Production of enterochelin by Escherichia coli 0111.

The major neutral iron-transporting compound produced by Escherichia coli 0111/K58/H2 has been isolated from iron-deficient cultures of the organism and compared with the corresponding compound, enterochelin, produced by E. coli K12. The product contained serine and 2,3-dihydroxybenzoic acid and formed a complex with Fe3+. Since the PMR spectra of the products from the two strains were identical, it was concluded that E. coli 0111 also secreted enterochelin under iron-deficient conditions. Although it was not possible to establish the optical configuration of the serine residues in the molecule, the CD spectra of the metal free and Fe3+, complexes were found to be of the same sign and magnitude. The spectra show that metal binding results in considerable conformational changes in the enterochelin molecule. The biological properties of the two compounds appear to be identical as judged by their ability to abolish the bacteriostatic effect of serum on E. coli 0111.     

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.     

Inhibition of NAD biosynthesis by paraquat in Escherichia coli

Niacin significantly reduced the bacteristatic effect of 1 mM paraquat for Escherichia coli. Without niacin (an intermediate in the salvage pathway for pyridine nucleotide coenzyme biosynthesis), the NAD concentration was decreased rapidly and significantly in E. coli during paraquat poisoning. Niacin prevented the decline in NAD in paraquat-poisoned cells; quinolinate (an intermediate in de novo NAD biosynthesis prior to the entry point of niacin) did not. These data suggest that paraquat poisons the de novo pathway of pyridine nucleotide coenzyme biosynthesis. Similar consequences have been reported to result from hyperbaric oxygen poisoning of E. coli; thus, there is growing evidence for a common mechanism of toxicity for hyperoxia and paraquat.     

Counter-transport mediated by the lactose permease of Escherichia coli

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of pre-loaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H⁺ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide $\text{m}$ chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP⁺). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP⁺ had no inhibitory effect on the overshoot at pH 6 and below.     

Genetic activity of bleomycin in Escherichia coli

The induction of umuC gene expression, cell lethality, induction of W-reactivation of UV-irradiated λ-phage and the induction of mutagenesis caused by bleomycin (Blm) were studied in Escherichia coli K-12 strains with special references to the effects of SOS repair deficiencies. (1) The umuC gene is inducible by Blm and the induction is regulated by the lexA and recA genes. (2) The lexA and recA mutants are slightly more sensitive to Blm-killing than wild-type strain. (3) The plating efficiency of UV-irradiated λ-phage increased by Blm treatment of the host cell. This increase was not observed in the umuC mutant. The plating efficiency of UV-irradiated λ-phage was drastically reduced in the lexA and recA strains treated with Blm. (4) No significant increase of the reversion of nonsense mutation (his-4 to His⁺) in AB1157 by the treatment of Blm was observed. Possible implications of these results are discussed.     

Expression of maize prolamins in Escherichia coli

We have constructed a cDNA expression library of developing corn (Zea mays L.) endosperm using plasmid pUC8 as vector and Escherichia coli strain DH1 as host. The expression library was screened with non-radioactive immunological probes to detect the expression of gamma-zein and alpha-zein. When anti-gamma-zein antibody was used as the probe, 23 colonies gave positive reactions. The lengths of cDNA inserts of the 23 colonies were found to be 250–900 base pairs. When anti-alpha zein antibody was used, however, fewer colonies gave positive reactions. The library was also screened by colony-hybridization with ³²P-labeled DNA probes. Based on immunological and hybridization screening of the library and other evidence, we conclude that alpha-zein was either toxic to E. coli cells or rapidly degraded whereas gamma-zein and its fragments were readily expressed.     

Physical Properties of Escherichia coli Spheroplast Membranes

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.     

Ribonuclease I released from Escherichia coli by osmotic shock

Levels of Ribonuclease I (2.7.7.h) released from Escherichia coli K-37 by osmotic shock procedures were measured for cells grown under different conditions. In general, half the Ribonuclease I was recovered from E. coli K-37 by osmotic shock, but with cells grown under conditions of magnesium starvation much less activity was released.     

Counterflow efflux of thiamin in Escherichia coli

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated from an energy-dependent exit by a selective inhibition of exit by $\text{2 · 10}{}^{\mathrm{?2}}\text{M}$ NaN₃. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.     

Logarithmic Sensing in Escherichia coli Bacterial Chemotaxis

We studied the response of swimming Escherichia coli (E. coli) bacteria in a comprehensive set of well-controlled chemical concentration gradients using a newly developed microfluidic device and cell tracking imaging technique. In parallel, we carried out a multi-scale theoretical modeling of bacterial chemotaxis taking into account the relevant internal signaling pathway dynamics, and predicted bacterial chemotactic responses at the cellular level. By measuring the E. coli cell density profiles across the microfluidic channel at various spatial gradients of ligand concentration grad[L] and the average ligand concentration near the peak chemotactic response region, we demonstrated unambiguously in both experiments and model simulation that the mean chemotactic drift velocity of E. coli cells increased monotonically with grad [L]/ or ∼grad(log[L])—that is E. coli cells sense the spatial gradient of the logarithmic ligand concentration. The exact range of the log-sensing regime was determined. The agreements between the experiments and the multi-scale model simulation verify the validity of the theoretical model, and revealed that the key microscopic mechanism for logarithmic sensing in bacterial chemotaxis is the adaptation kinetics, in contrast to explanations based directly on ligand occupancy.     

The anaerobic oxidation of dihydroorotate by Escherichia coli K-12

The oxidation of dihydroorotate under anaerobic conditions has been examined using various mutant strains of Escherichia coli K-12. This oxidation in cells grown anaerobically in a glucose minimal medium is linked via menaquinone to the fumarate reductase enzyme coded for by the frd gene and is independent of the cytochromes. The same dihydroorotate dehydrogenase protein functions in both the anaerobic and aerobic oxidation of dihydroorotate. Ferricyanide can act as an artificial electron acceptor for dihydroorotate dehydrogenase and the dihydroorotate-menaquinone-ferricyanide reductase activity can be solubilised by 2 M guanidine · HCl with little loss of activity.     

Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis

A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pI) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxyl groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a pI of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a pK between 3.2 and 4.5 and the amino groups have a pK of 7.5. ε-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6′-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60 Å below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharide-phospholipid-protein complex. Since reactive groups of the phosphilipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 Å from the bacterial surface.     

Lateral phase separations in Escherichia coli membranes

Membranes from unsaturated fatty acid auxotrophs of Escherichia coli were studied by spin labeling and freeze-fracturing. From measurements of the partition of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) between the aqueous phase and fluid lipids in isolated membranes, temperatures, corresponding to the onset and completion of a lateral phase separation of the membrane phospholipids were determined. By freeze-fracture electron microscopy a change in the distribution of particle in the membrane was observed around the temperature of the onset of the lateral phase separation. When cells were frozen from above that temperature a netlike distribution of particles in the plasma membrane was observed for unfixed preparations. When frozen after fixing with glutaraldehyde the particle distribution was random. In membranes of cells frozen with or without fixing from a temperature below the onset of the phase separation, the particles were aggregated and large areas void of particles were present. This behavior can be understood in terms of the freezing rate with the aid of phase diagrams.     

Inhibition of Escherichia coli DNA polymerase I by rose bengal

The fluorescein dye, rose bengal, inhibits Escherichia coli DNA polymerase I reversibly in the dark and irreversibly in the light. The reversible inhibition, which occurs in the micromolar concentration range, is competitive with respect to the poly(dA-T) template/ primer and noncompetitive with respect to the complementary deoxynucleoside triphosphates. The Hill coefficient for the inhibition by rose bengal is 3.0. Equilibrium dialysis experiments using ¹³¹I-labeled rose bengal have demonstrated direct binding of the inhibitor to the enzyme. No dye binds to poly(dA-T) at concentrations where the inhibition is observed. There are 22 ± 3 rose bengal binding sites per polymerase which can be subdivided into a class of high affinity sites and one of low affinity sites. The high affinity sites (3 μm) bind rose bengal with a Hill coefficient of 1.7 and are responsible for the observed inhibition. The low affinity sites (7μm) are more numerous (about 16) and bind rose bengal in a noncooperative manner. The displacement of rose bengal from the enzyme by poly(dA-T) at equilibrium confirms the competition between poly(dA-T) and rose bengal inferred from the kinetic data for the polymerization reaction. The inhibition of the 3′,5′ exonuclease activity and the template-directed dATP ? P-P exchange reaction by rose bengal is fully consistent with the interaction of rose bengal at the polynucleotide binding site. The enzyme induces an extrinsic Cotton effect in the visible absorption of rose bengal. The abolition of this Cotton effect by poly(dA-T) further supports the proposed site of binding of the dye.     

Biosynthesis of enterochelin in Escherichia coli K-12 separation of the polypeptides for by the entD,E, F and G genes

Four enzymic components, coded for by the entD, entE, entF and entG genes, involved in the biosynthesis of enterochelin from 2,3-dihydroxybenzoate have been separated from cell extracts of mutant strains of Escherichia coli K-12.The starting material for fractionation of the E, F and G components was a cell extract of an entD mutant strain, which yielded the E, F and G enzymic components uncontaminated by a functional D component. The D component was isolated from cell extracts of an entE mutant strain. The conversion of 2,3-dihydroxybenzoate and l-serine into enterochelin is dependent on the presence of all four enzymic components.The E and F components were shown to catalyze ATP-pyrophosphate exchange reactions dependent on 2,3-dihydroxybenzoate and l-serine, respectively, whereas fractionated extracts of the entE and entF mutant strains lacked these reactions. These data provide firm evidence that the E and F components are involved in the initial activation of the substrates. The D and G components are necessary for subsequent and, as yet, undefined reactions.