Isolation of detergent-resistant membranes (DRMs) from Escherichia coli

Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells.     

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation.Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.     

Isolation,solubilization and reaggregation of outer membrane of Escherichia coli

Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.     

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extremely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Chlolesterol was shown to suppress the polymyxin-induced response in liposomes.     

Crystal structure of D-serine dehydratase from Escherichia coli

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5′-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97 Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55 Å resolution. The structure of DSD reveals a larger pyridoxal 5′-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.     

Nucleotide regulation of phosphoenolpyruvate carboxylase from Escherichia coli

Adenine, cytosine, guanine, and uracil nucleotides were surveyed as possible modulators of Escherichia coli phosphoenolpyruvate carboxylase. CMP, CDP, CTP, GDP, and GTP activate, ATP and GMP inhibit. The other nucleotides are without effect. Nucleotide activation is synergistic with acetyl-CoA or laurate. Cytosine nucleotide activation is also synergistic with fructose 1,6-diphosphate, whereas guanine nucleotide activation is not. The pH profiles for CMP and GDP activation, studied individually between pH 7.0 and 9.0, are similar to those for activation by fructose 1,6-diphosphate. ATP inhibits activation by acetyl-CoA, laurate, or fructose 1,6-diphosphate. Pairs of activators synergistically relieve the inhibition. Acetyl-CoA with laurate is most effective. Energy charge profiles suggest little sensitivity to charge fluctuation near 0.8. Ribose 5-phosphate also inhibits activation by acetyl-CoA, laurate, or fructose 1,6-diphosphate. GMP selectively inhibits fructose 1,6-diphosphate activation.     

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12

The thiol-oxidizing agent “diamide” (CH₃)₂NCON=NCON(CH₃)₂ was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic processes. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in γ-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide range of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.     

Phosphate transport in membrane vesicles from Escherichia coli

Escherichia coli strain AN710 possesses only the PIT system for phosphate transport. Membrane vesicles from this strain, which contain phosphate internally, perform exchange and active transport of phosphate. The energy for active transport is supplied by the respiratory chain with ascorbate-phenazine methosulphate as electron donor. To a lesser extent also the oxidation of d-lactate energizes phosphate transport; the oxidation of succinate is only marginally effective. Phosphate transport is driven by the proton-motive force and in particular by the pH gradient across the membrane. This view is supported by the observation that phosphate transport is stimulated by valinomycin, inhibited by nigericin and abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Neither inhibitor affects phosphate exchange. The phosphate analogue arsenate inhibits both the exchange reaction and active transport. Both processes are stimulated by K⁺ and Mg²⁺, the highest activities being observed with both ions present.Membrane vesicles have also been isolated from Escherichia coli K10, a strain which possesses only a functional PST phosphate transport system. These vesicles perform neither exchange nor active transport of phosphate, although active transport of amino acids is observed in the presence of ascorbate-phenazine methosulphate or d-lactate.     

Transport of uridine in Escherichia coli B and A showdomycin-resistant mutant

The mechanism of uridine transport in Escherichia coli B cells was studied using experimental approaches designed to limit possible ambiguities in interpretation of data obtained previously. For this purpose, the transport of [2-¹⁴C]uridine and [U-¹⁴C]uridine was determined in E. coli B and an E. coli B mutant which is resistant to the inhibitory effects of the nucleoside antibiotic, showdomycin.The majorty of the uridine transported as the intact nucleoside is cleaved to uracil and ribose l-phosphate. The uracil, in large part, is excreted, while ribose l-phosphate is retained. In addition, uridine is also rapidly cleaved to uracil and ribose l-phosphate in the periplasmic space. The uracil moiety may enter the cell, whereas ribose l-phosphate is not transported. The showdomycin-resistant mutant transports the intact nucleoside inefficiently, or not at all, but retains its ability to convert uridine to uracil in the periplasmic space.     

The purification of methionine sulfoxide reductase from Escherichia coli

A sensitive assay, based on the acylation of tRNA^Met, has been developed to measure the enzymatic reduction of methionine sulfoxide to methionine. Using this assay, methionine sulfoxide reductase has been purified to near homogeneity from extracts of Escherichia coli.     

Rapid purification of highly active ribosomes from Escherichia coli

Highly active salt-washed ribosomes from Escherichia coli are isolated by gel filtration on Sephacryl S-200 in a buffer containing 1 m ammonium chloride and putrescine, spermidine, calcium, and magnesium ions. Up to several hundred grams of cells can be processed in less than 24 h. The ribosome solution made 30% (v/v) in methanol is best stored as a liquid at ?20°C. An improved poly(U)-dependent peptide synthesis assay is described in which phenylalanine polymerization is greatly enhanced while the misincorporation of leucine is kept at in vivo levels.     

Crystal structure of non-redox regulated SSADH from Escherichia coli

SSADH is involved in the final step of GABA degradation, converting SSA to succinic acid in the human mitochondrial matrix, and its activity is known to be regulated via ‘redox-switch modulation’ of the catalytic loop. We present the crystal structure of EcSSADH, revealing that the catalytic loop of EcSSADH, unlike that of human SSADH, does not undergo disulfide bond-mediated structural changes upon changes of environmental redox status. Subsequent redox change experiments using recombinant proteins confirm the non-redox regulation of this protein. Detailed structural analysis shows that a difference in the conformation of the connecting loop (β15-β16) causes the formation of a water molecule-mediated hydrogen bond network between the connecting loop and the catalytic loop in EcSSADH, making the catalytic loop of EcSSADH more rigid compared to that of human SSADH. The cytosolic localization of EcSSADH and the cellular function of the GABA shunt in E. coli might result in the non-redox mediated regulatory mechanisms of the protein.     

Genome-scale genetic engineering in Escherichia coli

Genome engineering has been developed to create useful strains for biological studies and industrial uses. However, a continuous challenge remained in the field: technical limitations in high-throughput screening and precise manipulation of strains. Today, technical improvements have made genome engineering more rapid and efficient. This review introduces recent advances in genome engineering technologies applied to Escherichia coli as well as multiplex automated genome engineering (MAGE), a recent technique proposed as a powerful toolkit due to its straightforward process, rapid experimental procedures, and highly efficient properties.     

ATP-binding cassette transporters in Escherichia coli

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.     

Regulation of the intracellular potassium concentration in Escherichia coli B 525

The mutant Escherichia coli B 525 requires histidine, leucine and methionine and an elevated extracellular K⁺ concentration for growth, and is unable to retain K⁺ tightly inside the cells when incubated in media supplemented with glucose, arabinose, galactose or lactose as the sole energy and carbon source. The loss of K⁺ from the cells of B 525 can be prevented by adding histidine and leucine, which react specifically and only in combination. In media supplemented with glycerol as the substrate, with glucose and NH₄⁺, or with glucose under anaerobic conditions, a stationary level of K⁺ inside the cells can be obtained without the addition of histidine-leucine.On the addition of ribose to glycerol-adapted cells of B 525 preincubated in glycerol media, the intracellular K⁺ decreased immediately and markedly. This decrease can be overcome by the addition of histidine-leucine.