Isolation of a Potential Anchoring Motif Based on Proteome Analysis of Escherichia coli and Its Use for Cell Surface Display

For bacterial cell surface display, the target protein needs to be linked to an anchoring motif, and it is essential to choose an appropriate anchoring motif for efficient and stable display of the protein on the cell surface. To isolate a potential anchoring motif that would allow a stable and enhanced display of target proteins on the surface of an Escherichia coli host, we analyzed the outer membrane proteome of E. coli. On the basis of this proteomic analysis, the outer membrane protein X (OmpX), which has a small, monomeric β-barrel structure and is highly expressed, was selected as a potential anchoring motif. The role of OmpX as an anchoring motif for cell surface display was demonstrated using three important industrial enzymes: endoxylanase, lipase, and alkaline phosphatase. Two different positions (Lys¹²², Val¹⁶⁰) in the extracellular loops of OmpX were examined for C-terminal fusion, and the biological activities and localization of the displayed enzymes were analyzed. All three enzymes examined were efficiently displayed on the E. coli cell surface with high activity. These results reveal that the use of OmpX as an anchoring motif is an efficient method to display functional enzymes on the surface of an E. coli host.     

Structural and physico-chemical characteristics of tetraethylammonium tetrachloridoferrate(III)

The crystal structure of tetraethylammonium tetrachloridoferrate(III) was determined. The crystals are hexagonal, space group P6₃ mc, a = 8.198(1) Å, b = 8.198(1) Å, c = 13.183(3) Å, V = 767.3(2) Å³, Z = 2. The asymmetric unit of [(C₂H₅)₄N][FeCl₄] consists of half each of a tetraethylammonium cation and a tetrachloridoferrate(III) anion. Tetrachloridoferrate(III) ion adopts almost ideal tetrahedral geometry. Structural characterization of the compound is supplemented by the results of its magnetic susceptibility measurements and electron paramagnetic resonance (EPR) spectra. Magnetic measurements of a powdered [(C₂H₅)₄N][FeCl₄] sample gave a negative Weiss constant of ?1.57, which suggests antiferromagnetic coupling. The susceptibility curve of [(C₂H₅)₄N][FeCl₄] against temperature exhibits a maximum, indicating the presence of antiferromagnetic ordering with a Neel temperature of approximately 2.9 K.     

Nonthermal Atmospheric Argon Plasma Jet Effects on Escherichia coli Biomacromolecules

Nonthermal atmospheric plasma jet, a promising technology based on ionized gas at low temperatures, can be applied for disinfection of contaminated surfaces. In this study, Escherichia coli cells and their macromolecules were exposed to the nonthermal atmospheric argon plasma jet for different time durations. Total protein, genomic DNA, and malondialdehyde (MDA) levels of E. coli were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining; agarose gel electrophoresis; and measurement of absorbance at 534 nm, respectively. After exposure, the spectroscopic results of liquid samples indicated that the survival reduction of E. coli can reach to 100 % in an exposure time of 600 s. Moreover, inactivation zones of E. coli, DNA degradation, and MDA levels were significantly increased. Additionally, banding patterns of total protein were changed and amino acid concentrations increased following ninhydrin test. The experimental results suggest that the nonthermal plasma could serve as an effective instrument for both sterilizing E. coli and degrading macromolecules from the surface of the objects being sterilized.     

Antimicrobial Activity of Electrospun Poly(butylenes succinate) Fiber Mats Containing PVP-Capped Silver Nanoparticles

In this study, biodegradable poly(butylenes succinate) (PBS) fiber mats containing silver nanoparticles (AgNPs) were prepared by the electrospinning process. Small AgNPs (<10 nm) were simply synthesized using polyvinylpyrrolidone as the capping agent as well as the reductant. The morphology of the PBS-AgNPs fiber mats and the distribution of the AgNPs were well characterized by TEM and SEM. The release of Ag from the PBS fiber mats was quantitively determined by ICP. The PBS fiber mats with 0.29 % AgNPs content showed strong antimicrobial activity against both gram-positive Staphylococcus aureus and gram-negative Escherichia coli with the efficacy as high as 99 %. The effective bactericidal activity on E. coli was demonstrated for a short contacting time with the PBS-AgNPs fiber mats. In addition, the long-term release performance of Ag from the fiber mats can keep inhibiting the bacterial growth in the mats over a long period of time.     

Growth of Escherichia coli under extremely low-frequency electromagnetic fields

The inflence of extremely low-frequency (ELF) electromagnetic fields on Escherichia coli cultures in submerse fermentation was studied. The fermentation processes were carried out recycling the culture medium externally through a stainless steel tube inserted in a magnetic field generator (solenoid). The exposure time and electromagnetic induction were varied in a range of 1 to 12 h and 0.010 to 0.10 T, respectively, according to a Box-Wilson Central Composite Designs of face centered with five central points. Growth of E. coli could be altered (stimulated or inhibited) under magnetic fieldinduced effects. E. coli culturesexposed at 0.1 T during 6.5 h exhibited changes in its viability compared to unexposed cells, which was 100 times higher than the control. The magnetic field generator associated with the cellular suspension recycle is a new way of magnetic treatment in fermentation processes and could be appropriate to industrial scale up.     

Evaluation of ferricyanide effects on microorganisms with multi-methods

In this study, we report the effects of ferricyanide on organisms based on the changes in physiological state and morphology of Escherichia coli (E. coli) DH 5 α after being pretreated by ferricyanide. The impact on bacterial cell growth and viable rate of exposure to different concentrations of ferricyanide was determined, and the morphology change of E. coli was studied by atomic force microscopy (AFM). Finally, recovery test was used to evaluate the recovery ability of injured cells. The results showed that the effects on growth and morphology of E. coli were negligible when the concentration of ferricyanide was below 25.0 mM. While the results showed 50.8% inhibition of growth in the presence of 50.0 mM ferricyanide for 3 h, 89.6% viability was detected by flow cytometry (FCM) assay. AFM images proved that compact patches appeared on the bacterial surface and protected the bacterial viability. Furthermore, the results revealed that deterioration of bacterial surface closely related to the incubation time from 0.5 to 3 h at 100.0 mM ferricyanide. In the recovery test, microbial cell population and dissolved oxygen individually decreased 36.7% and 28.3% with 25.0 mM ferricyanide. These results clearly demonstrated that ferricyanide indeed affected viability of cells than morphology damaged, and the effects of toxin on bacteria were not reversible.     

Comparison of two innovatives approaches for bacterial detection: paramagnetic nanoparticles and self-assembled multilayer processes

Transfusion medicine is a field that has developed in the second half of the last century. Very rapidly, however, it became clear that this approach also carried its problems, such as the incompatibility of red blood cells and plasma between donors and recipients, and the possibility of transmitting viral and bacterial infections. An immunomagnetic biosensor for the label-free detection of a bacterial model, Escherichia coli, is described and compared to a self assembled multilayer system reported previously. The paramagnetic nanoparticles layer attracted to, and formed on, the gold electrode surface via a magnetic field up to 300 mT is not totally blocking for the redox probe comparing to the thiol self assembled monolayer (a biotin thiol and a spacer thioalcohol). Moreover, the modeling of the Nyquist spectra obtained by electrochemical impedance spectroscopy for increasing concentrations of E. coli shows for both system a sigmoid variation of the polarization resistance with increasing logarithmic concentration of bacteria. A sensitivity slope of 10.675 was obtained for the immunomagnetic sensor compared to 6.832 for the self assembled multilayer process, this indicating the higher sensitivity of the paramagnetic nanoparticles biosensor.     

Self-supported bacterial cellulose polyaniline conducting membrane as electromagnetic interference shielding material: effect of the oxidizing agent

Conducting bacterial cellulose (BC) membranes coated with a high proportion of polyaniline (PAni) were prepared through in situ oxidative polymerization of aniline on the surface of the BC in the presence of acetic acid as the protonating agent. The effect of two different oxidizing agents, ammonium persulfate (APS) or iron(III) chloride (FeCl₃), on the mechanical performance, electrical conductivity, crystallinity, morphology and ability to absorb the electromagnetic radiation was investigated. BC/PAni membranes prepared with FeCl₃ displayed higher conductivity and better mechanical performance than those observed for pure BC or the BC/PAni membranes prepared with APS. Experiments related to the electromagnetic absorbing properties revealed that BC/PAni membranes prepared with FeCl₃ also present improved absorbing properties in the frequency range of 8–12 GHz. The morphology of the membranes, observed by field emission gun-scanning electron microscopy, is strongly affected by the oxidizing agent. Whereas the BC/PAni membranes prepared with APS present PAni nanoparticles attached on the fiber surface as agglomerates in the form of flakes, those prepared with FeCl₃ display a uniform and smooth coating of PAni on the BC fibers as hierarchical mode.     

Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions

The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO₃ concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C–H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag⁺-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag⁺ concentrations (<10 μM AgNO₃) tested under “dynamic” growth mode: early addition vs. pulse in the mid-exponential phase, and compared to simultaneously batch-grown untreated bacteria or cells sampled just before the pulse, respectively. sFTIR microspectroscopy and TEM imaging were performed in close relation with growth kinetics characterization. No significant effect of the Ag⁺ pulses was detected, in accordance with macrokinetics data. For early-treated cells, effects on fatty acid composition were shown, although no major alteration of protein secondary structure was noticed. These partial effects were consistent with TEM observations and growth kinetics.

Effect of adenine on bacterial translocation using technetium-99m labeled E. coli in an intestinal obstruction model in rats

This study aims to investigate effects of adenine on bacterial translocation (BT) using ^99mTc-labeled E. coli in an intestinal obstruction rat model. In the study twenty-one rats were used. The rats were divided into three groups according to different feeding patterns. The control group (CG) was fed with a standard chow diet for 7 days. Group A1 and group A2 were fed with adenine supplemented chow diet for 7 days. At the end of the feeding period, after all groups was submitted intestinal obstruction. ^99mTc-E. coli was injected into the rats’ terminal ileum under anesthetic. The rats were sacrificed under aseptic conditions at 24th h after the surgery. The uptake of ^99mTc-E. coli was determined in organs such as the liver, mesenteric lymph nodes, spleen and ileum. Group A1 and group A2 results show that the uptake of ^99mTc-E. coli decreased in the blood and organs comparing to the CG. As a result, it was observed that adenine reduced the level of BT when compared with CG. The beneficial effect of adenine on BT in intestinal obstruction was observed. However, further studies are needed to more clearly assess how this benefit can be achieved.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Microcalorimetric investigation on antibacterial activity of the peptide from Plutella xylostella

The antibacterial activities of a kind of novel peptide from Plutella xylostella (pxCECA1) on methicillin-resistant staphylococcus aureus (MRSA) and Escherichia coli (E. coli) growth were investigated by microcalorimetry. The heat flow power–time curves of MRSA and E. coli growth in the presence of pxCECA1 were recorded using the 3114/3115 Thermal Activity Monitor Air Isothermal Calorimeter based on ampoule mode at 37 °C. Some parameters including growth rate constant k, maximum power output P _max, total heat output Q_t, generation time t _g, growth inhibitory ratio I, and half-inhibitory concentration of the drugs IC₅₀ were obtained to elucidate the antibacterial activity of pxCECA1. The results showed that k, P _max, and Q _t decreased, but I and t _g increased or delayed with the increase in pxCECA1 concentration. The IC₅₀ of pxCECA1 on E. coli was 6.122 μg mL^?1 and MRSA was 7.809 μg mL^?1. It could be concluded that pxCECA1 had stronger inhibitory effect on E. coli than MRSA. In vivo test was simultaneously performed using an E. coli and MRSA infection model to validate the antibacterial activity of pxCECA1. The results revealed that pxCECA1 with broad spectrum antimicrobial activities hopefully represented a class of promising substitute of antimicrobial agents.     

Influence of pH on the Molecular Weight of Poly-3-hydroxybutyric Acid (P3HB) Produced by Recombinant Escherichia coli

The production of ultrahigh molecular weight poly-3-hydroxybutyric acid (P3HB) from carbohydrates by recombinant Escherichia coli harboring genes from Ralstonia eutropha was evaluated. In shaken-flask experiments, E. coli XL1 Blue harboring plasmid pSK::phaCAB produced P3HB corresponding to 40 and 27 % of cell dry weight from glucose and xylose, respectively. Cultures in bioreactor using glucose as the sole carbon source at variable pH values (6.0, 6.5, or 7.0) allowed the production of P3HB with molecular weight varying between 2.0 and 2.5 MDa. These figures are significantly higher than the values often obtained by natural bacterial strains (0.5–1.0 MDa). Contrary to reports of other authors, no influence of pH was observed on the molecular weight of the polymer produced. Using xylose, P3HB with high molecular weight was also produced, indicating the possibility to produce these polymers from lignocellulosic materials.     

Expression and Characterization of Hyperthermotolerant Xylanases,SyXyn11P and SyXyn11E,in Pichia pastoris and Escherichia coli

Both Syxyn11P and Syxyn11E, two codon-optimized genes encoding glycoside hydrolase (GH) family 11 hyperthermotolerant xylanases (designated SyXyn11P and SyXyn11E), were synthesized and inserted into pPIC9K^M and pET-28a(+) vectors, respectively. The resulting recombinant expression vectors, pPIC9K^M-Syxyn11P and pET-28a(+)-Syxyn11E, were transformed into Pichia pastoris GS115 and Escherichia coli BL21, respectively. The maximum activities of two recombinant xylanases (reSyXyn11P and reSyXyn11E) expressed in P. pastoris and E. coli reached 30.9 and 17.8 U/ml, respectively. The purified reSyXyn11P and reSyXyn11E displayed the same pH optimum at 6.5 and pH stability at a broad range of 4.5–9.0. The temperature optimum and stability of reSyXyn11P were 85 and 80 °C, higher than those of reSyXyn11E, respectively. Their activities were not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of reSyXyn11P toward birchwood xylan were 4.3 mg/ml and 694.6 U/mg, whose K _m was close to that (4.8 mg/ml), but whose V _max was much higher than that (205.6 U/mg) of reSyXyn11E. High-performance liquid chromatography analysis indicated that xylobiose and xylotriose as the major products were excised from insoluble corncob xylan by reSyXyn11P.     

Coexpression of the Pyrroloquinoline Quinone and Glucose Dehydrogenase Genes from Serratia marcescens CTM 50650 Conferred High Mineral Phosphate-Solubilizing Ability to Escherichia coli

The genes gdh and pqqABCDE encoding glucose dehydrogenase and its pyrroloquinoline quinone cofactor were cloned from the mineral phosphate-solubilizing (MPS) bacterium Serratia marcescens CTM 50650. We investigated, for the first time, the impact of their coexpression in Escherichia coli on MPS ability. The production of recombinant PQQGDH conferred high MPS activity to the engineered E. coli. In fact, the amounts of soluble phosphorus (P) produced from tricalcium phosphate, hydroxyapatite, and Gafsa rock phosphate (GRP) were 574, 426, and 217 mg/L, respectively. In an attempt to increase the soluble P concentration, the E. coli strain coexpressing the gdh and pqqABCDE genes was immobilized in agar, calcium alginate, and k-carrageenan and was then further applied in a repeated batch (six batches) fermentation process to solubilize GRP. Compared to other encapsulated systems, alginate cell beads were noted to yield the highest concentration of soluble P, which attained 300 mg/L/batch. MPS efficiency was maximal in the presence of 5 and 40 g/L of GRP and glucose, respectively.     

Immunomagnetic nanoparticle-based sandwich chemiluminescence-ELISA for the enrichment and quantification of E. coli

Rapid methods for the quantification of Escherichia coli are required for the monitoring of faecal contamination in water to secure public health. The immunomagnetic separation (IMS) offers rapid enrichment and purification of bacteria in complex matrices and is compatible with immunoassays. By means of this technique, non-target cells and matrix components which might interfere with subsequent analytical methods are removed. We present the synthesis of magnetic nanoparticles (MNPs) and covalent coupling to antibodies against the enterobacterial common antigen (ECA) for use with IMS. Quantification was carried out with a chemiluminescence-based sandwich enzyme-linked immunosorbent assay (ELISA). Our anti-ECA-MNPs allow for a group-specific enrichment of bacterial cells, which can be combined with a species-specific analytical method. The particles were used along with commercially available magnetic columns for the selective enrichment of E. coli from 10-mL water samples. The volumetric enrichment factor was 9. For enriched samples, the limit of detection was reduced from 5.0?×?10⁶ cells·mL^-1 to 2.6?×?10⁵ cells·mL^-1. Using 200 µL anti-ECA-MNPs, we determined a recovery of 97?±?6% for a sample containing 10⁶ cells·mL^-1 and 89?±?2% for a sample containing 10⁷ cells·mL^-1. The overall time for cell enrichment and detection was 3 h 45 min.     

Comparison of the low-frequency magnetic field effects on bacteria Escherichia coli, Leclercia adecarboxylata and Staphylococcus aureus

This work studies biological effects of low-frequency electromagnetic fields. We have exposed three different bacterial strains-Escherichia coli, Leclercia adecarboxylata and Staphylococcus aureus to the magnetic field (t<30 min, B(m)=10 mT, f=50 Hz) in order to compare their viability (number of colony-forming units (CFU)). We have measured the dependence of CFU on time of exposure and on the value of the magnetic field induction B(m). Viability decreases with longer exposure time and/or higher induction B(m) for all strains, but the quantity of the effect is strain-dependent. The highest decrease of the viability and the biggest magnetic field effect was observed with E. coli. The smallest magnetic field effect appears for S. aureus. From the measurement of the growth dynamics we have concluded that the decrease of the CFU starts immediately after the magnetic field was switched on.     

Synthesis,thermal decomposition and dielectric behavior of bis(thiourea)silver(I)nitrate

New semi-organic bis(thiourea)silver(I)nitrate (TuAgN) single crystals have been grown from slow evaporation solution growth technique. Single crystal X-ray diffraction study reveals that the crystal belongs to orthorhombic system with the non-centrosymmetric space group C2221 and the calculated cell parameters are a = 33.3455 (6) Å, b = 45.2957 (7) Å, c = 20.3209 (5) Å, α = β = γ = 90°, and V = 30692.8 (10) Å ³. The thermal stability and decomposition behavior of TuAgN compound have been studied by thermogravimetric analysis at three different heating rates 5, 10, and 15 °C min^?1. The effective activation energy (E _a) and pre-exponential factor (ln A) of thermal decomposition of thiourea from TuAgN compound at three different heating rates are estimated by model free methods: Arrhenius, Flynn–Wall, Kissinger, and Kim–Park. The calculated effective activation energies were found to vary with the fraction (α) reacted. The compensation effect between the (ln A) and (E _a) has also been studied. Dielectric properties of TuAgN crystal have been studied in a wide range of frequencies and temperatures. AC conductivity has also been carried out.     

Influences of levofloxacin salts on the metabolism of Escherichia coli by microcalorimetry

A microcalorimetric technique was used to evaluate the influence of both Levofloxacin lactate in sodium chloride injection (drug A) and Levofloxacin hydrochloride in sodium chloride injection (drug B) on the metabolism of Escherichia coli. By means of an isothermal calorimeter and ampoule method at 37 °C, the power-time curves of E. coli growth were obtained under different conditions. The parameters such as the growth rate constant k, maximum power output P _m, time t _m corresponding to the maximum power output and inhibitory ratio I of these two drugs were obtained. The results reveal that the inhibitory abilities enhance with increasing concentrations of the two drugs. The critical growth concentration and the half-inhibitory concentration IC₅₀ were 0.15 and 0.079 μg mL^?1 (for drug A), 0.13 and 0.061 μg mL^?1 (for drug B), respectively. These results show the drug A has slightly better inhibitory effect on E. coli than that of drug B.     

Magnetic history of hexanediammonium tetrachloroferrate(II)

The magnetic susceptibility of the two-dimensional antiferromagnet (CH₂)₆(NH₃)₂ FeCl₄ is reported. The compound has unusual dependence on its magnetic history below its Neel temperature T_N. Though the magnetic ordering is associated with a structural distortion, this distortion does not seem to explain the magnetic behaviour of the compound. The results have been interpreted in terms of mixed magnetic phases which are likely to exist when the sample is cooled in a magnetic field.