Enhancement of α-methylglucoside efflux by respiration in respiratory mutants of Escherichia coli K-12

The rate of α-methylglucoside efflux from wild-type cells of Escherichia coli K-12 is enhanced by different substrates, as long as they are readily respired. A similar enhancement takes place in strains with impaired oxidative phosphorylation (unc mutants), regardless of their being able (strains AN120, N₁₄₄, and AN382) or unable (strain NR70) to energize the membrane through respiratory electron flow. The uncouplers carbonylcyanide-m-chlorophenylhydrazone and tetrachlorosalicylanilide do not diminish the efflux acceleration in wild-type strains or unc mutants. However, the stimulation of α-methylglucoside efflux does not occur in the mutant AN59 which cannot perform a normal respiratory electron transport due to a defective synthesis of ubiquinone. The failure to stimulate the efflux is observed with succinate, which is a typical substrate of respiration, as well as with substrates which can yield ATP both at respiratory and substrate levels such as gluconate or glycerol. Moreover, potassium cyanide nullifies the acceleration of α-methylglucoside efflux caused in any type of strain and by any substrate. These results show that neither ATP nor an energized state of the membrane appears to be needed for respiration to accelerate α-methylglucoside release from E. coli cells, and question the existence of any energy-requiring reaction for αMG exit, previously proposed by other authors.     

ATP synthesis by an artificial proton gradient in right-side-out membrane vesicles of Escherichia coli.

Membrane vesicles formed from spheroplasts of E. coli lysed in the presence of ADP and P_i produced ATP when an artificial proton gradient (acid outside) was formed across the membrane. ATP synthesis required Mg²⁺ and ADP, was inhibited by dicyclohyxylcarbodiimide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by valinomycin in the presence of KCl. Synthesis was absent in a mutant lacking the Mg²⁺-ATPase. The optimum external pH was 2.5 when the internal pH was 8.2. Oxidative phosphorylation driven by D-lactate or succinate was also observed.     

Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-octaprenyl-4-hydroxybenzoate carboxy-lyase

A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lyase was shown to require Mn²⁺, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.     

Partial characterization of anEscherichia coll strain harboring a xylanase encoding plasmid

Summary The plasmid pRH271, harboring a xylanase gene cioned fromBacilius subtilis, has been transferred into a mutant ofE. coli SK2284 which allowed the release of part of the xylanase in the culture supernatant. Kinetic parameters of this recombinantE. coll strain were determined in microscale batch culture with and without the selective pressure of antibiotics. No significant difference in µ_max was observed for the nontransformedE. coli strain when compared to the recombinant strain. However, K₅ values for glucose were two times higher in the case of the recombinant strain. Preliminary study of xylanase production in a large batch farmenter was also described.     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Beta-galactoside transport and proton movements in an adenosine triphosphatase-deficient mutant of Escherichia coli

A mutant of Escherichia coli lacking the Mg²⁺-adenosine triphosphatase (E.C.3.6.1.3.) has been previously shown to lack active transport of amino acids and sugars. It is shown that this mutant can couple proton movements to β-galactoside uptake, but that the rate of passive proton permeation is greatly increased. N,N′-dicyclohexylcarbodiimide simultaneously reduces the passive proton release and increases the β-galactoside transport. It is concluded that the mutant is uncoupled for active transport due to inability to establish a proton gradient.     

Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli

Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli B^E host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution.     

Relative substrate affinities of wild-type and mutant forms of the Escherichia coli sugar transporter GalP determined by solid-state NMR

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of ¹³C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly ¹³C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.     

Low Ubiquinone Content in Escherichia coli Causes Thiol Hypersensitivity

Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb⁻ phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery.     

Improved L-lysine production by the amplification of theCorynebacterium glutamicum dapA gene encoding dihydrodipicolinate synthetase inE. coli

Summary ThedapA gene (L-2,3-dihydrodipicolinate synthetase: DHDP synthetase) ofCorynebacterium glutamicum JS231, a lysine overproducer, cloned and subcloned inE. coli/C. glutamicum shuttle vector pECCG117 was used to transformE. coli threonine producer and threonine and lysine coproducer. The plasmid pDHDP5812 carryingdapA gene ofC. glutamicum led to increase in lysine production in theseE. coli strains. Threonine and lysine co-producerE. coli TF1 with pDHDP5812 produced lysine with small amount of threonine. The DHDP synthetase activity ofE. coli TF1 carrying pDHDP5812 showed high resistance toward inhibition by lysine.     

Genome sequence analyses of two isolates from the recent <Emphasis Type="Italic">Escherichia coli</Emphasis> outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EAHEC)

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).     

Is ubiquinone diffusion rate-limiting for electron transfer?

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 10⁹ M^–1 sec^–1 corresponding to a diffusion coefficient in the range of 10^–6 cm²/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA 81, 2606–2610, 1984) is 3×10^–9 cm²/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theK _m of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.     

Energization of phenylalanine transport and energy-dependent transhydrogenase by ATP in cytochrome-deficient Escherichia coli K12

$\text{E. coli}$ SASX76 does not form cytochromes unless supplemented with 5-aminolevulinic acid. Uptake of [¹⁴C]phenylalanine into cytochrome-deficient cells of this mutant was energized by glucose but not by endogenous substrates or D-lactate with or with or without fumarate. In contrast, uptake of this amino acid was supported in cytochrome-containing cells of this strain by oxidation of D-lactate or endogenous substrates. It is concluded that ATP can energize phenylalanine transport in cytochrome-deficient cells. Cytochrome-deficient cells lacked eńergy-dependent transhydrogenase activity driven by oxidation of NADH but ATP-driven transhydrogenation was unimpaired. Both transhydrogenase activities were present in cytochrome-containing cells.     

Point mutations change specificity and kinetics of metal uptake by ZupT from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.     

Selection aid properties of hyperproducing D-serine deaminase mutants of Escherichia coli

Summary Hyperproducing constitutive D-serine deaminase mutant E. coli were selected by a chemostat method. The specific activity of the enzyme in these mutants is 25fold higher in comparison with the fully induced parental strain.     

Restoration of active calcium transport in vesicles of an Mg2+-ATPase mutant of Escherichia coli by wild-type Mg2+-ATPase.

Strain NR70, a mutant of $\text{E. coli}$ lacking the Mg²⁺-adenosine triphosphatase (E.C. 3.6.1.3.) was previously shown to be defective in amino acid and sugar transport in whole cells and right-side-out membrane vesicles. It is shown here that the mutant is also deficient in the uptake of calcium into inverted membrane vesicles. Treatment of inverted vesicles from the wild-type strain with ethylenediamine tetraacetate removes the Mg²⁺-adenosine triphosphatase and results in an inability to transport calcium. Addition of a crude fraction containing the wild-type Mg²⁺-adenosine triphosphatase restores active uptake of calcium both to vesicles from the mutant and depleted vesicles from the wild-type.     

A high molecular weight plasmid ofZymomonas mobilis harbours genes for HgCl2 resistance

Summary Plasmids fromZ. mobilis could be stably maintained inE. coli HB101 in which the expression of various drug resistance markers could be monitored. A large molecular weight plasmid (5.2 kbp) ofZ. mobilis was found to harbour the genes for mercuric chloride degradation and to confer uponE. coli, resistance to a higher mercuric chloride concentration as compared toZ. mobilis. The introduction of this plamsid madeE. coli sensitive to concentrations of cadmium acetate which were originally non-inhibitory to it.     

GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of <Emphasis Type="Italic">E. coli</Emphasis> F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP synthase in LPPG micelles

G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F₁F₀ ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC ^αβ C ^α, L-(4,3)D HNN(CO)C ^αβ C ^α, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D ¹⁵N, ¹³C^aliphatic, ¹³C^aromatic-resolved [¹H,¹H]-NOESY with a total measurement time of ∼43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and ¹³C^β shifts, and for 97% of the side chain shifts. Moreover, the use of two G²FT NMR experiments, that is, (5,3)D HN{N,CO}{C ^αβ C ^α} and (5,3)D {C ^αβ C ^α}{CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G²FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins. Qi Zhang, Hanudatta S. Atreya, Douglas E. Kamen, Mark E. Girvin and Thomas Szyperski—New York Consortium on Membrane Protein Structure.     

Insulin decreases bacterial membrane fluidity. Is it a general event in its action?

The influence of insulin on the Hill coefficient during inhibition by Na⁺ of Escherichia coli membrane-bouna (Ca²⁺)ATPase was studied in vitro. In the presence of insulin, the values of n change from 1.9 to 1.1. Half-maximal effect was obtained at 1 × 10^?9M (140 units/ml). The hormone did not affect the allosteric behavior of the soluble enzyme. The hormone-induced-changes in the Hill coefficients are interpreted as a decrease in membrane fluidity produced by insulin. The general occurrence of this event in insulin action is suggested in this paper.