Monomeric Porin Research Articles

Improving Single-Molecule Antibody Detection Selectivity through Optimization of Peptide Epitope Presentation in OmpG Nanopore.

Outer membrane protein G (OmpG) is a monomeric porin found in Escherichia coli, which possesses seven flexible loops. OmpG has been engineered as a nanopore sensor, where its loops can host affinity epitopes for selective detection of biological molecules. In this study, we investigated various loop positions to incorporate a FLAG peptide antigen epitope in the most flexible loop 6 and tested the efficacy and sensitivity of these nanopore constructs in antibody detection. We observed an OmpG construct containing inserted FLAG sequence, which exhibited strong interaction with anti-FLAG antibodies in flow cytometry; however, it could not translate molecule interactions into a readable signal in current recordings. Further optimization of the peptide presentation strategy by replacing specific sections of loop 6 sequences with the FLAG tag created a construct capable of generating unique and distinct signals when interacting with various monoclonal or polyclonal anti-FLAG clones IgG antibodies in the mixture. The peptide display scheme demonstrated in this study can be generalized for the engineering of OmpG sensors, which can be used for screening and validating positive clones during antibody development, as well as for real-time quality control of cell cultures in monoclonal antibody production.

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single‐Molecule Multiplex Protein Detection

AbstractMolecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single‐channel current recordings. We exploited these motif‐displaying loops concurrently to facilitate single‐molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection.

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

Single-channel characterization of the chitooligosaccharide transporter chitoporin (SmChiP) from the opportunistic pathogen Serratia marcescens

Serratia marcescens is an opportunistic pathogen that can utilize chitin as a carbon source, through its ability to produce chitin-degrading enzymes to digest chitin and membrane transporters to transport the degradation products (chitooligosaccharides) into the cells. Further characterization of these proteins is important to understand details of chitin metabolism. Here, we investigate the properties and function of the S. marcescens chitoporin, namely SmChiP, a chitooligosaccharide transporter. We show that SmChiP is a monomeric porin that forms a stable channel in artificial phospholipid membranes, with an average single-channel conductance of 0.5 ± 0.02 nS in 1 M KCl electrolyte. Additionally, we demonstrated that SmChiP allowed the passage of small molecules with a size exclusion limit of <300 Da and exhibited substrate specificity toward chitooligosaccharides, both in membrane and detergent-solubilized forms. We found that SmChiP interacted strongly with chitopentaose (Kd = 23 ± 2.0 μM) and chitohexaose (Kd = 17 ± 0.6 μM) but did not recognize nonchitose oligosaccharides (maltohexaose and cellohexaose). Given that S. marcescens can use chitin as a primary energy source, SmChiP may serve as a target for further development of nutrient-based antimicrobial therapies directed against multidrug antibiotic-resistant S. marcescens infections.

Full-length TprK of Treponema pallidum subsp. pallidum in lipid nanodiscs is a monomeric porin

TprK is a key virulence factor of Treponema pallidum subsp. pallidum (T. pallidum) due to its ability to undergo intra-strain antigenic variation through gene conversion. This mechanism can generate millions of tprK gene and protein variants to allow immune evasion and pathogen persistence during infection. In silico structural modeling supports that TprK is an outer membrane β-barrel with porin function and with several surface-exposed loops, seven of which corresponding to the variable regions. No definitive structural of functional data, however, exist for this protein aside from its role in immune evasion. Studies to elucidate TprK biological function as a porin, are hindered by the evidence that TprK is not abundant on T. pallidum outer membrane, and by the fragility of T. pallidum envelope. To gain insight onto TprK structure and possible function as a porin, we used an Escherichia coli - based expression system that yielded highly pure full-length TprK without any intermediate denaturation step, and proceeded to reconstitute it in detergents and lipid nanodiscs. Visualization of TprK in nanodiscs using negative staining electron microscopy supported that TprK is a monomeric porin in an artificial lipid environment mimicking T. pallidum membrane. Our work provided evidence that TprK is a possible porin transporter of T. pallidum, a biological function compatible with its structural models. These results bring us closer to a comprehensive understanding of the function of this important virulence factor in syphilis pathogenesis and T. pallidum biology.

Elucidating the pH Dependent Mechanism of OmpG Gating

Protein pores are steadily emerging as valuable stochastic sensors for single- molecule detection. Over the decade, developments in stochastic sensing have been based on single channel recording in planar lipid bilayer. Historically, nanopores with rigid structures have been frequently used for sensing molecules and biological species. However, we have chosen a nanopore with a flexible structure for our protein nanopore: monomeric β-barrel porin outer membrane protein G (OmpG). This monomeric porin is composed of 14- s- strands connected by seven flexible extracellular loops. Wild-type OmpG spontaneously gates during an applied potential as revealed by planar bilayer studies. Strong evidence suggests that the dynamic movement of OmpG's loop 6 is responsible for the gating mechanism of the porin. The unusual flexible architecture of this porin not only successfully binds protein targets, but also provides unique detection. Our engineered OmpG sensor is sensitive enough to provide, for the first time, the ability to resolve homologous protein analytes.The ability of our biosensor to discriminate between homologous proteins reveals the important need in better understanding the underlying mechanism of OmpG gating. This understanding will allow us to further engineer OmpG and tailor it for sensing an array of protein analytes. In addition, this knowledge will aid in a deeper understanding of the biology of this membrane protein itself. In this work, we highlight the pH-dependent gating mechanism of OmpG by investigating repulsive forces of OmpG's extracellular loops.

The influence of monogalactosyldiacylglycerols from different marine macrophytes on immunogenicity and conformation of protein antigen of tubular immunostimulating complex

The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A2-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid “nanofluidics” as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems.

Analyzing the Interaction of RseA and RseB, the Two Negative Regulators of the σE Envelope Stress Response, Using a Combined Bioinformatic and Experimental Strategy

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, sigma(E), and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onsigma(E) activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.

Orientation of the Monomeric Porin OmpG in Planar Lipid Bilayers

Outer membrane protein G (OmpG) is a non-selective porin from Escherichia coli. OmpG is a monomer, which makes it unusual among porins, and suggests that it may be useful in biotechnology. In planar lipid bilayers, individual OmpG pores reconstituted by insertion from detergent exhibit pronounced asymmetry in current-voltage relationships and voltage-dependent gating. Here, this asymmetry is used to deduce the orientation of OmpG in the bilayers. We introduced two cysteines into the extracellular loops of OmpG. Cleavage of the disulfide bond formed by these residues significantly increases spontaneous gating of the pore. By adding DTT to one side of the bilayer or the other, we demonstrated that pores showing a quiet trace at negative potentials have a "trans" conformation (extracellular loops on the trans side of the bilayer), while pores showing a quiet trace at positive potentials have a "cis" conformation (extracellular loops on the cis side). With this knowledge, we examined the binding of a cyclodextrin to OmpG. When the cyclodextrin was presented to the extracellular face of the pore, transient multisite interactions were observed. In contrast, when the cyclodextrin was presented to the periplasmic face, a more stable single-site interaction occurred. Because the cyclodextrin can act as a molecular adapter by binding analytes, this information serves to advance the use of OmpG as a biosensor.

The Role of Lipids for the Functional Integrity of Porin: An FTIR Study Using Lipid and Protein Reporter Groups

We have investigated the temperature-dependent interaction of the porins OmpF from Paracoccus denitrificans and OmpG from Escherichia coli with lipid molecules after reconstitution in lecithin. Effects of incubation at increased temperatures on activity were tested by functional experiments for OmpG and compared with previously published results of OmpF in order to understand the activity loss of OmpF with monomerization. Protein-lipid interaction was monitored by different reporter groups both from lipid molecules and from protein. OmpF loses its activity by approximately 90% at 50 degrees C while OmpG does not show a temperature-dependent change in activity between room temperature and 50 degrees C. The interaction between OmpF and lipid molecules is severely altered in a two-step mechanism at 55 and approximately 75 degrees C for OmpF. The first step is attributed to changes in the degree of interaction between the aromatic girdle of OmpF and the interfacial region of the lipid bilayer, leading to monomerization of this trimeric porin. The second step at 75 degrees C is attributed to the changes in lipid-porin monomer interaction. Around 90 degrees C, reconstituted porin aggregates. For OmpG, changes in lipid-protein interaction were observed starting from approximately 80 degrees C because of temperature-induced breakdown of its folding. This study provides deeper understanding of porin-lipid bilayer interaction as a function of temperature and can explain the functional breakdown by monomerization while porin secondary structure is still preserved.

Outer membrane protein G: Engineering a quiet pore for biosensing

Bacterial outer membrane porins have a robust beta-barrel structure and therefore show potential for use as stochastic sensors based on single-molecule detection. The monomeric porin OmpG is especially attractive compared with multisubunit proteins because appropriate modifications of the pore can be easily achieved by mutagenesis. However, the gating of OmpG causes transient current blockades in single-channel recordings that would interfere with analyte detection. To eliminate this spontaneous gating activity, we used molecular dynamics simulations to identify regions of OmpG implicated in the gating. Based on our findings, two approaches were used to enhance the stability of the open conformation by site-directed mutagenesis. First, the mobility of loop 6 was reduced by introducing a disulfide bond between the extracellular ends of strands beta12 and beta13. Second, the interstrand hydrogen bonding between strands beta11 and beta12 was optimized by deletion of residue D215. The OmpG porin with both stabilizing mutations exhibited a 95% reduction in gating activity. We used this mutant for the detection of adenosine diphosphate at the single-molecule level, after equipping the porin with a cyclodextrin molecular adapter, thereby demonstrating its potential for use in stochastic sensing applications.

Isolation and characterization of the Omp-PA porin from Porphyromonas asaccharolytica, determination of the omp-PA gene sequence and prediction of Omp-PA protein structure

A single monomeric porin, Omp-PA (37 kDa), was isolated from the outer membrane of the gram-negative anaerobic rod Porphyromonas asaccharolytica. Further characterization revealed that this porin consists of two different fractions: a heat-modifiable fraction which in its denatured form migrated on SDS-PAGE as a protein with a molecular weight of 41 kDa and a heat-resistant fraction which did not change its migration on SDS-PAGE after boiling. A liposome swelling assay revealed that only the heat-resistant fraction was able to transport sugars after its incorporation into the liposomes, although it did not discriminate between differently sized sugars. We hypothesize that the heat-modifiable fraction corresponds to the “closed” conformer of Omp-PA, whereas the heat-resistant fraction corresponds to the “open” conformer of the protein. Cloning of the omp-PA gene revealed an open reading frame of 1161 bases, with a predicted protein sequence of 387 amino acids. The mature protein consists of 366 amino acids with a calculated MW of 41,102 Da and an estimated pI of 7.24. The C-terminal domain of Omp-PA is homologous to the characteristic OmpA signature domain (71% similarity with the OmpA consensus domain). Sequence comparison with other anaerobes from the Bacteroides family demonstrated homology across the entire ORF. Digestion of the P. asaccharolytica outer membrane analysis of trypsin-digested Omp-PA yielded two proteins migrating with apparent molecular weights of 37 and 27 kDa. These data fully supported our hypothesis that the C-terminal domain of the two-domain “closed” conformer of Omp-PA was digested by trypsin, whereas the single domain β-barrel “open” conformer was inaccessible to trypsin.

Crystal Structure of the Monomeric Porin OmpG

The outer membrane (OM) of Gram-negative bacteria contains a large number of channel proteins that mediate the uptake of ions and nutrients necessary for growth and functioning of the cell. An important group of OM channel proteins are the porins, which mediate the non-specific, diffusion-based passage of small (<600 Da) polar molecules. All porins of Gram-negative bacteria that have been crystallized to date form stable trimers, with each monomer composed of a 16-stranded β-barrel with a relatively narrow central pore. In contrast, the OmpG porin is unique, as it appears to function as a monomer. We have determined the X-ray crystal structure of OmpG from Escherichia coli to a resolution of 2.3 Å. The structure shows a 14-stranded β-barrel with a relatively simple architecture. Due to the absence of loops that fold back into the channel, OmpG has a large (∼13 Å) central pore that is considerably wider than those of other E. coli porins, and very similar in size to that of the toxin α-hemolysin. The architecture of the channel, together with previous biochemical and other data, suggests that OmpG may form a non-specific channel for the transport of larger oligosaccharides. The structure of OmpG provides the starting point for engineering studies aiming to generate selective channels and for the development of biosensors.

Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 ( E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the β-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.

Molecular and structural characterization of the HMP-AB gene encoding a pore-forming protein from a clinical isolate of Acinetobacter baumannii.

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.

Folding of a monomeric porin, OmpG, in detergent solution.

OmpG, a porin from E. coli, has been examined in planar lipid bilayers and in detergent solution. First, bilayer recordings were used to reinforce the evidence that the functional form of OmpG is a monomer. Both pH-dependent gating and blockade by covalent modification add support to this proposal. The findings contrast with the properties of the classical porins, which function as trimers. Second, the folding of OmpG in detergent solution was examined. A water-soluble form of OmpG was obtained by dialysis from denaturant into buffer. Incubation of water-soluble OmpG in detergent results in conversion to a form that possesses the hallmarks of a beta barrel. The folding of water-soluble OmpG in detergent was monitored by circular dichroism, protease resistance, and heat modifiability. OmpG is first transformed into an intermediate with increased beta-sheet content on the time scale of minutes at 23 degrees C. This is followed by the slow acquisition of heat modifiability and protease resistance over several hours. The formation of a beta barrel during this period was demonstrated in a double cysteine mutant by using intramolecular disulfide bond formation to report N and C terminus proximity. Finally, conditions are presented for folding OmpG with greater than 90% efficiency, thereby paving the way for structural studies.

Topology of the Erwinia chrysanthemi oligogalacturonate porin KdgM.

The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure. A model of this protein, based on sequence similarity and the amphipathy profile, was constructed. The model depicts a beta-barrel composed of 14 antiparallel beta-strands. The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis. The protein has seven surface-exposed loops. They are rather small with the exception of one, loop L6. Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM. Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins. With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria.

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12. Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.

Projection structure of the monomeric porin OmpG at 6 å resolution

The Escherichia coli porin OmpG, which acts as an efficient unspecific channel for mono-, di- and trisaccharides, has been purified and crystallized in two dimensions. Projection maps of two different crystal forms of OmpG at 6 Å resolution show that the protein has a β-barrel structure characteristic for outer membrane proteins, and that it does not form trimers, unlike most other porins such as OmpF and OmpC, but appears in monomeric form. The size of the barrel is ∼2.5 nm, indicating that OmpG may consist of 14 β-strands. The projection map suggests that the channel is restricted by internal loops.

Purification, characterization and sequence analysis of Omp50,a new porin isolated from Campylobacter jejuni

A novel pore-forming protein identified in Campylobacter was purified by ion-exchange chromatography and named Omp50 according to both its molecular mass and its outer membrane localization. We observed a pore-forming ability of Omp50 after re-incorporation into artificial membranes. The protein induced cation-selective channels with major conductance values of 50-60 pS in 1 M NaCl. N-terminal sequencing allowed us to identify the predicted coding sequence Cj1170c from the Campylobacter jejuni genome database as the corresponding gene in the NCTC 11168 genome sequence. The gene, designated omp50, consists of a 1425 bp open reading frame encoding a deduced 453-amino acid protein with a calculated pI of 5.81 and a molecular mass of 51169.2 Da. The protein possessed a 20-amino acid leader sequence. No significant similarity was found between Omp50 and porin protein sequences already determined. Moreover, the protein showed only weak sequence identity with the major outer-membrane protein (MOMP) of Campylobacter, correlating with the absence of antigenic cross-reactivity between these two proteins. Omp50 is expressed in C. jejuni and Campylobacter lari but not in Campylobacter coli. The gene, however, was detected in all three species by PCR. According to its conformation and functional properties, the protein would belong to the family of outer-membrane monomeric porins.