Subunit constituent of the porin trimers that form the permeability channels in the outer membrane of Salmonella typhimurium.

The polypeptide composition of the functional porin trimers that produced the permeability channels in the outer membrane of Salmonella typhimurium was examined on two-dimensional slab gels. The results suggested that the majority of porin trimers from strains producing mixed species of porin polypeptides consisted of homologous subunit polypeptides. The present results do not exclude the possibility that a small fraction of porin trimer is constructed from heterologous subunit polypeptides.     

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.     

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

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

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.     

Outer membrane of Salmonella XIV. Reduced transmembrane diffusion rates in porin-deficient mutants

Rates of diffusion of a β-lactam antibiotic, cephaloridine, across the outer membrane of $\text{Salmonella typhimurium}$ cells were measured by determining the rates of its hydrolysis by β-lactamases located in the periplasmic space. It was shown that the permeability coefficient of the outer membrane toward cephaloridine decreased to about one-tenth of that in the wild type, in mutant strains deficient in two “porin” proteins, previously shown to produce transmembrane pores in in vitro reconstitution experiments. In contrast, the loss of the 33,000 dalton outer membrane protein did not have any noticeable effect on the permeability coefficient.     

Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease.

Saccharomyces cerevisiae strains are often host to several types of cytoplasmic double-stranded RNA (dsRNA) genomes, some of which are encapsidated by the L-A dsRNA product, an 86,000-dalton coat protein. Here we present the finding that nuclear recessive mutations in the NUC1 gene, which encodes the major nonspecific nuclease of yeast mitochondria, resulted in at least a 10-fold increase in amounts of the L-A dsRNA and its encoded coat protein. The effect of nuc1 mutations on L-A abundance was completely suppressed in strains that also hosted the killer-toxin-encoding M dsRNA. Both NUC1 and nuc1 strains containing the L-A genome exhibited an increase in coat protein abundance and a concomitant increase in L-A dsRNA when the cells were grown on a nonfermentable carbon source rather than on glucose, an effect independent of the increase in coat protein due to nuc1 mutations or to the absence of M. The increase in L-A expression in nuc1 strains was similar to that observed in strains with mutations in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. nuc1 mutations did not affect the level of porin in the mitochondrial outer membrane. Since the effect of mutations in nuc1 was to alter the copy number of the L-A coat protein genome rather than to change the level of the M toxin genome (as do mak and ski mutations), these mutations define a new class of nuclear genes affecting yeast dsRNA abundance.     

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

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

Role of phosphorylation of porin (VDAC) in regulation of mitochondrial outer membrane under normal conditions and alcohol intoxication

The present work is an overview of the factors regulating permeability of the outer membrane of mitochondria and the state of the channels formed by porin (voltage-dependent anion channels, VDAC). According to the accumulated data, modulation of the outer membrane permeability can be induced by endogenous phosphorylation of VDAC channels. Different protein kinases, such as protein kinase A, protein kinase C, tyrosine protein kinase, hexokinase, glycogen synthetase kinase-3β (GSK-3β), Akt and p38 kinases, were shown to be involved in VDAC phosphorylation. Among these protein kinases, alcohol-induced stress-kinases, GSK-3β, Akt, and p38 identified in mitochondria may participate in phosphorylation of porin, modulation of VDAC conductance, and regulation of the outer membrane permeability.     

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

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

Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate

In whole-cell biocatalysis, cell envelopes represent a formidable barrier for substrates to permeate. The present research addresses this critical issue by investigating the effects of outer membrane mutation on uridine diphosphate (UDP)-glucose-utilizing enzymes in whole-cell systems. Owing to the severe limitation in substrate permeability, the wild-type Escherichia coli cells only exhibited as low as 4% of available enzyme activities. The reduction of the barriers of the outer membrane permeability (by mutations in its structure) led to a striking acceleration (up to 14-fold) of the reaction rate in cells expressing UDP-glucose dehydrogenase. Mutations in the lipopolysaccharide synthesis pathway or Braun’s lipoprotein are both effective. The acceleration was dependent upon the substrate concentrations as well as the enzyme expression level. In addition, the mutation has been demonstrated to be much more effective than the freeze–thaw permeabilizing method. An application of outer membrane mutants was illustrated with the synthesis of a disaccharide (N-acetyllactosamine) from UDP-glucose. Both reaction rate and product yield were enhanced significantly (more than twofold) in the lipoprotein mutant, demonstrating the importance of the outer membrane permeability barrier and the advantages of using outer membrane mutants in synthesis. This research and the results outlined in this paper point to a valid strategy in addressing permeability issues in whole-cell biocatalysis. It also highlights a need for an assessment of substrate permeability in biocatalysis research and development.     

Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.     

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.     

The barrier function of the outer membrane of Pseudomonas maltophilia in the diffusion of saccharides and β-lactam antibiotics

This paper reports that the efficiency of solute diffusion through the outer membrane of Pseudomonas maltophilia is roughly 3 to 5% of that of Escherichia coli. This is despite the fact that the outer membrane pore(s) is only a little smaller than that of E. coli. These results suggest that P. maltophilia has a low copy number of porin(s). The outer membrane of antibiotic resistant clinical isolates showed even less efficient permeability towards saccharides and antibiotics than the laboratory strains.     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000     

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.     

Subunit structure of functional porin oligomers that form permeability channels in the other membrane of Escherichia coli.

Oligomers of a protein, porin, form permeability channels in the outer membrane of Escherichia coli B. A functional porin oligomer was identified and was purified to homogeneity by gel filtration in the presence of salts and sodium dodecyl sulfate. Molecular weights of purified porin oligomer and heat-dissociated monomer appeared to be 102,900 and 32,600, respectively, when determined by sedimentation equilibrium in the presence of sodium dodecyl sulfate. We concluded that the porin oligomer thus consists of three identical subunits. These data and results from other laboratories suggest porin trimers exist also in the outer membrane of intact cells, and participate in the formation of permeability channels. It was found that porin trimer bound less sodium dodecyl sulfate than the porin monomer.