JOURNAL OF BACTERIOLOGY, JUIY 1991, P. 4182-4187
Vol. 173, No. 13
0021-9193/91/134182-06$02.00/0
Copyright C 1991, American Society for Microbiology
The Major Outer Membrane Protein of Acidovorax delafeldii Is an Anion-Selective Porin MANFRED BRUNEN,' HARALD ENGELHARDT,'* ANGELA SCHMID,2 AND ROLAND BENZ2 Abteilung fur Molekulare Strukturbiologie, Max-Planck Institut fur Biochemie, D-8033 Martinsried,' and Lehrstuhlffur Biotechnologie, Universitat Wurzburg, D-8700 Wiirzburg, Germany Received 30 January 1991/Accepted 26 April 1991
The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldit) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70°C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% i sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore-forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth.
characterization, and functional properties of a second major outer membrane protein of A. delafieldii, the integral outer membrane protein Omp34. We show that it is a typical porin, forming anion-selective channels with an unusual kind of dependence of the channel conductance on ion concentration that has not been observed to date for bacterial porins.
One of the major functions of the bacterial outer membrane is to control the flux of molecules between the environment and the periplasmic space. This is achieved by pore-forming proteins (porins), which provide channels about 1 to 2 nm in diameter (for recent reviews, see references 6 and 24). Two types of porins may be distinguished. The general diffusion pores control the passage of molecules by their pore size and exhibit some selectivity for cations or anions. OmpF and PhoE of Escherichia coli are examples of this type. The specific porins that possess binding sites for certain molecules, e.g., sugars, enable the cell to select specific solutes from dilute media at the cell surface-environment border. A well-characterized specific porin is the LamB protein (maltoporin) of E. coli (12, 18, 30). The porins of E. coli and other members of the family Enterobacteriaceae have been intensively studied by functional and structural analyses, whereas only a few investigations of porins from bacteria more closely related to Acidovorax delafieldii have been published. A. delafieldii (formerly Pseudomonas delafieldii [38]) is a member of the a subdivision of the class Proteobacteria and belongs to the "acidovorans complex," which comprises bacteria closely related to Comamonas acidovorans (38). A. delafieldii and C. acidovorans appear to possess relatively simple outer membranes, containing only a limited number of protein species. Most of them have already been assigned to certain structures or functions (15, 21). Electron microscopic investigations showed that one of the major outer membrane proteins of each species forms a crystalline layer that covers the whole cell (29) and is reminiscent of surface layers to some degree (5, 21). This surface protein is in intimate contact with the outer membrane (16) and can be expected to affect both its structural and functional properties. The outer membranes of A. delafieldii and C. acidovorans are suitable model systems for studying such protein-membrane interactions and their functional consequences. In this report we describe the purification, biochemical *
MATERIALS AND METHODS
Bacterial strains and chemicals. A. delafieldii (P. delafieldii DSM 64, identical with ATCC 17505) was obtained from the German Collection of Microorganisms, Braunschweig, Germany. Most of the chemicals were purchased from Merck; lysozyme, lithium dodecyl sulfate (LDS), and DNase were from Sigma; and N,N'-methylenebisacrylamide, acrylamide, and Triton X-100 were from Serva. Diphytanoyl phosphatidylcholine was purchased from Avanti Biochemicals Inc. (Birmingham, Ala.), n-decane was from Fluka, and octylpolyoxyethylene was from Bachem. Purification of the porin from A. delafieldii. Cells were grown overnight in nutrient broth (5 g of peptone and 3 g of meat extract per liter [pH 7]) in batch cultures, harvested, and broken in a cell mill. Incubation with DNase (10 ng/ml, 1 h, 30°C) was followed by treatment with lysozyme (100 ,ug/ml, 8 h). The cytoplasmic membrane was solubilized by incubation with 2% Triton X-100 at 25C for 30 min. All steps were carried out in a buffer containing 50 mM sodium phosphate (pH 7.5), 1 mM MgCl2, and 3 mM sodium azide. The outer membrane (5 mg of protein per ml) was solubilized at 30°C for 2 h in a solution containing 5 mM EDTA, 150 mM KCl, 10% octylpolyoxyethylene (protein-detergent, 1:20 [wt/ wt]), and 100 mM phosphate (pH 10 to 11). Insoluble material was removed by centrifugation at 100,000 x g for 30 min; the supernatant was diluted 1:10 with bidistilled water to reduce the salt and detergent concentrations and immediately applied to a Mono Q HR 5/5 ion-exchange chromatography column (Pharmacia Fine Chemicals, Uppsala, Sweden). The chromatography buffer solution consisted of 10 mM triethanolamine (pH 7.5), 3 mM sodium azide, and 1% octylpolyoxyethylene. Bound protein was eluted with an NaCl gradient (0 to 700 mM). The pooled fractions were
Corresponding author. 4182
VOL. 173, 1991
ANION-SELECTIVE PORIN OF ACIDOVORAX DELAFIELDII
concentrated either by mixing with dry Sephadex G 25 or by ultrafiltration. LDS-PAGE. Samples for polyacrylamide gel electrophoresis (PAGE) were mixed with sample buffer (pH 6.8) containing 4% LDS, 100 mM Na2CO3, 100 mM dithiothreitol, and 20% sucrose and incubated for 15 min at 100°C or as indicated. LDS-PAGE was performed as described by Delepelaire and Chua (19) with either homogeneous 12% acrylamide gels or with gradient gels (7 to 15% acrylamide) in a Pharmacia midget gel electrophoresis system. LPS staining. The outer membrane and the purified porin (80 jig of protein each) were solubilized in sample buffer at 100°C and digested by treatment with proteinase K (1 mg of enzyme per 4 mg of protein) for 2 h at 60°C. Portions of the samples were analyzed by LDS-PAGE and stained for lipopolysaccharide (LPS) by the silver stain method of Tsai and Frasch (37) and then stained with Coomassie brilliant blue for protein. Detection of sugars was also performed with isolated membranes and purified 0mp34 by using a glycan detection kit (Boehringer Mannheim). Attenuated total reflection infrared spectroscopy. Spectroscopic analysis of the purified porin was performed by using the attenuated total reflection technique. The spectra were recorded with a Nicolet 740 FT-IR spectrometer. The protein (50 to 100 ,ug, dialyzed against bidistilled water) was dried under a nitrogen stream on a germanium crystal. Two hundred scans were accumulated at a resolution of better than 2 cm-1. The amide I and II bands were subjected to band shape analysis to assess the secondary structure composition as described in detail by Kleffel et al. (28). Conductance measurements. The porin function was investigated by the black lipid membrane technique (6, 9). For single-channel conductance measurements, the purified protein was reconstituted into membranes made of diphytanoyl phosphatidylcholine in n-decane, painted over a circular hole of 0.1 mm2. The membrane separated two compartments of a Teflon chamber filled with KCl or another salt solution. All experiments were carried out at room' temperature and at a membrane potential of 20 mV. Porin was added to a final concentration of 10 to 100 ng/ml to one or both compartments, and the current was measured via two calomel electrodes. The signal was amplified (109 times) with a current-to-voltage converter and monitored with a storage oscilloscope and a strip chart recorder. For zero-current measurements, membranes of 2 mm2 were used, containing about 103 channels. Concentrated salt solution was added to one compartment in several steps to apply a salt gradient across the membrane. After equilibrium conditions were established, the zero-current potential was measured with a Keithley 610 C electrometer connected to the electrodes. The method was previously described in detail by Benz et al. (10).
Mr x103 9
68
-__ ___0
45
f
29
OV
21
_ -
"
1
3
2
6
5
4
7
8
FIG. 1. Temperature modification of Omp34 from A. delafieldii in LDS-PAGE. Lanes: 1, Mr marker proteins; 2 through 8, purified Omp34 solubilized at 60, 64, 68, 72, 76, 80, and 100°C, respectively. The 7 to 12% polyacrylamide-LDS gel was stained with Coomassie brilliant blue.
gradient. In the presence of detergent the Omp34 forms stable oligomers that dissociate at around 70°C into the 34,000-Mr monomers (34K proteins) (Fig. 1). This is a common property among the pore-forming outer membrane proteins (31). Minor bands that occur only at low temperatures (Fig. 1, lanes 2 through 5) may represent the oligomers in other conformations or configurations that differ in their mobility characteristics, or, more likely, they may originate from complexes that are still associated with outer membrane components such as lipids or LPSs. There is indeed LPS in the purified porin fraction. Carbohydrate-specific immunolabeling showed a strong reaction with the outer membrane fraction and a weak reaction with the' purified porin 'fraction. We could demonstrate three major bands in preparations of the solubilized outer membrane by means of an LPS-sensitive silver stain (37) after the protein had completely been digested by treatment with proteinase K (Fig. 2). Only the band running at the position of Mr 29,000 was found in the preparation of purified Omp34 as well (Fig. 2, lane 4). The amount of LPS per unit of protein was much higher in intact membranes than in the isolated 34K protein. Probably less than 5% of the LPS is contained'in the protein preparation, as judged from the staining intensities. The fact that only a small portion of one particular LPS component out of three was present in the MrxlO3 95 68 45
m
29
RESULTS Purification and characterization of the major outer membrane protein. The outer membrane was solubilized by using high pHs and high detergent and salt concentrations as specified in Materials and Methods. Ion-exchange chromatography was sufficient to separate the major intrinsic membrane protein with an Mr of 34,000 (Omp34) from the accompanying polypeptides. Omp34 exhibited a very low binding affinity to the column under the conditions applied, and most of it was eluted in the void volume. Only a small portion was retained on the column and could be eluted together with the other proteins by using a linear NaCl
4183
21 12.5
6.5
1
2
3
4
5
6
7
8
FIG. 2. LPS content of the outer membrane and of Omp34 from A. delafieldii. The LDS gel (7 to 15% polyacrylamide) was stained with the LPS-sensitive silver stain first (lanes 1 through 4). Lanes 5 through 8 represent the same gel stained with Coomassie brilliant blue. Lanes: 3 and 7, outer membrane preparation; 4 and 8, purified Omp34 (samples in lanes 3, 4, 7, and 8 were run after digestion of the protein); 1, 2, 5, and 6, Mr marker proteins. The outer membrane preparation and the purified protein fraction contained about 40 ,ug of protein before proteinase K treatment.
4184
J. BACTERIOL.
BRUNEN ET AL.
TABLE 1. Average single-channel conductance (X) of Omp34 from A. delafieldii in different salt solutionsa Salt
LiCl KF KCI KBr KJ KNO2 KNO3 KHCO KCH3CO
K2SO4
Concn (N)
A (nS)
A/a (108 cm)
1 1 1 0.1 1 0.1 0.1 1 1 1 1 0.1 1
0.86 1.25 1.21 0.34 1.4 0.36 0.33 1.5 1.4 1.1 0.43 0.15 1.4
1.2 1.7 1.1 2.8 1.2 2.7 2.5 1.6 1.5 1.4 0.62 1.7
a Membranes were formed from diphytanoyl phosphatidylcholine-n-decane. Experiments were run at 20 mV, pH 6, and 25°C. The conductivity values are averages of at least 100 single steps. a, bulk conductivity of each salt.
FIG. 3. Stepwise increase of the membrane conductivity after addition of 1 to 10 ng of the purified A. delafieldii porin to the aqueous phase containing 0.3 M KCl. The membranes were made from 1% diphytanoyl phosphatidylcholine in n-decane. The applied potential was 20 mV, the temperature was 25°C, and the octylpolyoxyethylene concentration was
Vol. 173, No. 13
0021-9193/91/134182-06$02.00/0
Copyright C 1991, American Society for Microbiology
The Major Outer Membrane Protein of Acidovorax delafeldii Is an Anion-Selective Porin MANFRED BRUNEN,' HARALD ENGELHARDT,'* ANGELA SCHMID,2 AND ROLAND BENZ2 Abteilung fur Molekulare Strukturbiologie, Max-Planck Institut fur Biochemie, D-8033 Martinsried,' and Lehrstuhlffur Biotechnologie, Universitat Wurzburg, D-8700 Wiirzburg, Germany Received 30 January 1991/Accepted 26 April 1991
The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldit) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70°C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% i sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore-forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth.
characterization, and functional properties of a second major outer membrane protein of A. delafieldii, the integral outer membrane protein Omp34. We show that it is a typical porin, forming anion-selective channels with an unusual kind of dependence of the channel conductance on ion concentration that has not been observed to date for bacterial porins.
One of the major functions of the bacterial outer membrane is to control the flux of molecules between the environment and the periplasmic space. This is achieved by pore-forming proteins (porins), which provide channels about 1 to 2 nm in diameter (for recent reviews, see references 6 and 24). Two types of porins may be distinguished. The general diffusion pores control the passage of molecules by their pore size and exhibit some selectivity for cations or anions. OmpF and PhoE of Escherichia coli are examples of this type. The specific porins that possess binding sites for certain molecules, e.g., sugars, enable the cell to select specific solutes from dilute media at the cell surface-environment border. A well-characterized specific porin is the LamB protein (maltoporin) of E. coli (12, 18, 30). The porins of E. coli and other members of the family Enterobacteriaceae have been intensively studied by functional and structural analyses, whereas only a few investigations of porins from bacteria more closely related to Acidovorax delafieldii have been published. A. delafieldii (formerly Pseudomonas delafieldii [38]) is a member of the a subdivision of the class Proteobacteria and belongs to the "acidovorans complex," which comprises bacteria closely related to Comamonas acidovorans (38). A. delafieldii and C. acidovorans appear to possess relatively simple outer membranes, containing only a limited number of protein species. Most of them have already been assigned to certain structures or functions (15, 21). Electron microscopic investigations showed that one of the major outer membrane proteins of each species forms a crystalline layer that covers the whole cell (29) and is reminiscent of surface layers to some degree (5, 21). This surface protein is in intimate contact with the outer membrane (16) and can be expected to affect both its structural and functional properties. The outer membranes of A. delafieldii and C. acidovorans are suitable model systems for studying such protein-membrane interactions and their functional consequences. In this report we describe the purification, biochemical *
MATERIALS AND METHODS
Bacterial strains and chemicals. A. delafieldii (P. delafieldii DSM 64, identical with ATCC 17505) was obtained from the German Collection of Microorganisms, Braunschweig, Germany. Most of the chemicals were purchased from Merck; lysozyme, lithium dodecyl sulfate (LDS), and DNase were from Sigma; and N,N'-methylenebisacrylamide, acrylamide, and Triton X-100 were from Serva. Diphytanoyl phosphatidylcholine was purchased from Avanti Biochemicals Inc. (Birmingham, Ala.), n-decane was from Fluka, and octylpolyoxyethylene was from Bachem. Purification of the porin from A. delafieldii. Cells were grown overnight in nutrient broth (5 g of peptone and 3 g of meat extract per liter [pH 7]) in batch cultures, harvested, and broken in a cell mill. Incubation with DNase (10 ng/ml, 1 h, 30°C) was followed by treatment with lysozyme (100 ,ug/ml, 8 h). The cytoplasmic membrane was solubilized by incubation with 2% Triton X-100 at 25C for 30 min. All steps were carried out in a buffer containing 50 mM sodium phosphate (pH 7.5), 1 mM MgCl2, and 3 mM sodium azide. The outer membrane (5 mg of protein per ml) was solubilized at 30°C for 2 h in a solution containing 5 mM EDTA, 150 mM KCl, 10% octylpolyoxyethylene (protein-detergent, 1:20 [wt/ wt]), and 100 mM phosphate (pH 10 to 11). Insoluble material was removed by centrifugation at 100,000 x g for 30 min; the supernatant was diluted 1:10 with bidistilled water to reduce the salt and detergent concentrations and immediately applied to a Mono Q HR 5/5 ion-exchange chromatography column (Pharmacia Fine Chemicals, Uppsala, Sweden). The chromatography buffer solution consisted of 10 mM triethanolamine (pH 7.5), 3 mM sodium azide, and 1% octylpolyoxyethylene. Bound protein was eluted with an NaCl gradient (0 to 700 mM). The pooled fractions were
Corresponding author. 4182
VOL. 173, 1991
ANION-SELECTIVE PORIN OF ACIDOVORAX DELAFIELDII
concentrated either by mixing with dry Sephadex G 25 or by ultrafiltration. LDS-PAGE. Samples for polyacrylamide gel electrophoresis (PAGE) were mixed with sample buffer (pH 6.8) containing 4% LDS, 100 mM Na2CO3, 100 mM dithiothreitol, and 20% sucrose and incubated for 15 min at 100°C or as indicated. LDS-PAGE was performed as described by Delepelaire and Chua (19) with either homogeneous 12% acrylamide gels or with gradient gels (7 to 15% acrylamide) in a Pharmacia midget gel electrophoresis system. LPS staining. The outer membrane and the purified porin (80 jig of protein each) were solubilized in sample buffer at 100°C and digested by treatment with proteinase K (1 mg of enzyme per 4 mg of protein) for 2 h at 60°C. Portions of the samples were analyzed by LDS-PAGE and stained for lipopolysaccharide (LPS) by the silver stain method of Tsai and Frasch (37) and then stained with Coomassie brilliant blue for protein. Detection of sugars was also performed with isolated membranes and purified 0mp34 by using a glycan detection kit (Boehringer Mannheim). Attenuated total reflection infrared spectroscopy. Spectroscopic analysis of the purified porin was performed by using the attenuated total reflection technique. The spectra were recorded with a Nicolet 740 FT-IR spectrometer. The protein (50 to 100 ,ug, dialyzed against bidistilled water) was dried under a nitrogen stream on a germanium crystal. Two hundred scans were accumulated at a resolution of better than 2 cm-1. The amide I and II bands were subjected to band shape analysis to assess the secondary structure composition as described in detail by Kleffel et al. (28). Conductance measurements. The porin function was investigated by the black lipid membrane technique (6, 9). For single-channel conductance measurements, the purified protein was reconstituted into membranes made of diphytanoyl phosphatidylcholine in n-decane, painted over a circular hole of 0.1 mm2. The membrane separated two compartments of a Teflon chamber filled with KCl or another salt solution. All experiments were carried out at room' temperature and at a membrane potential of 20 mV. Porin was added to a final concentration of 10 to 100 ng/ml to one or both compartments, and the current was measured via two calomel electrodes. The signal was amplified (109 times) with a current-to-voltage converter and monitored with a storage oscilloscope and a strip chart recorder. For zero-current measurements, membranes of 2 mm2 were used, containing about 103 channels. Concentrated salt solution was added to one compartment in several steps to apply a salt gradient across the membrane. After equilibrium conditions were established, the zero-current potential was measured with a Keithley 610 C electrometer connected to the electrodes. The method was previously described in detail by Benz et al. (10).
Mr x103 9
68
-__ ___0
45
f
29
OV
21
_ -
"
1
3
2
6
5
4
7
8
FIG. 1. Temperature modification of Omp34 from A. delafieldii in LDS-PAGE. Lanes: 1, Mr marker proteins; 2 through 8, purified Omp34 solubilized at 60, 64, 68, 72, 76, 80, and 100°C, respectively. The 7 to 12% polyacrylamide-LDS gel was stained with Coomassie brilliant blue.
gradient. In the presence of detergent the Omp34 forms stable oligomers that dissociate at around 70°C into the 34,000-Mr monomers (34K proteins) (Fig. 1). This is a common property among the pore-forming outer membrane proteins (31). Minor bands that occur only at low temperatures (Fig. 1, lanes 2 through 5) may represent the oligomers in other conformations or configurations that differ in their mobility characteristics, or, more likely, they may originate from complexes that are still associated with outer membrane components such as lipids or LPSs. There is indeed LPS in the purified porin fraction. Carbohydrate-specific immunolabeling showed a strong reaction with the outer membrane fraction and a weak reaction with the' purified porin 'fraction. We could demonstrate three major bands in preparations of the solubilized outer membrane by means of an LPS-sensitive silver stain (37) after the protein had completely been digested by treatment with proteinase K (Fig. 2). Only the band running at the position of Mr 29,000 was found in the preparation of purified Omp34 as well (Fig. 2, lane 4). The amount of LPS per unit of protein was much higher in intact membranes than in the isolated 34K protein. Probably less than 5% of the LPS is contained'in the protein preparation, as judged from the staining intensities. The fact that only a small portion of one particular LPS component out of three was present in the MrxlO3 95 68 45
m
29
RESULTS Purification and characterization of the major outer membrane protein. The outer membrane was solubilized by using high pHs and high detergent and salt concentrations as specified in Materials and Methods. Ion-exchange chromatography was sufficient to separate the major intrinsic membrane protein with an Mr of 34,000 (Omp34) from the accompanying polypeptides. Omp34 exhibited a very low binding affinity to the column under the conditions applied, and most of it was eluted in the void volume. Only a small portion was retained on the column and could be eluted together with the other proteins by using a linear NaCl
4183
21 12.5
6.5
1
2
3
4
5
6
7
8
FIG. 2. LPS content of the outer membrane and of Omp34 from A. delafieldii. The LDS gel (7 to 15% polyacrylamide) was stained with the LPS-sensitive silver stain first (lanes 1 through 4). Lanes 5 through 8 represent the same gel stained with Coomassie brilliant blue. Lanes: 3 and 7, outer membrane preparation; 4 and 8, purified Omp34 (samples in lanes 3, 4, 7, and 8 were run after digestion of the protein); 1, 2, 5, and 6, Mr marker proteins. The outer membrane preparation and the purified protein fraction contained about 40 ,ug of protein before proteinase K treatment.
4184
J. BACTERIOL.
BRUNEN ET AL.
TABLE 1. Average single-channel conductance (X) of Omp34 from A. delafieldii in different salt solutionsa Salt
LiCl KF KCI KBr KJ KNO2 KNO3 KHCO KCH3CO
K2SO4
Concn (N)
A (nS)
A/a (108 cm)
1 1 1 0.1 1 0.1 0.1 1 1 1 1 0.1 1
0.86 1.25 1.21 0.34 1.4 0.36 0.33 1.5 1.4 1.1 0.43 0.15 1.4
1.2 1.7 1.1 2.8 1.2 2.7 2.5 1.6 1.5 1.4 0.62 1.7
a Membranes were formed from diphytanoyl phosphatidylcholine-n-decane. Experiments were run at 20 mV, pH 6, and 25°C. The conductivity values are averages of at least 100 single steps. a, bulk conductivity of each salt.
FIG. 3. Stepwise increase of the membrane conductivity after addition of 1 to 10 ng of the purified A. delafieldii porin to the aqueous phase containing 0.3 M KCl. The membranes were made from 1% diphytanoyl phosphatidylcholine in n-decane. The applied potential was 20 mV, the temperature was 25°C, and the octylpolyoxyethylene concentration was
