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.     

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Polarity-dependent voltage-gated porin channels from Escherichia coli in lipid bilayer membranes.

A porin preparation from Escherichia coli 0111:B4 consisting of Omp F and Omp C (with Omp F in excess) was purified by salt extraction procedures and investigated in bilayer lipid membranes formed according to the Montal-Mueller technique. The porin preparation was added to the KCl electrolyte compartment of the Montal-Mueller cell which was connected to the voltage source. As the porin incorporated into the membrane, asymmetric, voltage-gated ion channels were formed. Transmembrane voltages greater than +50 mV (measured with respect to the side of porin addition) caused channel closing, while negative voltages, on the other hand, had no effect on channel behaviour but did increase the rate of porin incorporation at higher voltages. With porin added to both compartments voltage gating no longer occurred. Single-channel conductances corresponded to effective pore diameters of 1.5 nm for opening events and 1.18 nm for channel closing events. The number of charges involved in gating was approximately 2.     

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.     

Polyamines decrease Escherichia coli outer membrane permeability.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.     

Subunit structure of Escherichia coli exonuclease VII

Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure. The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons. This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits. The structure of the native enzyme consists of one large subunit and four small subunits. We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci. Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes.     

Computer simulations of the OmpF porin from the outer membrane of Escherichia coli.

Molecular dynamics simulations were used to study the structure and dynamics of the Escherichia coli OmpF porin, which is composed of three identical 16-stranded beta-barrels. Simulations of the full trimer in the absence of water and the membrane led to significant contraction of the channel in the interior of each beta-barrel. With very weak harmonic constraints (0.005 kcal/mol A2/atom) applied to the main-chain C alpha atoms of the beta-barrel, the structure was stabilized without alteration of the average fluctuations. The resulting distribution of the fluctuations (small for beta-strands, large for loops and turns) is in good agreement with the x-ray B factors. Dynamic cross-correlation functions showed the importance of coupling between the loop motions and barrel flexibility. This was confirmed by the application of constraints corresponding to the observed temperature factors to the barrel C alpha atoms. With these constraints, the beta-barrel fluctuations were much smaller than the experimental values because of the intrinsic restrictions on the atomic motions, and the loop motions were reduced significantly. This result indicates that considerable care is required in introducing constraints to keep proteins close to the experimental structure during simulations, as has been done in several recent studies. Loop 3, which is thought to be important in gating the pore, undergoes a displacement that shifts it away from the x-ray structure. Analysis shows that this arises from the breakdown of a hydrogen bond network, which appears to result more from the absence of solvent that from the use of standard ionization states for the side chains of certain beta-barrel residues.     

Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin.

Deep rought mutants, which produce very defective lipopolysaccharides, are unable to export normal levels of porins into the outer membrane. In this study, we showed that lipopolysaccharides from such mutants were also unable to facilitate the trimerization, in vitro, of monomeric OmpF porin secreted by spheroplasts of Escherichia coli B/r. In contrast, lipopolysaccharides containing most or all of the core oligosaccharides were able to facilitate trimerization.     

Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo.

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.     

Arabidopsis thaliana vacuolar TPK channels form functional K+ uptake pathways in Escherichia coli

Very few vacuolar two pore potassium channels (TPKs) have been functionally characterized. In this paper we have used complementation of K⁺ uptake deficient Escherichia coli mutant LB2003 to analyze the functional properties of Arabidopsis thaliana TPK family members. The four isoforms of AtTPKs were cloned and expressed in LB2003 E. coli background.The expression of channels in bacteria was analyzed by RT-PCR. Our results show that AtTPK1, AtTPK2 and AtTPK5 are restoring the LB2003 growth on low K⁺ media. The analysis of potassium uptake exhibited elevated level of K⁺ uptake in the same three types of AtTPKs transformants.     

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.     

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.     

Plasmid genes increase membrane permeability in Escherichia coli

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.     

Subunit b-dimer of the Escherichia coli ATP synthase can form left-handed coiled-coils

One remaining challenge to our understanding of the ATP synthase concerns the dimeric coiled-coil stator subunit b of bacterial synthases. The subunit b-dimer has been implicated in important protein interactions that appear necessary for energy conservation and that may be instrumental in energy conservation during rotary catalysis by the synthase. Understanding the stator structure and its interactions with the rest of the enzyme is crucial to the understanding of the overall catalytic mechanism. Controversy exists on whether subunit b adopts a classic left-handed or a presumed right-handed dimeric coiled-coil and whether or not staggered pairing between nonhomologous residues in the homodimer is required for intersubunit packing. In this study we generated molecular models of the Escherichiacoli subunit b-dimer that were based on the well-established heptad-repeat packing exhibited by left-handed, dimeric coiled-coils by employing simulated annealing protocols with structural restraints collected from known structures. In addition, we attempted to create hypothetical right-handed coiled-coil models and left- and right-handed models with staggered packing in the coiled-coil domains. Our analyses suggest that the available structural and biochemical evidence for subunit b can be accommodated by classic left-handed, dimeric coiled-coil quaternary structures.