In vitro assembly of the functional porin trimer from dissociated monomers in Pseudomonas aeruginosa

The molecular weights of monomeric and oligomeric forms of the newly identified porins, protein D2, of the outer membrane of Pseudomonas aeruginosa appeared to be 47,000 and 137,000, respectively, as determined by the light scattering technique. Presence of the trimeric aggregates of the homologous subunits in the intact outer membrane, the liposome membrane, and the non-ionic surfactant were confirmed through cross-linking experiments and immunoblotting techniques. The protein D2 monomers prepared in 0.1% of sodium dodecyl sulfate at 23 degrees C spontaneously reassembled into the trimeric aggregate when the surfactant dropped below critical concentration. The diffusion rates of saccharides and beta-lactam antibiotics through the liposome membranes reconstituted from the reassembled protein D2 trimers were indistinguishable from those of the native protein D2. This study shed some light on the porin trimer assembly as well as on the mechanism of carbapenem diffusion through the protein D2 pores.     

Outer membrane porin protein of Haemophilus influenzae type b: pore size and subunit structure

The 40-kDa porin protein of Haemophilus influenzae type b was reconstituted into proteoliposomes. The relative rates of diffusion of small uncharged sugars across the channels formed by this protein were determined by measuring the rates of osmotic swelling of the liposomes. From these rates, a pore diameter of 1.8 nm was estimated using the Renkin equation. A chemical cross-linking technique was used to investigate the oligomeric structure of the 40-kDa porin. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis revealed the presence of porin dimers and trimers after reaction of the protein with dithio-bis-(succinimidyl propionate). These results confirmed that the porin of H. influenzae forms large water-filled channels and indicated that it probably exists as trimers in the outer membrane.     

The motion of a single molecule,the lambda-receptor,in the bacterial outer membrane

Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo. The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model that allows extraction of the motion of the protein from measurements of the mobility of the bead-molecule complex; these results are equally applicable to analyze bead-protein complexes in other membrane systems. Within a domain of radius approximately 25 nm, the receptor diffuses with a diffusion constant of (1.5 +/- 1.0) x 10(-9) cm(2)/s and sits in a harmonic potential as if it were tethered by an elastic spring of spring constant of ~1.0 x 10(-2) pN/nm to the bacterial membrane. The purpose of the protein motion might be to facilitate transport of maltodextrins through the outer bacterial membrane.     

Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl.     

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.     

Evidence for the Presence of a Porin in the Membrane of Glyoxysomes of Castor Bean

Glyoxysomes of endosperm tissue of castor bean (Ricinus communis L.) seedlings were solubilized in a detergent and added to a lipid bilayer. Conductivity measurements revealed that the glyoxysomal preparation contained a porin-like channel. Using an electrophysiological method, which we established for semiquantitative determination of porin activity, we were able to demonstrate that glyoxysomal membranes purified by sucrose density gradient centrifugation contain an integral membrane protein with porin activity. The porin of glyoxysomes was shown to have a relatively small single-channel conductance of about 330 picosiemens in 1 M KCl and to be strongly anion selective. Thus, the glyoxysomal porin differs from the other previously characterized porins in the outer membrane of mitochondria or plastids, but is similar to the porin of spinach (Spinacia oleracea L.) leaf peroxisomes. Our results suggest that, in analogy to the porin of leaf peroxisomes, the glyoxysomal porin facilitates the passage of small metabolites, such as succinate, citrate, malate, and aspartate, through the membrane.     

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.     

Export of porin to the outer membrane of the phototrophic bacterium Rhodobacter capsulatus 37B4

Export of porin to the outer membrane of the phototrophic purple bacterium Rhodobacter capsulatus was studied with the use of the uncoupler of the electron transport chain, carbonylcyanide-m-chlorophenylhydrazone (CCCP). The agent reversibly blocked the transport of porin across the cytoplasmic membrane. By means of radioactive labeling and immunoprecipitation, porin was found to occur in two forms: (i) the exported form that was extractable from the outer membrane without disrupting the cells, and (ii) a pre-form with a slightly higher apparent molecular mass which accumulated in the cells during the block of the export process. Proteolysis studies revealed that the preform was highly sensitive to added proteases, whereas the exported form was resistant.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - OMP outer membrane porin; pre-OMP, form of outer membrane porin before export - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Molecular and Structural Characterization of the HMP-AB Gene Encoding a Pore-Forming Protein from a Clinical Isolate of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.     

Purification, amino acid composition and N-terminal sequence of the major protein (protein H) of the outer membrane of Pasteurella multocida]

The major protein (protein H) of the outer membrane of Pasteurella multocida was purified by size-exclusion chromatography after selective extraction with detergents. The protein forms homotrimers which are stable in the presence of SDS at room temperature. Upon treatment at 100 degrees C, the protein is fully dissociated by the detergent into monomers exhibiting an apparent molecular mass of 37 kDa as estimated by electrophoresis. The amino acid composition of protein H is characterized by a low hydropathy index (HI = -0.40) and is strongly related to the compositions of bacterial porins, notably porins P2 (Haemophilus influenzae), PIA (Neisseria gonorrhoeae) and Cl.2 ("class 2 porin" of N. meningitidis). The N-terminal amino acid sequence of protein H shares a strong homology with those of porins OmpC (Escherichia coli) and P2. These data indicate that protein H of P. multocida is a porin belonging to the superfamily of the non-specific porins of Gram-negative eubacteria outer membrane.     

Use of a nonpenetrating substrate analogue to study the molecular mechanism of the outer membrane dicarboxylate transport system in Escherichia coli K12

We have recently demonstrated that a cell-surface dicarboxylate-binding protein (DBP) is involved in the outer membrane dicarboxylate transport system in Escherichia coli K12. The present report deals with our findings relating to the mode of action of this protein, and the identity and properties of the outer membrane integral protein which is involved in the translocation of dicarboxylic acids across the hydrophobic regions of the outer membrane. By the use of a nonpenetrating succinate analogue, aspartate-dextran, and through reconstitution studies with purified DBP, the cell-surface DBP is found to play an important role in succinate influx but not efflux. Transport studies with major outer membrane protein mutants indicate that the matrix protein (also referred to as protein I or porin) is the only outer membrane integral protein actively involved in the outer membrane dicarboxylate transport system. In the absence of a functional DBP, porin translocates succinate in a relatively less efficient and nonspecific manner. A tentative working model is proposed for this transport system. In this model, the cell-surface DBP is depicted as the substrate recognition component of the otherwise nonspecific porin channel. Together, this "DBP-porin channel complex" forms an efficient, specific transport channel for dicarboxylic acids.     

A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin

In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling beta-lactam diffusion via porins.     

Description of Complex Forms of a Porin in Bacteroides fragilis and Possible Implication of this Protein in Antibiotic Resistance

Periodic surveys of antibiotic susceptibility patterns among anaerobes have emphasized that new mechanisms of resistance have emerged, especially in the Bacteroides fragilis group. Resistance to the combination of amoxicillin and clavulanic acid among some imipenem-susceptible Bacteroides fragilis strains has been associated with modifications in outer membrane protein electrophoretic patterns with the loss of some porin-like proteins. Porins are outer membrane proteins that play a major part in membrane permeability; if they are under-expressed, they can be responsible for antibiotic resistance. In a previous work, we isolated one outer membrane protein of 45 kDa from Bacteroides fragilis and showed its porin activity. In the present study, we aim to isolate the different complex forms of this protein and to underline their possible role in antibiotic resistance. We therefore compared the electrophoretic patterns of the outer membrane proteins of several strains of Bacteroides fragilis. Although these patterns are similar to each other, some proteins, especially those of high molecular weight, are less visible in the samples heated before electrophoresis. We targeted these high molecular weight proteins (which appeared sensitive to heat) and isolated them by electro-elution. We thus identified two high molecular weight proteins (210 and 130/135 kDa) which seemed to be components of a complex including the 45 kDa outer membrane protein formerly identified by us as a porin protein. Their porin activities were tested by the swelling assay of proteoliposomes which showed that the 210 kDa protein behaved like the 45 kDa protein whereas the 130/135 kDa protein had less porin activity. Furthermore, swelling assays with antibiotic solutions made it possible to compute the role of this protein complex in antibiotic resistance.     

Characterization of Porins Isolated from the Outer Membrane of Serratia liquefaciens

A major outer membrane protein with an apparent molecular weight of 42 kDa was purified from Serratia liquefaciens grown on Brain Heart Infusion medium. The same protein was obtained when the cells were grown on a synthetic medium supplemented with 2% glucose. The amino acid composition of this protein revealed it to be hydrophilic. The pore-forming ability of the 42-kDa protein was determined by the liposome swelling assay. This assay demonstrated that the protein forms nonspecific channels with a diameter between 1.16 and 1.6 nm. An additional protein with a molecular weight of 47 kDa was obtained on synthetic medium supplemented with maltose. This protein exhibited specific pore-forming ability to maltose and maltodextrins, but was also permeable to other compounds, according to their size. When bacteria were grown on Nutrient Broth medium, two outer membrane proteins with molecular weights of 41 kDa and 42 kDa were produced by the bacteria. All three types of proteins represent monomers of respective oligomers. The monomers did not exhibit pore-forming ability when incorporated into liposomes. We, therefore, propose that the oligomer is the functional unit of a porin capable of forming permeability channels in the outer membrane of Serratia liquefaciens. These results indicate that S. liquefaciens contains several porins exhibiting specific osmoregulation or that are induced by a specific nutrient, where the 42-kDa outer membrane protein of this bacterium is certainly a major porin. Received: 6 July 1998 / Accepted: 19 August 1998     

Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).     

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila.

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled [( 35S]cysteine or [35S]methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid per mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.     

Biosynthesis of mitochondrial porin and insertion into the outer mitochondrial membrane of Neurospora crassa

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4 degrees C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane.     

Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers.

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.     

Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.     

The functional properties of Ompβ, the regularly arrayed porin of the hyperthermophilic bacterium Thermotoga maritima

Abstract The regularly arrayed outer membrane protein, Ompβ, of Thermotoga maritima was purified to homogeneity and was characterized functionally by incorporation into artificial lipid bilayers. The polypeptide has an apparent molecular mass ( M _r) of approx. 40 000 and forms stable trimers in the presence of 1% octyl-polyoxyethylene or 2% SDS which dissociate when boiling the sample. The protein has a secondary structure (predominantly β-sheet) and an amino acid composition characteristic for porins. Pore-forming activity was demonstrated by porin incorporation into artificial bilayers proving that Ompβ is a true porin: selectivity measurements showed a 4.4-fold selectivity for cations over anions. Conductivity of the porin is influenced by surface charges and also depends on the applied voltage.