end labeled and used as a probe in Southern blot hybridization of HindIII-digested V. cholerae genomic DNA. A single band in the 1 kb region of the gel lighted up in the autoradiogram. A minibank was prepared in pUC18 by eluting DNA from this region of the gel, and the recombinant clones were screened using the 32-mer probe by dot-blot hybridization. A recombinant clone carrying a 0.8-kb V. cholerae DNA fragment has been identified. Dot-blot hybridization using the 0.8-kb DNA fragment as the probe revealed that all strains of V. cholerae examined so far belonging to different serovars and biotypes have this gene. E. coli cells used as control did not hybridize with the 0.8-kb DNA fragment.
The outer membrane of the hypertoxinogenic strain 569B of V. cholerae has smooth type LPS (Chakrabarti and Chatterjee,
1984). Sensitivity of these cells to -lactam antibiotics suggests
that the outer membrane with its complement of smooth type LPS fails to
restrict permeation by these antibiotics. One of the remarkable
features in the interaction of
-lactam antibiotics with V.
cholerae is the emergence of resistant cells at a high frequency
and represents an example of adaptive mutation (Sengupta et
al., 1992). The 25-kDa outer membrane protein described in this
article was induced within 3 h following the exposure of V.
cholerae cells to
-lactam antibiotics and was detected as a
12-kDa protein in SDS-PAGE. Of all
-lactam-resistant mutants
isolated and analyzed so far, this protein is synthesized
constitutively. To investigate whether this protein has any direct role
in conferring resistance to
-lactam antibiotics in V.
cholerae, the protein was purified to homogeneity and its possible
function(s) searched for.
The results presented here show that the
-lactam-induced protein is a dimer with identical subunits of 12
kDa. Unlike most outer membrane proteins which are acidic, the 25-kDa
protein is basic (pI 8.5). The hydrophobic coefficient of the protein
is low, and the amino acid composition shows dominance of hydrophilic
residues over the hydrophobic ones. A large part of the 25-kDa protein
is exposed on the surface of the outer membrane.
The presence of a
hydrophobic core in the 25-kDa protein might explain its structural
stability. High salt concentration destroyed the -sheet structure
and removal of salt, at least in vitro, restored the secondary
structure. The core hydrophobic region might be involved in dimer
formation through interactions with LPS of the outer membrane located
at the outer leaflet in Gram-negative organisms. E. coli porins are known to refold into
-sheeted conformation through
interactions with LPS (Sen and Nikaido, 1991).
The 25-kDa protein can be selectively extracted by LiCl solution and not by NaCl or KCl. The optimum time of LiCl extraction is about 20-30 min. It is possible that the 25-kDa protein in contact with other Omps might occupy a crevice from where it can be easily extracted by LiCl compared to other outer membrane-spanning proteins which require longer time for extraction. The probability of the presence of hydrophobic transmembrane segments in the 25-kDa protein is expected to be low because of the dominance of hydrophilic amino acid residues.
While
searching for the role of the 25-kDa protein in conferring -lactam
resistance to V. cholerae cells, it was observed that the
protein is not a
-lactamase itself nor can it induce chromosomal
-lactamase activity. The protein is not an autolysin or a
penicillin-binding protein. The liposome swelling assay described here
suggested that the 25-kDa protein possibly conferred
-lactam
resistance to V. cholerae cells by interfering with the entry
of the antibiotic through the porin OmpF through which
-lactam
antibiotics normally enter the cell. Considering that the Stoke's
radius of the protein (26 Å) and the pore size of V. cholerae OmpF are comparable and that the nature of charge on the two
proteins are opposite, interaction between OmpF and 25-kDa protein can
lead to blocking of the pores and thereby restrict the passage of the
antibiotic.
An 18-kDa cytosolic protein is coinduced with the 25-kDa
protein following exposure of cells to -lactam antibiotics. This
protein is not a precursor of the 12-kDa monomer. The role of this
protein, if any, in conferring
-lactam resistance to V.
cholerae is not known.
Surprisingly, all -lactam-resistant
mutants examined so far are resistant to inhibitors of the B-subunit of
DNA gyrase, like novobiocin and coumermycin. Preliminary studies using
a reporter plasmid have indicated that the superhelix density of
plasmids isolated from the mutant cells is less than those isolated
from the wild type cells. Dot-blot hybridization using a 32-mer
oligonucleotide comprising part of the DNA segment encoding the 25-kDa
protein has shown that all strains of V. cholerae belonging to
different serovars and biotypes examined possess the gene. Whether
local change in superhelix density in the DNA is responsible for the
induction of the 25-kDa protein needs to be investigated. Evidence has
now accumulated that changes in the superhelix density of DNA are
responsible for osmoregulation of OmpF and OmpC in E. coli.
The synthesis of the 40- and 38-kDa outer membrane proteins of V.
cholerae are regulated by the osmolarity of the growth medium
(Lohia et al., 1985). The lack of osmoregulation of these
proteins in
-lactam-resistant mutants along with their novobiocin
resistance also points toward the possibility of a change in the
superhelix density leading to the derepression of the 25-kDa protein
synthesis in the mutant cells. Although indirectly, this hypothesis is
supported by the isolation of novobiocin-resistant mutants which were
also
-lactam-resistant.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |