Purification and characterization of protein H, the major porin of Pasteurella multocida.

Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude from this set of structural and functional data that protein H of P. multocida is a pore-forming protein related to the superfamily of the nonspecific bacterial porins.     

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 beta-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 <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.     

Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures.

The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.     

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73.

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.     

Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions.

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.     

Cloning and Characterization of the Gene Encoding for OMP-PD Porin: The Major <Emphasis Type="Italic">Photobacterium damsela</Emphasis> Outer Membrane Protein

The outer membrane protein of Photobacterium damsela (OMP-PD) and the gene encoding for this porin protein were isolated and characterized. The deduced amino acid sequence of the OMP-PD monomer has 338 amino acids and a calculated molecular weight of 36,951 Da. This sequence includes a 22-amino acid signal peptide at the N-terminal, which is not found when the monomer is located in the outer membrane. Native OMP-PD protein forms a trimeric structure of approximately 110 kDa. It exhibits resistance to proteases, and it can be cleaved only following denaturation by SDS. The degree of identity of the OMP-PD amino acid sequence to porins from the Enterobacteriaceae was only 24%. Identity to Vibrio or Photobacterium porins was 38% and 48%, respectively. Nevertheless, the multiple alignment of this sequence with other structurally defined Enterobacteria porins demonstrated that the location of the 16 beta-strands and eight external loops, including a larger external L3 loop, are conserved in OMP-PD. These results, together with the previously known ability of OMP-PD to form an ion channel in artificial liposomes, strongly support its role as a porin in P. damsela and will help further investigations into the role of OMP-PD in P. damsela pathogenicity.     

Isolation and characterization of a conserved porin protein from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.     

Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics

Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.     

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.     

The bacterial porin superfamily: sequence alignment and structure prediction

The porins of Gram-negative bacteria are responsible for the 'molecular sieve' properties of the outer membrane. They form large water-filled channels which allow the diffusion of hydrophilic molecules into the periplasmic space. Owing to the strong hydrophilicity of their amino acid sequence and the nature of their secondary structure (beta strands), conventional hydropathy methods for predicting membrane topology are useless for this class of protein. The large number of available porin amino acid sequences was exploited to improve the accuracy of the prediction in combination with tools detecting amphipathicity of secondary structure. Using the constraints of beta-sheet structure these porins are predicted to contain 16 membrane-spanning strands, 14 of which are common to the two (enteric and the neisserial) porin subfamilies.     

Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.     

The TolB protein interacts with the porins of Escherichia coli.

TolB is a periplasmic protein of the cell envelope Tol complex. It is partially membrane associated through an interaction with the outer membrane lipoprotein PAL (peptidoglycan-associated lipoprotein), which also belongs to the Tol system. The interaction of TolB with outer membrane porins of Escherichia coli was investigated with a purified TolB derivative harboring a six-histidine tag. TolB interacted with the trimeric porins OmpF, OmpC, PhoE, and LamB but not with their denatured monomeric forms or OmpA. These interactions took place both in the presence and in the absence of lipopolysaccharide. TolA, an inner membrane component of the Tol system, also interacts with the trimeric porins via its central periplasmic domain (R. Dérouiche, M. Gavioli, H. Bénédetti, A. Prilipov, C. Lazdunski, and R. Lloubès, EMBO J. 15:6408-6415, 1996). In the presence of the purified central domain of TolA (TolAIIHis), the TolB-porin complexes disappeared to form TolAIIHis-porin complexes. These results suggest that the interactions of TolA and TolB with porins might take place in vivo and might be concomitant events participating in porin assembly. They also suggest that the Tol system as a whole may be involved in porin assembly in the outer membrane.     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.     

Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.     

The major outer membrane protein of Acidovorax delafieldii is an anion-selective porin.

The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldii) 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 degrees C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% beta 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.