©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 25-kDa -Lactam-induced Outer Membrane Protein of Vibrio cholerae
PURIFICATION AND CHARACTERIZATION (*)

(Received for publication, July 6, 1994; and in revised form, November 14, 1994)

Amitabha Deb (§) Debasish Bhattacharyya (1) Jyotirmoy Das (¶)

From the Department of Biophysics and Division of Protein Engineering, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A 25-kDa outer membrane protein, induced following treatment of Vibrio cholerae cells with beta-lactam antibiotics and constituting about 8-10% of the total outer membrane proteins of beta-lactam-resistant mutants, has been purified to homogeneity. It is a basic (pI 8.5) protein rich in beta-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 beta-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 beta-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 beta-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 beta-lactam-resistant mutants examined are resistant to novobiocin.


INTRODUCTION

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) (^1)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 beta-lactam antibiotics, which inhibit the final step of murein biosynthesis, has recently been examined primarily for two reasons. First, although beta-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 beta-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 beta-lactam antibiotics (Sengupta et al., 1992). Prolonged treatment with beta-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 beta-lactam-resistant and wild type cells were identical (Sengupta et al., 1990; 1992). Cells resistant to a beta-lactam antibiotic exhibited broad cross-resistance to other beta-lactam antibiotics. A new 12-kDa outer membrane protein was detected in SDS-PAGE from both beta-lactam-resistant cells and wild type cells grown in presence of beta-lactam antibiotics (Sengupta et al., 1992). The protein constituted about 8-10% of the total outer membrane proteins of beta-lactam-resistant cells. Since the beta-lactam-induced protein was found to be associated with every type of beta-lactam resistant mutant of V. cholerae, studies were undertaken to investigate its role in conferring beta-lactam resistance. The protein has been purified to homogeneity and characterized. Results presented here suggest that the beta-lactam-induced protein, a homodimer of 12-kDa monomers, might block the porin OmpF through which beta-lactam antibiotics normally enter the cell.


EXPERIMENTAL PROCEDURES

Materials

1-Anilino 8-naphthalene sulfonic acid, sodium salt (1-ANS), dansyl chloride, Tris, HCl, LiCl, all electrophoretic reagents, standard proteins, DNase, and RNase were purchased from Sigma. Sarkosyl NL97 was purchased from Geigy Industrial Chemicals. Radiochemicals used were either from Bhabha Atomic Research Centre, Trombay, India or from Amersham Inc. HPLC buffers were degassed and purged with nitrogen to prevent oxidation of proteins particularly at high or low pH.

Bacterial Strains and Growth Conditions

The hypertoxinogenic strain 569B of V. cholerae (biotype, classical; serotype, Inaba) which carries a streptomycin resistance marker (50 µg/ml) was used in this study. Cells were grown in a gyratory shaker at 37 °C in nutrient broth (NB) containing 0.18 M NaCl at pH 8.0 and maintained as described previously (Roy et al., 1982). The E. coli strains C-600, K-12, and DH-5alpha were grown in LB at pH 7.4. Cell viability was assayed as colony forming units on nutrient agar plates, and growth was assayed by measuring absorbance at 585 nm (A). 8 times 10^8 colony forming units/ml corresponded to A of 1.0. The V. cholerae strains were stored at room temperature and the E. coli strains at 4 °C.

Preparation of Crude Cell Envelope and Other Membranes

V. cholerae cells at different stages of growth were harvested by centrifugation (6,000 times g, 5 min) and suspended in 50 mM potassium phosphate buffer (pH 7.2) containing 5 mM phenylmethylsulfonyl fluoride. Cells were disrupted in an ultrasonic disintegrator by a number of 30-s pulses at 55 watts until the cell suspension became translucent. The cell lysate was treated with DNase and RNase (100 µg/ml) for 20 min at 37 °C, and the unbroken cells were removed by centrifugation. The cell lysate was centrifuged in a Beckman Ti50 rotor for 30 min at 105,000 times g. The supernatant was discarded, the pellet constituting the crude cell envelope was washed with cold phosphate buffer, suspended in the same buffer, and kept at -20 °C.

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 times 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 beta-lactam-induced protein from the outer membrane, outer membrane from beta-lactam-resistant cells was suspended in 2.5 M LiCl solution in water. The suspension was briefly homogenized, centrifuged at 105,000 times 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 Purification

The supernatant of LiCl extract was passed through a Sephadex G-75 column (1 times 90 cm) equilibrated with 20 mM Tris-Cl (pH 8.0) containing 20 mM EDTA, and 0.4 M LiCl. 0.8-ml fractions were collected, and absorbance at 280 nm of each fraction was measured and analyzed by SDS-PAGE. The fractions containing the 12-kDa protein in SDS-PAGE were pooled and concentrated using Centrikon-3 (Amicon Inc.) concentrator. The trace amount of high molecular mass impurities which coeluted with the 12-kDa fractions from gel filtration was removed by size exclusion HPLC using a TSK G3000 SW column (0.75 times 30 cm, Pharmacia). The column was equilibrated with 20 mM sodium phosphate buffer (pH 7.2) and was precalibrated with standard gel filtration markers. The major protein band corresponding to a molecular mass of 25 kDa was pooled and stored in the presence of 0.4 LiCl. To check the purity of the protein about 20 µg of the protein was analyzed by reverse phase high performance liquid chromatography (RP-HPLC) (µ-bondapak C(18), 3.9 times 300 mm; Waters), and the chromatogram was developed using a linear gradient of the solvents A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in 100% acetonitrile) in 40 min. The RP-HPLC analysis was carried out in a HP-1090 liquid chromatograph (Hewlett Packard, model 1090A) with an integrator (model 3392A, Hewlett Packard).

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).

Electrophoresis

SDS-PAGE was carried out following the method of Laemmli(1970) except that 10-18% gradient gels were used. For a better resolution of the OmpC- and OmpF-like proteins of V. cholerae, the method of Lugtenberg et al.(1975) was used. Electrophoresis was carried out at a constant current (15 mA/cm) and the gel stained with either 0.25% Coomassie Blue or autoradiographed for resolving radiolabeled proteins.

Isoelectric Focusing

Isoelectric focusing was carried out in nondenaturing slab gels of 12.5% polyacrylamide containing ampholines (pH 3.0-10.0, Pharmacia) (O'Farell, 1975). The pH gradient was calibrated using isoelectric point markers (Pharmacia). The gels were fixed (12% trichloroacetic acid, 30% methanol, and 3.5% sulfosalicylic acid) and stained by silver staining (Sammons et al., 1981; Schoenle et al., 1984).

Amino Acid Analysis

For amino acid analysis, 20 µmol of the protein was hydrolyzed with 6 N HCl at 110 °C for 24 h in a sealed glass tube flushed with nitrogen with traces of phenol.

Spectroscopic Methods

For CD spectroscopy the purified 25-kDa protein in 20 mM potassium phosphate buffer (pH 7.0) (with or without 0.4 M LiCl) was taken in quartz cuvettes of 1-cm path length. CD spectra between 190-250 nm were recorded using a JASCO V500C spectropolarimeter at 20 °C. Molar ellipticity was calculated using the equation, [] = []/[10 {MRC}.l ] where [] is the molar ellipticity in degrees Cm^2/dmol, [] is ellipticity recorded by the instrument in millidegrees, MRC is the mean residue concentration of the protein and is equal to the number of amino acid residues times molar concentration of the protein and l is the path length in centimeters. The calculations for alpha-helix content was done according to the relation, [] = -30,300 f(H) - 2340 (Chen and Yang, 1971), where [] is molar ellipticity at 222 nm and f(H) (0 leq f(H) < 1) is the fraction of alpha-helix content.

Interaction with ANS

50 µg of the 25-kDa protein was treated with increasing concentrations of ANS (150-500 µM), and the interaction was monitored spectrofluorimetrically in a Hitachi F4020 spectrofluorometer with a standard 700-µl cuvette using an excitation wavelength of 375 nm. The emission spectra was obtained by scanning the wavelength from 400-600 nm. Whenever necessary, the concentration of the protein or ANS was varied. The number of binding sites were calculated from the Scatchard plot (Scatchard, 1949). The effective hydrophobicity was determined by titrating different concentrations of protein with fixed concentration of ANS and monitoring the fluorescence intensity (Kato and Nakai, 1980). The initial slope (S(o)) of the titration curve was taken as a measure of hydrophobicity.

Radioiodination

0.4 ml of cells in the logarithmic phase of growth in phosphate-buffered saline (pH 7.5), containing 400 µCi of I (specific activity 17.0 Ci/mg) and 200 µg of freshly prepared chloramine T were incubated at room temperature for 30 min. The reaction was stopped by adding 0.15 ml of sodium metabisulfate (2.4 mg/ml in 0.05 M phosphate buffer (pH 7.5)), and residual iodide was diluted by adding 0.2 ml of KI (10 mg/ml in 0.05 M phosphate buffer (pH 7.5)). Cells were centrifuged at 10,000 times g for 10 min, and the pellet was suspended in phosphate-buffered saline, pH 7.5. The outer membrane was isolated and analyzed by SDS-PAGE followed by autoradiography.

Sequencing of N-terminal End

To 2 mg of the 25-kDa protein in 0.5 ml of 0.2 M NaHCO(3) (pH 9.0), 0.1 ml of dansyl chloride (10 mg/ml) in acetone was added. The reaction mixture was incubated in the dark at 37 °C for 16 h, dialyzed exhaustively, and dried in vacuum. The dansyl chloride-protein complex was hydrolyzed with 1 ml of 5.7 N HCl at 105 °C for 22 h. After removal of HCl, the hydrolysate was taken in a few drops of 50% pyridine and was subjected to thin layer chromatography. The standard dansylated amino acids used as markers were spotted on the same plate. The chromatography was performed using either benzene/pyridine/acetic acid (16:4: 1) or n-butanol/pyridine/acetic acid/water (30:20:6:24) solvents in the dark. The plates were dried and fluorescence was viewed under UV light. Sequencing was done in an automated amino acid sequencer (Applied Biosystem, model 473A).

Biological Assays

The beta-lactamase activity was assayed iodometrically (Tai et al., 1985). Under the conditions used, the limit of detection was 0.1 unit of the enzyme/ml. The autolytic activity was assayed using the method of Prestidge and Pardee (1957) using Bacillus megaterium protoplasts. The protoplasts were stable for 1 h.

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 times g for 1 h. The supernatant and the pellet were analyzed by SDS-PAGE followed by autoradiography.

Liposome Swelling Assay

This was carried out following the method of Nikaido et al.(1991). To prepare liposomes or proteoliposomes, 2 ml of benzene was added to 2.4 µmol of acetone-washed egg phosphatidylcholine and 0.2 µmol of diacetyl phosphate and dried under a stream of nitrogen. To the dried sample 2 ml of diethyl ether was added and dried in nitrogen atmosphere. The dried sample was suspended either in water or in the solution of desired protein in water. The sample was dried and finally suspended in 0.6 ml of 17% (w/v) dextran T-40 in 5 mM Tris-Cl (pH 7.5).


RESULTS

beta-Lactam-induced Protein

The 12-kDa outer membrane protein detected in SDS-PAGE following treatment of V. cholerae cells with beta-lactam antibiotics can be selectively extracted by treatment of whole cells or isolated outer membrane with LiCl (Sengupta et al., 1992). Taking advantage of this observation, the protein was purified to homogeneity. Gel filtration through Sephadex G-75 of the LiCl extract of the outer membrane of beta-lactam resistant mutant of V. cholerae revealed three protein fractions (Fig. 1a). Peak III contained primarily the 25-kDa protein which was resolved as a 12-kDa protein in SDS-PAGE with minor contamination of the 40-kDa outer membrane protein (Fig. 2A, lane a). The fractions 35-40 of peak III were pooled, concentrated, and analyzed by size exclusion HPLC (Fig. 1b), whereby the 40-kDa contaminant was removed. A single peak appeared (retention time = 13.52 min) whose molecular mass was estimated to be 25 kDa (Fig. 1b, upper inset). The homogeneity of the protein was confirmed by RP-HPLC (Fig. 1c) and by 10-18% gradient SDS-PAGE (Fig. 2B, lane b). In SDS-PAGE, the protein is resolved as a 12 kDa band indicating that the beta-lactam-induced protein is a homodimer. The Stoke's radius of the protein was found to be 26 Å (Fig. 1b, lower inset) with frictional coefficient (f/f(o)) equal to 1 suggesting that the protein is spherical in nature. The possibility of any glycosylic linkages in the 25-kDa protein was eliminated using periodic acid Schiff's base staining (Zacharius et al., 1969). The isoelectric point of the 25-kDa protein was 8.5, and the gels showed a single band confirming the purity of the protein (data not shown). In contrast to majority of the outer membrane proteins which are acidic in nature, the 25-kDa protein is basic.


Figure 1: Chromatograms of 25-kDa beta-lactam-induced protein at different steps of purification. a, elution profile of the LiCl extract of the outer membrane of V. cholerae penG^r mutants from a Sephadex G-75 column (1 times 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 beta-lactam-induced protein. Inset B, dependence of Stoke's radius ((s)) on V. The column was calibrated with catalase ((s) = 52 Å), hemoglobin ((s) = 33.2 Å), ovalbumin ((s) = 31.2 Å), and cytochrome c ((s) = 17 Å) (Corbette and Roche, 1984). The arrow indicates the position of the beta-lactam induced protein. c, reverse phase HPLC of the 25-kDa protein. 20 µg of the purified protein was loaded on a C(18)-µ Bondapak RP-HPLC column (3.9 times 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^r 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 alpha-lactalbumin (14,000); Lane b, purified beta-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 beta-sheeted structure. The alpha-helix content of the 25-kDa protein was estimated to be 6-7%. In the presence of 0.4 M LiCl, the beta-sheeted structure is destroyed, and the protein assumes largely random coil conformation (Fig. 3B). The transition is reversible. The beta-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.

Interactions Involved in Dimer Formation

To investigate whether the dimers of 12-kDa monomeric units of the 25-kDa protein are formed by covalent linkages, the 25-kDa protein was analyzed in SDS-PAGE in the presence and absence of 2-mercaptoethanol in the sample buffer. In both the cases, only the 12-kDa protein could be resolved in the gel with no trace of the 25-kDa protein. This observation excludes the possibility of -S-S- linkages between the two monomers to form the dimer.

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.



Localization of the 25-kDa Protein

To examine whether the 25-kDa protein is exposed in the outer membrane, V. cholerae cells were labeled with chloramine T and I as described under ``Experimental Procedures.'' The outer membrane was isolated and analyzed by SDS-PAGE followed by autoradiography. The 12-kDa monomer of the 25-kDa protein was intensely labeled with I (Fig. 5, lane a). Thus, a major part of the 25-kDa protein is exposed on the outer leaflet of the outer membrane. The other outer membrane proteins that are known to be exposed, namely 40, 38, 35, 27, and 23 kDa, were also labeled (Fig. 5).


Figure 5: Radioiodination of the outer membrane of wild type and penG^r mutants. Cells were iodinated as described under ``Experimental Procedures,'' and outer membrane was isolated and analyzed by 15% SDS-PAGE. Lane a, penG^r mutants; lane b, wild type cells. The numbers in the margin indicate molecular size in kDa.



Searching for Function(s) of the 25-kDa Protein

No plasmid could be detected in either the wild type or in the beta-lactam-resistant mutants thereby eliminating the possibility of induction of plasmid borne beta-lactamase function which might have been responsible for conferring resistance to beta-lactam antibiotics. The purified 25-kDa protein does not have any beta-lactamase activity as detected by microiodometry even in the presence of imipenem which is known to be an inducer of penicillinase activity (Tai et al., 1985). All attempts to induce chromosomal beta-lactamase using a variety of beta-lactams have been unsuccessful in V. cholerae. The absence of beta-lactamase activity in the beta-lactam-resistant mutants was also confirmed by bioassays based on the conversion of penicillin to penicilloic acid by penicillinase which reduces I(2) to I and examining the color change of starch-I(2) blue paper. The beta-lactamase-producing E. coli cells were used as control in these experiments.

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 beta-lactam-induced autolysin, B. megaterium protoplasts were exposed to crude lysates of beta-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 beta-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 beta-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, (^2)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 beta-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.



beta-Lactam-resistant Mutants Are Novobiocin-resistant

A unique feature of all the beta-lactam-resistant mutants isolated so far is that they are also resistant to novobiocin and coumermycin, the inhibitors of the B-subunit of DNA gyrase. The minimum inhibitory concentrations of novobiocin and coumermycin for the wild type cells are 2 and 10 µg/ml, respectively. The mutant cells can grow in the presence of 10 µg/ml of novobiocin and 20 µg/ml of coumermycin. The sensitivity of the mutant cells to nalidixic acid, an inhibitor of the A subunit of DNA gyrase, is the same as wild type cells. To examine whether the reverse is also true, mutants resistant to novobiocin (nov^r) were isolated. Two classes of mutants were obtained. The first group of mutants was resistant up to 10 µg of novobiocin/ml and the second group up to 50 µg of novobiocin/ml. 99% of the group I mutants were resistant to beta-lactams (penG^r). On the other hand, none of the group II mutants examined was resistant to penicillin or other beta-lactam antibiotics. When the cellular proteins of the nov^rpenG^r mutants were analyzed by SDS-PAGE, induction of 12-kDa protein could be detected (Fig. 7, lane a). In contrast, in the cellular proteins of nov^r penG^s from any of the two groups of novobiocin-resistant mutants, the 12-kDa protein could not be detected in the gel (Fig. 7, lane b). Thus, the 12-kDa protein is expressed in beta-lactam-resistant V. cholerae cells irrespective of whether these cells were previously exposed to beta-lactams or not, and these proteins apparently have no role in conferring novobiocin resistance to these cells.


Figure 7: SDS-PAGE of cell lysates of novpenG^r mutant (lane a), and nov mutant (lane b) of V. cholerae 569B. Numbers in the margin indicate molecular mass in kDa.



Osmoregulation in beta-Lactam-resistant Cells

It has been reported that the outer membrane proteins of sizes 40 and 38 kDa in V. cholerae are osmoregulated (Lohia et al., 1985). These two proteins have recently been identified as the OmpC- and OmpF-like proteins, respectively, of V. cholerae.^2 Surprisingly, the synthesis of both 38- and 40-kDa proteins in the beta-lactam-resistant mutants are totally insensitive to the osmolarity of the growth medium (Fig. 8). Thus, the osmosensing mechanism is affected in the beta-lactam-resistant mutants.


Figure 8: SDS-PAGE of the outer membrane proteins of 569B (lanes a-d) and penG^r 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.



Sequencing of the N-terminal End and Identification of the Gene Encoding the 25-kDa Protein

The sequence of the first 22 amino acid residues from the N-terminal end of the protein is SIDTNHKARSINAGVYASQEQA. The oligonucleotide sequence deduced from the amino acid sequence using E. coli codon bias showed more than 65% homology with a Pseudomonas aeruginosa phosphate-specific porin OprP which is induced when the cells are grown in phosphate-depleted medium (Siehnel et al., 1990). The synthesis of the 25-kDa protein was, however, insensitive to phosphate ion concentration in the growth medium.

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.


DISCUSSION

The outer membrane of the hypertoxinogenic strain 569B of V. cholerae has smooth type LPS (Chakrabarti and Chatterjee, 1984). Sensitivity of these cells to beta-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 beta-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 beta-lactam antibiotics and was detected as a 12-kDa protein in SDS-PAGE. Of all beta-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 beta-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 beta-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 beta-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 beta-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 beta-lactam resistance to V. cholerae cells, it was observed that the protein is not a beta-lactamase itself nor can it induce chromosomal beta-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 beta-lactam resistance to V. cholerae cells by interfering with the entry of the antibiotic through the porin OmpF through which beta-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 beta-lactam antibiotics. This protein is not a precursor of the 12-kDa monomer. The role of this protein, if any, in conferring beta-lactam resistance to V. cholerae is not known.

Surprisingly, all beta-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 beta-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 beta-lactam-resistant.


FOOTNOTES

*
This work was supported by Research Grant BT/TF/15/03/91 from the Department of Biotechnology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a predoctoral fellowship from the Council of Scientific and Industrial Research, India. Present address: Dept. of Cancer Biology, NN 1-12, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.

To whom correspondence should be addressed. Tel.: 91-33-473-0350; Fax: 91-33-473-0284 or 473-0350; iicb%sirnetc{at}sirnetd.ernet.in.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; PAGE, polyacrylamide gel electrophoresis; 1-ANS, 1-Anilino 8-naphthalene sulfonic acid, sodium salt; RP-HPLC, reverse phase-high performance liquid chromatography; kb, kilobase(s).

(^2)
S. Chakrabarti, K. Chaudhuri, and J. Das, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Dr. R. Nagraj of the Centre for Cellular and Molecular Biology, Hyderabad, for providing one of us (A. D.) the training and facilities for amino acid sequencing.


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