Synthetic hydraphile channels of appropriate length kill Escherichia coli

Crown ether-based synthetic cation conducting channels called hydraphiles show clear ionophoretic activity in phospholipid vesicles. These compounds are shown to be active against the bacterium E. coli. Disk diffusion assays were performed to assess the toxicity of different hydraphile derivatives. Liquid culture tests were conducted to quantitate the dependence of bacterical activity on channel length. It is proposed that hydraphiles are toxic to bacteria as a result of channel formation in the membrane. The bactericidal activity is found to depend at least on the presence of a functional central relay and proper channel length. It is speculated that hydraphiles insert into the bilayer and disrupt the cell's osmotic balance, leading to cell death.     

Electrochemical attachment of motile bacterial cells to gold

Selective attachment of Escherichia coli K-12 bacterial cells to charged gold surfaces was demonstrated. Electrostatic binding of E. coli K-12 bacterial cells to positively charged surfaces was observed starting at +750 mV. The binding of E. coli K-12 cells to positively charged gold surfaces is proposed to occur due to long-range electrostatic interactions between the negatively charged O-chain of lipopolysaccharide (LPS) molecules protruding the bacterial cell body and the electrode surface. Removing LPS alters the cellular surface charge and results in cellular attachment to negatively charged surfaces. Thus, applying an electrical potential allows for the direct, real time detection of live, dead or damaged bacterial cells. The attachment of E. coli K-12 bacterial cells to surfaces with an applied potential substantiates the hypothesis that an electrostatic interaction is responsible for the binding of bacterial cells to positively charged molecular assemblies on surfaces used for building bacterial microarrays.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

Attachment of motile bacterial cells to prealigned holed microarrays

Construction of biomotors is an exciting area of scientific research that holds great promise for the development of new technologies with broad potential applications in areas such as the energy industry and medicine. Herein, we demonstrate the fabrication of prealigned microarrays of motile Escherichia coli bacterial cells on SiOx substrates. To prepare these arrays, holed surfaces with a gold layer on the bottom of the holes were utilized. The attachment of bacteria to the holes was achieved via nonspecific interactions using poly-l-lysine hydrobromide (PLL). Our data suggest that a single motile bacterial cell can be selectively attached to an individual hole on a surface and bacterial cell binding can be controlled by altering the pH, with the greatest occupancy occurring at pH 7.8. Cells attached to hole arrays remained motile for at least 4 h. These data indicate that holed surface structures provide a promising footprint for the attachment of motile bacterial cells to form high-density site-specific functional bacterial microarrays.     

Biotransformation of Benzoate to 2,4,6-Trihydroxybenzophenone by Engineered Escherichia coli

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.     

Synthesis of Ferulenol by Engineered Escherichia coli: Structural Elucidation by Using the In Silico Tools

4-Hydroxycoumarin (4HC) has been used as a lead compound for the chemical synthesis of various bioactive substances and drugs. Its prenylated derivatives exhibit potent antibacterial, antitubercular, anticoagulant, and anti-cancer activities. In doing this, E. coli BL21(DE3)pLysS strain was engineered as the in vivo prenylation system to produce the farnesyl derivatives of 4HC by coexpressing the genes encoding Aspergillus terreus aromatic prenyltransferase (AtaPT) and truncated 1-deoxy-D-xylose 5-phosphate synthase of Croton stellatopilosus (CstDXS), where 4HC was the fed precursor. Based on the high-resolution LC-ESI(±)-QTOF-MS/MS with the use of in silico tools (e.g., MetFrag, SIRIUS (version 4.8.2), CSI:FingerID, and CANOPUS), the first major prenylated product (named compound-1) was detected and ultimately elucidated as ferulenol, in which information concerning the correct molecular formula, chemical structure, substructures, and classifications were obtained. The prenylated product (named compound-2) was also detected as the minor product, where this structure proposed to be the isomeric structure of ferulenol formed via the tautomerization. Note that both products were secreted into the culture medium of the recombinant E. coli and could be produced without the external supply of prenyl precursors. The results suggested the potential use of this engineered pathway for synthesizing the farnesylated-4HC derivatives, especially ferulenol.     

Extending carbon chain length of 1-butanol pathway for 1-hexanol synthesis from glucose by engineered Escherichia coli

An Escherichia coli strain was engineered to synthesize 1-hexanol from glucose by extending the coenzyme A (CoA)-dependent 1-butanol synthesis reaction sequence catalyzed by exogenous enzymes. The C4-acyl-CoA intermediates were first synthesized via acetyl-CoA acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and trans-enoyl-CoA reductase (Ter) from various organisms. The butyryl-CoA synthesized was further extended to hexanoyl-CoA via β-ketothiolase (BktB), Hbd, Crt, and Ter. Finally, hexanoyl-CoA was reduced to yield 1-hexanol by aldehyde/alcohol dehydrogenase (AdhE2). Enzyme activities for the C6 intermediates were confirmed by assays using HPLC and GC. 1-Hexanol was secreted to the fermentation medium under anaerobic conditions. Furthermore, co-expressing formate dehydrogenase (Fdh) from Candida boidinii increased the 1-hexanol titer. This demonstration of 1-hexanol production by extending the 1-butanol pathway provides the possibility to produce other medium chain length alcohols using the same strategy.     

Synthesis of DnaK and GroEL in Escherichia coli cells exposed to different magnetic field signals

The effects of extremely low frequency magnetic field (ELF-MF)(1 mT, 50 Hz) on the heat shock protein (HSP) synthesis in Escherichia coli were investigated. Two magnetic field signals were studied: sinusoidal (SMF) and pulsed square wave (PMF). It was found that bacteria exposed to SMF showed a significantly higher level of DnaK and GroEL proteins as compared to sham-exposed bacteria as revealed by Western blot, whereas a lower level was observed after PMF exposure. Similar results were obtained when bacterial cells were exposed to heat shock (HS) after ELF-MF exposure: again SMF and PMF resulted in an increase and in a reduction of HSP amount in comparison with sham control, respectively. In conclusion, the MF influences the synthesis of HSPs in E. coli in a way that critically depends on the signal characteristics.     

Direct electrochemistry and electrocatalytic mechanism of evolved Escherichia coli cells in microbial fuel cells

E. coli cells evolved under electrochemical tension in a microbial fuel cell possess direct electrochemical behavior due to the excretion of hydroquinone derivatives through a highly permeable outer membrane, and their catalyzed fuel cell demonstrates excellent performance.     

Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag

Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO₂) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO₂ capture and utilization. In particular, the CA of Thermovibrio ammonificans (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in Escherichia coli have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of Hydrogenovibrio marinus CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of Neisseria gonorrhoeae CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.     

Critical factors for reproducible capillary electrophoresis of Escherichia coli cells

Sharp peaks of Escherichia coli JM 109 (up to 1 300 000 theoretical plates) were recorded with either extremely diluted (< 5 mM) or extremely concentrated (ca. 150 mM) Tris-borate (TB) running buffers. However, under the conditions of yielding sharp peaks, migration time of E. coli was irreproducible. Critial factors influencing reproducibility were found to include bacterial growth phase, storage condition, cell pretreatment before injection, and concentration of running buffer. Buffer concentrations in the range of 20-100 mM TB were essential for reproducibility. E. coli JM 109 was shown to be sensitive to ultrasonification. Bacterial growth and storage conditions could be monitored by CE, with results comparable to those obtained with optical methods.     

蛋白折叠液相色谱法复性和纯化大肠杆菌表达的重组人粒细胞集落刺激因子

用蛋白折叠液相色谱法(PFLC)对大肠杆菌表达的包涵体形式的重组人粒细胞集落刺激因子(rhG-CSF)进行了复性并同时纯化。用Cu2+-亚氨基二乙酸（IDA） Sepharose作为固定化金属离子亲和色谱的固定相。在低浓度脲存在下,以咪唑为洗脱剂,采用线性梯度洗脱rhG-CSF。该法仅通过一步PFLC分离,减少了复性过程中rhG-CSF的聚集,复性后的rhG-CSF的比活性为1.8×108 IU/mg,纯度为97%,质量回收率为32%。     

Acetaldehyde Detoxification Using Resting Cells of Recombinant Escherichia coli Overexpressing Acetaldehyde Dehydrogenase

Acetaldehyde dehydrogenase (E.C. 1.2.1.10) plays a key role in the acetaldehyde detoxification. The recombinant Escherichia coli cells producing acetaldehyde dehydrogenase (ist-ALDH) were applied as whole-cell biocatalysts for biodegradation of acetaldehyde. Response surface methodology (RSM) was employed to enhance the production of recombinant ist-ALDH. Under the optimum culture conditions containing 20.68 h post-induction time, 126.75 mL medium volume and 3 % (v/v) inoculum level, the maximum ist-ALDH activity reached 496.65?±?0.81 U/mL, resulting in 12.5-fold increment after optimization. Furthermore, the optimum temperature and pH for the catalytic activity of wet cells were 40 °C and pH 9.5, respectively. The biocatalytic activity was improved 80 % by permeabilizing the recombinant cells with 0.075 % (v/v) Triton X-100. When using 2 mmol/L NAD⁺ as coenzyme, the permeabilized cells could catalyze 98 % of acetaldehyde within 15 min. The results indicated that the recombinant E. coli with high productivity of ist-ALDH might be highly efficient and easy-to-make biocatalysts for acetaldehyde detoxification.     

Optimization of Culture Conditions for Enhanced Lysine Production Using Engineered Escherichia coli

In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on l-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0–5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of l-lysine was obtained after 72 h of culture, with l-lysine yield and productivity of 45.4 % and 1.9 g/(L?·?h), respectively.     

Ultraviolet-B-Induced Damage to Escherichia coli Fpg Protein

We investigated the effect of UVB light (290 < or = lambda < or = 320 nm) on the structure and enzymatic activities of Escherichia coli Fpg protein (2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine-DNA glycosylase), a DNA repair enzyme containing a zinc finger motif and five chromophoric Trp residues. Irradiation with UVB light of air-saturated pH 7.4 buffered aqueous solutions of Fpg induces the formation of polymers as shown by sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis. In argon-saturated solutions, polymer formation produces a precipitate. The polymerization quantum yield is 0.07 +/- 0.01 and 0.15 +/- 0.02 in air- and argon-saturated solutions, respectively. In the polymerized Fpg protein, second-derivative absorption spectroscopy indicates that three and one Trp residues are destroyed in air- and argon-saturated solutions, respectively. Polymers are devoid of all three activities of the Fpg protein, whereas the unpolymerized protein retains full activities. Matrix-assisted laser desorption/ionization experiments demonstrate that polymer formation is accompanied by the formation of short polypeptides containing the first 32 or 33 residues of the N-terminal domain. Theses polypeptides are most probably formed by the photolytic cleavage of Fpg protein induced by light absorption by the adjacent Trp-34 residue.     

Discrimination between single Escherichia coli cells using time-resolved confocal spectroscopy

We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidic stream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopy are used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellow fluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliter detection volumes. The measured burst width characteristics are predominately governed by the fluorescence quantum yield and absorption cross section of the proteins used. It is these characteristics which were used to distinguish between cells with high precision. By utilizing scTCSPC individual fluorescence lifetimes originating from single cells could also be determined. Average fluorescence lifetimes are determined using standard deconvolution procedures. The simplicity of the approach for obtaining well-defined burst width distributions is expected to be extremely valuable for single-cell sorting experiments.     

Multi-walled carbon nanotubes for plasmid delivery into Escherichia coli cells

Introduction of foreign genes into bacterial cells (transformation) is used for supplementing defective genes or providing additional biological functions. Transformation can be achieved using either chemical or physical methods, e.g., electroporation. Bulk electroporation offers several advantages over chemical methods, including high transformation efficiency, but its application is limited due to the high numbers of cells and plasmids needed as a result of the high death rate of cells during this process, and the difficulty in electroporating single cells. Synthetic inorganic gene nanocarriers have received limited attention in the transformation of bacterial cells. Here we present a plasmid delivery system based on water dispersible multi-walled carbon nanotubes (CNTs) that can simultaneously target the bacterial surface and deliver the plasmids into the cells via temporary nanochannels across the cell envelope. Transformation experiments performed on E. coli provide evidence for the high potential of CNTs for nanoscale cell electroporation.     

Inhibition of sulfur incorporation to transfer RNA by ultraviolet-A radiation in Escherichia coli

tRNA sulfurtransferase activity was assayed in Escherichia coli cell extracts obtained from bacterial suspensions exposed to a sub-lethal dose of ultraviolet-A radiation (fluence 148 kJ m(-2)) imparted at a low fluence rate (41 W m(-2)). We found that the irradiation reduced the enzymatic activity to one fourth of the control value, indicating that ultraviolet-A exposure inhibits the synthesis of 4-thiouridine, the most abundant thionucleoside in E. coli tRNA. Changes in the tRNA content of 4-thiouridine and its derived photoproduct 5-(4'-pyrimidin 2'-one) cytosine were studied in bacteria growing under ultraviolet-A irradiation. In these conditions the accumulation of photoproduct was limited, and the kinetics of this process was non-coincident with disappearance of 4-thiouridine. The results, which are compatible with the fact that ultraviolet-A induces an inhibition of the 4-thiouridine synthesis, suggest that the effect of radiation on tRNA modification is relevant to tRNA photo-inactivation in growing bacteria.     

Asymmetric Baeyer-Villiger oxidations of 4-mono- and 4,4-disubstituted cyclohexanones by whole cells of engineered Escherichia coli.

Whole cells of an Escherichia coli strain that overexpresses Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase have been used for the Baeyer-Villiger oxidations of a variety of 4-mono- and 4,4-disubstituted cyclohexanones. In cases where comparisons were possible, this new biocatalytic reagent provided lactones with chemical yields and optical purities that were comparable to those obtained from the purified enzyme or a strain of bakers' yeast that expresses the same enzyme. The efficient production of cyclohexanone monooxygenase in the E. coli expression system (ca. 30% of total soluble protein) allowed these oxidations to reach completion in approximately half the time required for the engineered bakers' yeast strain. Surprisingly, 4,4-disubstituted cyclohexanones were also accepted by the enzyme, and the enantioselectivities of these oxidations could be rationalized by considering the conformational energies of bound substrates along with the enzyme's intrinsic enantioselectivity. The enzyme expressed in E. coli cells also oxidized several 4-substituted cyclohexanones bearing polar substituents, often with high enantioselectivities. In the case of 4-iodocyclohexanone, the lactone was obtained in > 98% ee and its absolute configuration was assigned by X-ray crystallography. The crystal belongs to the monoclinic P2(1) space group with a = 5.7400(10), b = 6.1650(10), c = 11.377(2) A, b = 99.98(2) degrees, and Z = 2. Taken together, these results demonstrate the utility of an engineered bacterial strain in delivering useful chiral building blocks in an experimentally simple manner.