Naturally crystalline porin in the outer membrane of Bordetella pertussis

The Gram-negative bacterium Bordetella pertussis is the agent responsible for whooping-cough, and much interest has focused on the functions, structures and immunological properties of the molecules exposed at its outer surface. We have found by electron microscopy that cells of two strains of B. pertussis are covered with a crystalline surface lattice. This lattice is not an extrinsic layer of high molecular weight glycoproteins, such as occur on many other bacteria, but is a natural crystal of an intrinsic membrane protein of 40,000 Mr. This molecule has been shown to be an anion-selective member of an extensive family of proteins ("porins") that render Gram-negative outer membranes permeable to solutes of up to approximately 650 Mr. Computer image processing reveals a trimeric channel-like structure that closely resembles other porins visualized in artificial arrays after treatment with detergents, but in a novel (p2) crystal form. This correlation provides a "missing link" between earlier structural studies based on artificial arrays of porins (of undefined physiological status), and membrane-permeabilization experiments with solubilized porins (in undefined structural states). For the strains characterized so far, crystallinity of the porin surface lattice shows an intriguing correlation with nonpathogenicity.     

Selection for mutants altered in the expression or export of outer membrane porin OmpF.

Strains in which the lacZ gene (which specifies beta-galactosidase) is fused to a gene encoding an envelope protein often exhibit a phenotype termed overproduction lethality. In such strains, high-level synthesis of the cognate hybrid protein interferes with the process of protein export, and this leads ultimately to cell death. A variation of this phenomenon has been discovered with lacZ fusions to the gene specifying the major outer membrane porin protein OmpF. In this case, we find that lambda transducing phage carrying an ompF-lacZ fusion will not grow on a host strain that constitutively overexpresses ompF. We have exploited this observation to develop a selection for ompF mutants. Using this protocol, we have isolated mutants altered in ompF expression and have identified mutations that block OmpF export. Our results suggest that it should be possible to adapt this selection for use with other genes specifying exported proteins.     

Orientation of porin channels in the outer membrane of Bordetella pertussis

We have examined the surface topography and channel connectivity of a naturally crystalline porin that is known to be functional, and whose structure has not been perturbed by detergent extraction, A three-dimensional density map, calculated from two independent tilt series of negatively stained cell envelopes, reveals three separate channels per trimer on one side (the ‘smooth’ side), and a single common opening at the other (‘rough’) side. This arrangement is consistent with the molecular structures recently determined at high resolution by X-ray crystallography for three other porins after detergent solubilization, and implies that the Bordetella pertussis porin may have the same kind of folding. Surface relief maps calculated from electron micrographs of cell envelopes contrasted by unidirectional shadowing clearly show that the side with single opening (i.e. the rough side) represents the external surface.     

Topology of the porin MspA in the outer membrane of Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.     

The outer membrane porin from Yersinia enterocolitica in yersiniosis diagnostics

The diagnostic test system based on a species-specific antigen, pore-forming protein from the outer membrane of Yersinia enterocolitica, for yersiniosis verification by the method of ELISA has been developed and approved. The proposed ELISA test system is characterized by high sensitivity (95%) and specificity (89%) and provides a differential diagnostics of yersiniosis from other acute enteric infections with similar clinical manifestations. In comparison with the commercial diagnostics based on indirect hemagglutination reaction, which is conventionally used in clinical practice, the porin-based ELISA provides the high level (90–95%) of yersiniosis identification at early (1st week) and late (2nd–4th week) stages of infection process. It has been found that the ELISA test system reveals antibodies to the Y. enterocolitica porin in patient’s serum irrespective of the serological variant of causative agent.     

Structure-function correlation of outer membrane protein porin from Paracoccus denitrificans

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.     

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE

Abstract Within gram-negative bacteria such as Escherichia coli , the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.     

Interaction between quinolones antibiotics and bacterial outer membrane porin OmpF

In these work, we try to establish a relation between the hydrophobicity of some quinolones and their interaction with OmpF. In order to do that, the values of the binding constant of some quinolones of different "generations" with OmpF were determined by UV-visible spectrophotometry and by fluorimetry. Our results show that there is a strong interaction between all the drugs and the protein and that it becomes larger for the last "generation" fluoroquinolones. These results were compared with previous ones obtained for the interaction of these drugs with simpler biomembrane models (liposomes) and it is possible to conclude that some of the quinolones associate preferably with the protein than with these models. This suggests that an interaction drug/porin is, probably, the preferentially used for the latest fluoroquinolones what makes reasonable to believe that a strong affinity for OmpF means a better capacity to transpose the barrier formed by the outer membrane.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF

Abstract The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae . Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.     

Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine

Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12. Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively. A third locus, ompB, is defined by mutations that map at 74 minutes. The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression. One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters. Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype. The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner. Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins. In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e. OmpF- OmpC+. Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations. One of these classes exhibits an OmpF+ OmpC- phenotype. The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions. Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor. These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.     

Isolation and characterisation of porin from the outer membrane of Synechococcus PCC 6301

Pore-forming protein (porin) was isolated from N,N-dimethyl-dodecylaminoxid (LDAO)-extracted outer membranes of Synechococcus PCC 6301 and purified by ion exchange chromatography on DEAE-Sephacel column. The apparent molecular mass on SDS-PAGE was determined to be about 52000. The native porin was reconstituted into black lipid bilayer membranes and showed a single-channel conductance of 5.5 nS in 1 M KCl. The porin was found to be N-terminally blocked. The C-terminal amino acid sequence was identified as Phe-Thr-Phe. Amino acid analysis suggested that the porin protein consists of about 420 amino acid residues, yielding a polarity of 43.6% and a molecular mass of 45000 in contrast to the mobility on SDS-PAGE.Abbreviations DEAE Diethylaminoethyl; M _r, relative molecular mass - LDAO N,N-Dimethyl-dodecylaminoxid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoretogram - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - UTEX Culture Collection of Algae at the University of Texas     

The protective properties of a porin from the outer membrane of Yersinia pseudotuberculosis

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions were selected for isolation and refolding of recombinant monomer and porin trimer. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that the recombinant porins are similar in the composition of secondary structure elements to the isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

In vitro interactions between lipopolysaccharides and heterologous outer membrane porin proteins

Smooth lipopolysaccharides fromEscherichia coli, Brucella, andPseudomonas solanacearum were studied for their ability to interact with porin proteins ofE. coli andB. ovis. Despite the differences in chemical composition and taxonomic origin, lipopolysaccharides and heterologous porins associated in vitro to form spherical bodies on whose surface antigenic determinants of the porins were exposed. No differences were observed between these materials and those obtained by homologous reassociations. The results demonstrated that no species-specific requirements exist for lipopolysaccharides-porin association and stress the physicochemical similarity in the outer membranes of bacteria without taxonomic relationship.     

Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.