Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes.

Protein K, a recently described outer membrane protein correlated with encapsulation in Escherichia coli (Paakkanen et al., J. Bacteriol. 139:835-841, 1979), has been purified to apparent homogeneity. Purification was based upon the noncovalent association of protein K with peptidoglycan, and the purified protein was shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. Incorporation of small amounts (10(-10) to 10(-11) M) of purified protein K into artificial lipid bilayers resulted in an increase, by many orders of magnitude, in membrane conductance. The increased conductance resulted from the formation of large, water-filled, ion-permeable channels exhibiting single-channel conductance in 1.0 M KCl of 1.83 nS. The membrane conductance showed a linear relationship between current and applied voltage and was not voltage induced or regulated. The channel was permeable to large organic ions (e.g., Tris+ Cl-) and, based upon a pore length of 7.5 nm, a minimum channel diameter of 1.2 nm was estimated; these properties resemble values for other enteric porins. The possible biological role of the pores produced by protein K is discussed.     

Identification of two porins in Pelobacter venetianus fermenting high-molecular-mass polyethylene glycols.

Porins were purified from cells of the anaerobic gram-negative bacterium Pelobacter venetianus grown with 20-kDa polyethylene glycol. After treatment of the cell envelope fraction with sodium dodecyl sulfate-containing solutions, the murein contained only two major peptidoglycan-associated proteins of 14 and 23 kDa. Both proteins were released from the peptidoglycan by the detergent Triton X-100. Genapol X-80 released only the 23-kDa protein. This protein was purified by chromatography on a hydroxyapatite column. It did not form sodium dodecyl sulfate-resistant oligomers. Reconstituted in lipid bilayer membranes, the 23-kDa protein formed cation-selective channels with a single-channel conductance of 230 pS in 1 M KCl. The channel is not a general-diffusion pore, since its conductance depends only moderately on the salt concentration. The channel conducted ammonium much better than potassium or rubidium ions, suggesting that it is probably involved in ammonium uptake. The outer membrane of P. venetianus contains a further, non-murein-associated pore with an unknown molecular mass. It is also cationically selective and has a single-channel conductance of 1.6 nS in 1 M KCl, which suggests that its effective diameter is similar to that of porins from enteric bacteria.     

Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes.

A new major outer membrane protein, P, was induced in Pseudomonas aeruginosa PAO1 upon growth in medium containing 0.2 mM or less inorganic phosphate. Studies with media containing different levels of phosphate and with mutants of PAO1 suggested that protein P was coregulated with alkaline phosphatase and phospholipase C. Protein P was substantially purified and shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. The incorporation of purified protein P into artificial lipid bilayers resulted in an increase of the membrane conductance by many orders of magnitude. Single-channel experiments demonstrated that protein P channels were substantially smaller than all previously studied porins from P. aeruginosa and enteric bacteria, with an average single-channel conductance in 1 M NaCl of 0.25 nS. The protein P channel was apparently not voltage induced or regulated. The results of single-channel conductance experiments, using a variety of different salts, allowed a minimum channel diameter estimate of 0.7 nm. Furthermore, from these results it was concluded that the protein P channel was highly specific for anions. Zero-current potential measurements confirmed that protein P was at least 30-fold more permeable for Cl- than for K+ ions. The possible biological role of the small, anion-specific protein P channels in phosphate uptake from the medium is discussed.     

Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes.

The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS.     

Identification of the outer membrane porin of Thermus thermophilus HB8: the channel-forming complex has an unusually high molecular mass and an extremely large single-channel conductance

The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.     

Interaction of the lamB protein with the peptidoglycan layer in Escherichia coli K12

In Escherichia coli K12 the product of gene lamB is an outer membrane protein involved in the transport of maltose and maltodextrins and serving as a receptor for several bacteriophages including lambda. About 30 to 40% of this protein can be recovered associated to peptidoglycan when the cells are dissolved in sodium dodecyl sulfate in the presence of 2 mM Mg2+ ions. The bound protein can then be quantitatively eluted from peptidoglycan by incubating the complex in Triton X-100 and EDTA, or sodium dodecyl sulfate and NaCl. The protein eluted in such ways is still totally active in its phage-neutralizing activity. Two other membrane proteins known to behave similarly to the lamB protein are proteins Ia and Ib. However the binding of these proteins to peptidoglycan appears tighter, in several respects, than that of the lamB protein. The lamB protein may span the outer membrane since it appears to interact with the peptidoglycan on the inner side of this membrane while it is known to be accessible to both phages and antibodies at the cell surface.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

Modification of positional distribution of fatty acids in phosphatidylinositol of rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the epsilon-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.     

Modification of the conductance,selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the ?-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris+, N(C2H5)4+, Hepes-) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.     

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers.

The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.     

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.     

The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.     

Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.     

The C-Terminal Domain of the Bordetella pertussis Autotransporter BrkA Forms a Pore in Lipid Bilayer Membranes

BrkA is a 103-kDa outer membrane protein of Bordetella pertussis that mediates resistance to antibody-dependent killing by complement. It is proteolytically processed into a 73-kDa N-terminal domain and a 30-kDa C-terminal domain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BrkA is also a member of the autotransporter family of proteins. Translocation of the N-terminal domain of the protein across the outer membrane is hypothesized to occur through a pore formed by the C-terminal domain. To test this hypothesis, we performed black lipid bilayer experiments with purified recombinant protein. The BrkA C-terminal protein showed an average single-channel conductance of 3.0 nS in 1 M KCl. This result strongly suggests that the C-terminal autotransporter domain of BrkA is indeed capable of forming a pore.     

An anion-selective channel from the Pseudomonas aeruginosa outer membrane

Protein P from Pseudomonas aeruginosa outer membrane was reconstituted in lipid bilayer membranes from diphytanoylphosphatidylcholine. The reconstitution resulted in the formation of anion-selective channels with a conductance of 160 pS for 0.1 M chloride solution. The channels were at least 100-times more selective for anions than for cations as judged from zero-current membrane potentials. The single-channel conductance was dependent on the size of the different anions and saturated at higher salt concentrations suggesting single ion occupancy of the protein P channel.     

A calcium-activated cation-selective channel in rat cultured Schwann cells

Calcium-activated channels, in the plasma membrane of rat cultured Schwann cells were studied in isolated 'inside-out' membrane patches. With identical (150 mM NaCl) solutions on either side of the membrane, a single channel conductance of 32 pS was calculated for inward current; the conductance was somewhat less for outward current. The channel is about equally permeable to sodium and potassium ions, but is not detectably permeable to either chloride or calcium. Under our experimental conditions the channel is activated by high (more than 10(-4) M) concentrations of calcium and is sensitive to voltage, channel activity increasing with membrane depolarization.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris⁺, N(C₂H₅)₄⁺, Hepes^?) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.     

Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis.

Important protein-based immunoreactivities have long been associated with the cell wall core of mycobacteria. In order to explore the molecular basis of such activities, purified cell walls of Mycobacterium tuberculosis were extracted with sodium dodecyl sulfate to produce an insoluble residue composed of the mycolylarabinogalactan-peptidoglycan complex and about 2% of unextractable protein. Treatment of the product from an avirulent strain of M. tuberculosis with trifluoromethanesulfonic acid released a single polypeptide with a molecular size of 23 kilodaltons, accounting for all of the insoluble cell wall protein. Extensive purification and then analysis of the 23-kilodalton protein demonstrated the absence of diaminopimelic acid, muramic acid, or other peptidoglycan components, pointing to either a novel linkage between protein and peptidoglycan or a noncovalent but tenacious association. The released 23-kilodalton protein showed amino acid homology and other similarities to the outer membrane protein OmpF of Escherichia coli. Although a similar product was released in small quantities from cell walls of the virulent M. tuberculosis Erdman and H37Rv by lysozyme treatment, the cell walls of virulent bacilli were dominated by the presence of poly-alpha-L-glutamine, accounting for as much as 10% of their weight. The poly-alpha-L-glutamine was successfully separated from the cell wall proper, demonstrating again the absence of a covalent association between peptidoglycan and the polymer. The antigenicity of these products is demonstrated, and their roles vis-a-vis analogous polypeptides from other bacteria in immunogenicity, pathogenicity, and bacterial physiology are discussed.