Proteins forming large channels from bacterial and mitochondrial outer membranes: porins and phage lambda receptor protein.
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Bacterial Outer Membrane Proteins↗
Biomedical subjects
H Nikaido
Publications and source records attributed to H Nikaido.
Porin channels in Escherichia coli: studies with beta-lactams in intact cells.
Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.
Bacterial Outer Membrane Proteins↗
Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.
Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.
Bacterial Outer Membrane Proteins↗
Bacteriophage lambda receptor protein in Escherichia coli K-12: lowered affinity of some mutant proteins for maltose-binding protein in vitro.
Mutant and wild-type LamB proteins (phage lambda receptor proteins) were purified by affinity chromatography with immobilized maltose-binding protein, and their transport functions were tested in reconstituted liposomes. Two mutant proteins exhibited a marked decrease in affinity for immobilized maltose-binding protein, as well as altered transport rates.
ATP-Binding Cassette Transporters↗
A mutant form of maltose-binding protein of Escherichia coli deficient in its interaction with the bacteriophage lambda receptor protein.
In one malE mutant known to be deficient in the transport of maltose and maltodextrins across the outer membrane, the altered MalE protein was shown to be defective in its interaction with the phage lambda receptor, or LamB protein, of the outer membrane.
ATP-Binding Cassette Transporters↗
Sequence of the malK gene in E.coli K12.
We present the sequence of gene malK which encodes a component of the system for maltose transport in E.coli K12. We also determined the position of deletion (S50) which fuses malK to the following gene lamB; the malK-lamB protein hybrid contains all of the malK protein. The mRNA corresponding to the last two thirds of gene malK could form stable stem and loop structures. The malK protein, as deduced from the gene sequence, would include 370 residues and correspond to a molecular weight of 40700. The sequence as well as sequence comparisons with the ndh protein of E.coli are discussed in terms of the location and function of the malK protein.
ATP-Binding Cassette Transporters↗
Extensive homology between membrane-associated components of histidine and maltose transport systems of Salmonella typhimurium and Escherichia coli.
A strong homology was found between the amino acid sequences, deduced from DNA nucleotide sequences, of cytoplasmic membrane-associated components of the high affinity histidine transport system of Salmonella typhimurium (coded by the hisP gene) and the maltose-maltodextrin transport system of Escherichia coli (coded by the malK gene). When the HisP protein sequence was aligned with that of the NH2-terminal two-thirds of the MalK protein, 32% of the positions were identical, and an additional 35% were occupied by functionally similar amino acid residues. These results suggest that some, and possibly many, "periplasmic-binding protein-dependent" transport systems have evolved from a common ancestral system.
ATP-Binding Cassette Transporters↗
Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.
Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.
Cell Membrane Permeability↗
Genetic analyses of the vertebral anomalies of the IS strain of rat and the development of a BN congenic line with the anomalies.
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Animals↗
Genetical studies of low plasma alkaline phosphatase (ALP) activity in the IS strain of rats.
The low plasma alkaline phosphatase (ALP) activity of the IS strain of rats was genetically analyzed by using the cross matings with the BN strain. In the F1 hybrids, the ALP activities were intermediate but largely biased to the low activity. In the (F1 x BN) back-cross progeny, the distributions of plasma ALP activities were apparently bimodal in both sexes. Alp-2 locus with two alleles of Alp-2h and Alp-2l is tentatively proposed. Alp-2 locus did not link to the rat linkage group (LG) I, II, IV, V, and LGs containing Amy-1 and Cs-1. In the (F1 x IS) back-cross progeny, the plasma ALP activities were not different from that of the F1 hybrids and the distribution was not bimodal. Alp-2l may be dominant over Alp-2h. One third of the back-cross progeny showed vertebral anomalies and the difference of plasma ALP activities was not detected between the rats with and without vertebral anomalies.
Alkaline Phosphatase↗
Purification and functional characterization of mutant LamB proteins.
The phage lambda receptor, or LamB, proteins have been purified from several missense lamB mutants, and the properties of the channel produced by these proteins were investigated in a reconstituted liposome system. In at least one mutant, a significant reduction in the sugar selectivity of the channel was observed, and another mutant protein interacted poorly with the maltose-binding protein immobilized on Sepharose beads.
Bacterial Outer Membrane Proteins↗
Physical interaction between the phage lambda receptor protein and the carrier-immobilized maltose-binding protein of Escherichia coli.
When Triton X-100/EDTA extracts of the outer membrane of Escherichia coli K12 were passed through a column containing maltose-binding protein covalently linked to Sepharose 6MB beads, the phage lambda receptor protein or LamB protein was quantitatively and specifically adsorbed to the column and was eluted with a solution containing 1 M NaCl, but not with that containing 0.5 M maltose. The binding did not take place when columns containing inactivated Sepharose beads alone, or Sepharose bound to histidine-binding protein of Salmonella typhimurium, were used. This interaction is consistent with the hypothesis that the periplasmic maltose-binding protein interacts with the part of the LamB protein exposed on the inner surface of the outer membrane, thereby increasing the specificity of the solute penetration process through the LamB channel.
ATP-Binding Cassette Transporters↗
Persistence of segregated phospholipid domains in phospholipid--lipopolysaccharide mixed bilayers: studies with spin-labeled phospholipids.
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Electron Spin Resonance Spectroscopy↗
Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli.
Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.
Cell Membrane↗
Mutants of Escherichia coli that are resistant to certain beta-lactam compounds lack the ompF porin.
Carbenicillin-resistant mutants of Escherichia coli K-12 and B/r were found to produce greatly diminished levels of the porin coded by the ompF gene. Physiological and ecological implications of these findings are discussed.
Anti-Bacterial Agents↗
Identification of a cytoplasmic membrane-associated component of the maltose transport system of Escherichia coli.
The maltose transport system of Escherichia coli contains at least five components, three of which, i.e. the products of lamB, malE, and malF genes, have so far been identified as constituents of the outer membrane, periplasmic space, and cytoplasmic membrane, respectively. We identified another component, a cytoplasmic membrane protein of an apparent molecular weight of 43,000, as the product of the malK gene on the basis of polyacrylamide gel electrophoretic analysis of various mutants and suppressed strains and by the incorporation of extra tyrosine residue into this proten in malK amber mutants containing the suppressor Su3+ allele. The transport of maltose thus appears to require at least two proteins associated with the cytoplasmic membrane.
Alleles↗
Diffusion of solutes through channels produced by phage lambda receptor protein of Escherichia coli: inhibition by higher oligosaccharides of maltose series.
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Bacterial Outer Membrane Proteins↗
Mitochondrial outer membrane contains a protein producing nonspecific diffusion channels.
Vesicles permeable to sucrose, but not to high molecular weight dextrans, were produced when fragments of mitochondrial outer membrane obtained from rat liver or mung bean seedlings were fused with liposomes of soybean phospholipids. By using this reconstitution assay, the channel-forming protein in the mung bean mitochondria was identified as protein(s) with an apparent molecular weight of 30,000 through differential detergent extraction and centrifugation in a sucrose density gradient. The channel was nonspecific, and allowed the diffusion of saccharides of up to 2000 to 8000 daltons.
Animals↗