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     

Kinetic Properties of Purified Pseudomonas aeruginosa Phosphorylcholine Phosphatase Indicated That This Enzyme May Be Utilized by the Bacteria to Colonize in Different Environments

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl–BHI broth and in 3% NaCl–nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl–nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl–nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins. Received: 16 June 1998 / Accepted: 18 July 1998     

Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24–70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250–430) likely forms a 16-strand β-barrel and appears to be related to the unusual N-terminal SLH-domain-containing β-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.     

Underexpression of porin protein OmpF in agar-entrapped, sessile-like Escherichia coli

The electrophoretic patterns of the outer membrane proteins of agar-entrapped Escherichia coli cells were found to be different from those of free organisms. In particular, the porin protein OmpF was underexpressed in immobilized bacteria, that displayed enhanced resistance to latamoxef.     

Isolation and characterization of a porin-like protein of 45 kilodaltons from Bacteroides fragilis

Resistance to the combination of amoxicillin and clavulanic acid in some Bacteroides fragilis strains may be associated with a lack of porin proteins. Comparison of outer membrane protein profiles from one resistant strain ( B. fragilis CFPL 358) and two susceptible strains of B . fragilis (ATCC 25285 and CFPL 92125) showed that a few proteins were missing in the resistant strain, especially a 45-kDa protein. To determine whether this protein was a porin-like protein, we attempted to isolate it from the two susceptible strains by using gel filtration (Sephacryl S-200, Superose 6) and ion exchange chromatographies (DEAE Trisacryl, DEAE Sepharose Fast Flow). Elution from DEAE resins was poor compared to the 60–67-kDa region, which suggested that the 45-kDa protein exhibited stronger cationic forms. The use of sodium dodecyl sulfate during elution improved the recovery of the 45-kDa protein, showing that detergent modified its conformation and its ionic bounds with the chromatographic matrices but it was not sufficient for good purification. Superose 6 gel filtration also failed to separate this protein from the 60–67-kDa region. The only method resulting in the positive recovery of a purified 45-kDa band from both susceptible B. fragilis strains was electroelution from SDS-PAGE. The swelling assay showed that the 45-kDa protein was a porin-like protein. From this study, we concluded that the 45-kDa protein from B. fragilis was a porin-like protein which might be involved in the antibiotic resistance of a strain in which this protein was missing.     

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 (37kDa), 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 41kDa 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 41,102Da 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 27kDa. 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 beta-barrel "open" conformer was inaccessible to trypsin.     

Characterisation of local isolates of Enterobacteriaceae from Turkey

20 local isolates of enterics belonging to the genera Salmonella, Enterobacter, Proteus, Citrobacter from human, chicken and/or egg were characterised for their antibiotic resistance patterns, plasmid profiles, phage types, outer membrane proteins, and lipopolysaccharide patterns. Relatedness of these characteristics for epidemiological analysis was assessed. 18 (90%) strains were resistant to at least one antibiotic and those (multi-drug resistant ones) resisting to two or more antibiotics constituted 50% of all isolates. A common 54 kb plasmid was harboured by 55% of the isolates. 14 isolates showed smooth type lipopolysaccharide. 60% of the 20 isolates contained outer membrane proteins in a molecular weight range of 34.6 to 30.6 kDa. The data reveal the lack of correlation between the characteristics investigated.     

Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni

The surfaces of the disrupted-cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative-staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center-to-center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS-PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS-PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer-assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.     

Isolation and characterization of outer membrane permeability mutants in Escherichia coli K-12.

Escherichia coli normally requires the lamB gene for the uptake of maltodextrins. We have identified and characterized three independent mutations that allow E. coli to grow on maltodextrin in the absence of a functional lamB gene by allowing maltodextrins with a molecular weight greater than 1,000 to cross the outer membrane barrier. Two of the mutations map to the structural gene for the outer membrane porin OmpF, and the remaining mutation maps to the structural gene for the second major outer membrane porin, OmpC. These mutations increase the permeability of the outer membrane to small hydrophilic substances, antibiotics, and detergents. These mutations alter the electrophoretic mobility of the respective porin proteins.     

A Proteomic Approach to Understand the Role of the Outer Membrane Porins in the Organic Solvent-Tolerance of Pseudomonas aeruginosa PseA

Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log P_ow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log P_ow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94–8.67 and the molecular weights were in the range of 19.5–104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure.     

Identification of an OprD homologue in Acinetobacter baumannii

With the increased number of resistant Acinetobacter baumannii strains, it is urgently required to decipher the molecular bases of outer membrane permeability. The analyses of the outer membrane from different A. baumannii strains indicated a modification in the expression of two proteins of 29 and 43 kDa, respectively. By electrophoresis and MALDI-MS analyses, the 43 kDa OMP was identified as a protein belonging to the OprD family, a basic amino acid and imipenem porin.     

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.     

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.     

Outer membrane proteins of thebacteroides fragilis group

The Triton-insoluble outer membrane proteins of members of the obligately anaerobicBacteroides fragilis group were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The application of previously published methods for the preparation of outer membranes from these anaerobes led to relatively low recoveries of outer membrane material. Additionally, the outer membrane recovered was not qualitatively or quantitatively representative of the total outer membrane. A method previously devised for the isolation of the outer membrane proteins fromEscherichia coli was used. By this method, the members of theB. fragilis group were found to possess a striking capacity to alter their cell-surface proteins in response to cultivation in different media.     

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.     

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.     

Recognition of immunologically important antigens in Bacteroides fragilis infection —an experimental study in mice

When the outer membrane proteins (OMP) of Bacteroides fragilis ATCC 23745 grown under seven different culture conditions were analysed by SDS-PAGE, five of the seven preparations showed similar protein profiles. When the three different OMP preparations were subsequently analysed by Western blotting, that from B. fragilis grown in brain heart infusion broth supplemented with haemin and menadione alone showed more immunodominant bands than those grown with additional cholesterol and methionine or cholesterol and cysteine. Only the former showed immunodominant proteins around 45.6 kDa and 66.2 kDa throughout the course of infection in mice. A protein corresponding to 97.4 kDa was found under all culture conditions. The results emphasise the importance of culture conditions in the expression of immunologically reactive antigens and also the possible use of such antigens in the serodiagnosis of B. fragilis infection.T. Khanna is and D. Panigrahi was with the Department of Medical Microbiology and N.K. Ganguly and S. Majumdar are with the Department of Experimental Medicine, at the Postgraduate Institute of Medical Education and Research, Chandigarh, India. D. Panigrahi is now with Kuwait University, Faculty of Alled Health Science, P.O. Box 31470 Sulaibikhat, 90805, Kuwait     

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.     

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.