Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> nicotinate mononucleotide adenylyltransferase mutants <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD⁺ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD⁺, especially for one of the alleles, nadD72.     

Quantum dot-based array for sensitive detection of <Emphasis Type="Italic">Escherichia coli</Emphasis>

A fluorescent quantum dot-based antibody array, used in sandwich format, has been developed to detect Escherichia coli O157:H7. Numerous parameters such as solid support, optimal concentration of immunoreagents, blocking reagents, and assay time were optimized for array construction. Quantum dot-conjugated anti-IgG was used as the detecting system. The array allows the detection of E. coli O157:H7 at concentrations below 10 CFU mL⁻¹ without sample enrichment, exhibiting an increase of three orders of magnitude in the limit of detection compared to ELISA. The interference caused by Gram (+) and Gram (−) bacteria was negligible at low concentrations of bacteria.     

Liquid chromatography/mass spectrometry characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> species

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.     

Functional Analysis of Ribonucleotide Reductase from <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cordyceps militaris produces cordycepin (3′-deoxyadenosine), which has various activities, including anti-oxidant, anti-tumoral, anti-viral, and anti-inflammatory. Ribonucleotide reductase (RNR) seems to be a candidate to produce cordycepin in C. militaris because RNR catalyzes the reduction of nucleotides to 2′-deoxynucleotides, whose structures are similar to that of cordycepin. However, the role of RNR has not been confirmed yet. In this study, complementary DNAs (cDNAs) of C. militaris RNR (CmRNR) large and small subunits (CmR1 and CmR2) were cloned from C. militaris NBRC9787 to investigate the function of CmRNR for its cordycepin production. C. militaris NBRC9787 began to produce cordycepin when grown in a liquid surface culture in medium composed of glucose and yeast extract for 15 days. CmR1 cDNA and CmR2 cDNA were obtained from its genomic DNA and from total RNA extracted from its mycelia after cultivation for 21 days, respectively. Recombinant CmR1 and CmR2 were expressed individually in Escherichia coli and purified. Purified recombinant CmR1 and CmR2 showed RNR activity toward adenosine diphosphate (ADP) only when two subunits were mixed but only show the reduction of ADP to 2′-deoxyADP. These results indicate that the pathway from ADP to 3′deoxyADP via CmRNR does not exist in C. militaris and cordycepin production in C. militaris may be mediated by other enzymes.     

Quercetin Glucoside Production by Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli strains expressing the O-glucosyltransferases UGT73B3 or UGT84B1 were compared for the production of glucosides from quercetin supplied into a defined medium. The formation of quercetin-3-glucoside (Q3G) by UGT73B3 showed a maximum at 33 °C, while the formation of quercetin-7-glucoside by UGT84B1 increased with increasing temperature to 37 °C. The highest concentrations of Q3G were attained by strains having a deletion in the pgi gene-coding phosphoglucose isomerase, which effectively blocked the entry of glucose-6P into the Embden–Meyerhof–Parnas pathway. Formation of Q3G was improved in 1-L controlled bioreactors compared to shake flask cultures, a result attributed to the greater oxygen transfer rate in bioreactors. Under batch conditions with 30 g/L glucose as the sole carbon source, E. coli MEC367 (MG1655 pgi) expressing UGT73B3 generated 3.9 g/L Q3G in 56 h.     

Strain-Dependent Carotenoid Productions in Metabolically Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids, also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion, we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information can serve as the basis for the subsequent development of microorganisms for carotenoids of interest.     

Determination of viable <Emphasis Type="Italic">Escherichia coli</Emphasis> using antibody-coated paramagnetic beads with fluorescence detection

A rapid and convenient assay system was developed to detect viable Escherichia coli in water. The target bacteria were recovered from solution by immunomagnetic separation and incubated in tryptic soy broth with isopropyl-β-d-thiogalactopyranoside, which induces formation of β-galactosidase in viable bacteria. Lysozyme was used to lyse E. coli cells and release the β-galactosidase. β-Galactosidase converted 4-methylumbelliferyl-β-d-galactoside to 4-methylumbelliferone (4-MU), which was measured by fluorescence spectrophotometry using excitation and emission wavelengths of 355 and 460 nm, respectively. Calibration graphs of 4-MU fluorescence intensity versus E. coli concentration showed a detection range between 8 × 10⁴ and 1.6 × 10⁷ cfu mL⁻¹, with a total analysis time of less than 3 h. The advantage of this method is that it detects viable cells because it is based on the activity of the enzyme intrinsic to live E. coli.     

Study of the growth of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>, <Emphasis Type="Italic">Escherichia coli</Emphasis> and their mixtures by microcalorimetry

In a majority of environments, microbes live as interacting communities. Microbial communities are composed of a mix of microbes with often unknown functions. Polymicrobial diseases represent the clinical and pathological manifestations induced by the presence of multiple infectious agents. These diseases are difficult to diagnose and treat and usually are more severe than monomicrobial infections. The interaction relationship between Enterococcus faecalis and Escherichia coli was researched using a Calvet calorimeter. Three mixtures of both bacteria were prepared in the following proportions: 20 + 80 % (0.2 mL E. faecalis + 0.8 mL E. coli), 50 + 50 % (0.5 mL E. faecalis + 0.5 mL E. coli) and 80 + 20 % (0.8 mL E. faecalis + 0.2 mL E. coli). Experiments were carried out at concentration of 10⁶ CFU mL^?1 and a constant temperature of 309.65 K. The differences in shape of graph of E. faecalis, E. coli and their mixtures were compared. Also, the thermokinetic parameters such as detection time (t _d), growth constant (k), generation time (G) and the amount of heat released (Q) were calculated.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Expression of a <Emphasis Type="Italic">hemA</Emphasis> Gene from <Emphasis Type="Italic">Agrobacterium radiobacter</Emphasis> in a Rare Codon Optimizing <Emphasis Type="Italic">Escherichia coli</Emphasis> for Improving 5-aminolevulinate Production

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.     

Rapid Determination of <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine using Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> Overexpressing Glutamine Synthetase

A genetically engineered Escherichia coli was developed as the source of enzyme for rapidly quantifying glutamine. E. coli BL21 (DE3) cells overexpressing a glutamine synthetase from Bacillus subtilis were prepared as tube aliquots and used in a small volume of nontoxic mixture. The current method was compared to high performance liquid chromatography analysis, Sigma kit (GLN-1) and Mecke method. The method is applicable to a wide range of glutamine concentrations (0.05–2.5 mM) and correlates well to the detection results obtained from high performance liquid chromatography (Pearson correlation is 0.978 at the 0.01 level). Moreover, the whole assay procedure takes less than 15 min and uses nontoxic reagents, so it can be applied to monitor glutamine production and utilization conveniently.     

Cloning of a Heat-Stable Chitin Deacetylase Gene from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and its Functional Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml⁻¹ for the glycol chitin substrates, and its specific activity was 4.17 U mg⁻¹ for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used as the substrate, K _m was 4.92 mg ml⁻¹, and K _cat showed 6.25 s⁻¹, thus the ratio of K _cat and K _m was 1.27 ml s⁻¹ mg⁻¹. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.     

Tandem Multimer Expression and Preparation of Hypoglycemic Peptide MC6 from <Emphasis Type="Italic">Momordica charantia</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Tandem repeat multimers of Momordica charantia (MC) peptide MC6 were designed and the recombinant plasmid containing 10 copies of MC6 gene was constructed to improve the expression level of MC6 in Escherichia coli. Under the selected conditions of cultivation and induction, the expression level of recombinant TrxA–MC6₁₀ protein was above 25% of total bacteria protein. This fusion protein was purified and cleaved with HCl (13%, w/v). Either the un-cleaved or cleaved recombinant proteins was analyzed pharmacological activity by alloxan-induced diabetic mice and only the cleaved products of the recombinant protein showed significant hypoglycemic effects. The study provides a convenient and economical method for the large-scale production of anti-diabetic medicines for pharmaceutical applications.     

Cloning and Expression of Che a 1, the Major Allergen of <Emphasis Type="Italic">Chenopodium album</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b (+) vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni–NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.     

Solid-phase microbead array for multiplex O-serotyping of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Typing and classification of Escherichia coli (E. coli) according to cell wall components, like polysaccharides, is routinely done by serotyping. Given the presence of 188 known O-antigens, this process is complex. The authors present a proof-of-concept planar microbead array for multiplexed O-serotyping. Ten clinically relevant E. coli serotypes associated with high risk for diarrhea in humans were examined (O26, O55, O78, O118, O124, O127, O128, O142, O145 and O157). Antisera were assigned to specific microbead populations, which can be differentiated by size and fluorescence color. Automatted image processing and data analysis were conducted by a microscopic interpretation platform. Homogenous antiserum coating of the microbeads was demonstrated by an intra-population CV that ranges from 3.3 to 6.3% and by an inter-population CV of 9.5%. Typical detections limits are in the range from 0.31 to 0.71 refMFI. Significantly elevated fluorescence signals revealed that E. coli of a certain serogroup bound specifically to microbeads with the matching antiserum (p < 0.001). In our perception, the method represents a viable diagnostic tool for automated multiplex serotyping of E. coli. It enables simultaneous and high-throughput screening for different O-antigens by a simple staining and binding protocol.

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Graphical abstract Schematic of a planar microbead array for the typing and classification of E. coli according to cell wall components. Based on coated fluorescent microbeads, multiplex O-serotyping of E. coli is accomplished via fluorescence imaging.

     

Copolymer of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride with maleic acid as drug carrier

A copolymer of N,N-diallyl-N,N-dimethylammonium chloride with maleic acid of constant composition was prepared under the conditions of radical initiation. The possibility of the functionalization of the copolymer with drugs containing amino groups by polymer-analogous transformations was examined. Conditions were found for preparing conjugates of the copolymer with isoniazid. The structures and the quantitative compositions of the conjugates were determined by ¹³С NMR spectroscopy, and the possibility of preparing conjugates with controlled drug content was demonstrated.     

Optimization of Fermentation Conditions for the Biosynthesis of <Emphasis Type="SmallCaps">l</Emphasis>-Threonine by <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this study, the fed-batch fermentation technique was applied to improve the yield of l-threonine produced by Escherichia coli TRFC. Various fermentation substrates and conditions were investigated to identify the optimal carbon source, its concentration and C/N ratio in the production of l-threonine. Sucrose was found to be the optimal initial carbon source and its optimal concentration was determined to be 70 g/L based on the results of fermentations conducted in a 5-L jar fermentor using a series of fed-batch cultures of E. coli TRFC. The effects of glucose concentration and three different feeding methods on the production of l-threonine were also investigated in this work. Our results showed that the production of l-threonine by E. coli was enhanced when glucose concentration varied between 5 and 20 g/L with DO-control pulse fed-batch method. Furthermore, the C/N ratio was a more predominant factor than nitrogen concentration for l-threonine overproduction and the optimal ratio of ammonium sulfate to sucrose (g/g) was 30. Under the optimal conditions, a final l-threonine concentration of 118 g/L was achieved after 38 h with the productivity of 3.1 g/L/h (46% conversion ratio from glucose to threonine).     

Biocatalytic Properties of a Recombinant <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> Lactonase with Significantly Enhanced Production by Optimal Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The levo-lactonase gene of Fusarium proliferatum ECU2002 (EC3.1.1.25) was cloned and expressed in Escherichia coli JM109 (DE3) for biocatalytic resolution of industrially important chiral lactones, including DL-pantoyl lactone which was a key precursor to calcium d-pantothenate. By increasing the biomass concentration and lowering the inducer (isopropyl-β-d-thiogalactoside) concentration and induction temperature, the lactonase production was significantly enhanced up to 20 kU/L, which was 20 times higher than that of wild-type strain F. proliferatum ECU2002. The recombinant Fusarium lactonase was purified using immobilized metal affinity chromatography, and its SDS-PAGE revealed a molecular mass of 50 kDa for the recombinant protein, suggesting that the enzyme was a simplex protein. Furthermore, biocatalytic properties of the recombinant lactonase were investigated, including kinetic parameters, additive’s effect, and substrate specificity. The results reported in this paper provide a feasible method to make the whole cells of E. coli JM109 (DE3) expressing lactonase gene to be a highly efficient and easy-to-make biocatalyst for asymmetric synthesis of chiral compounds.     

Inhibitory Effect of Biosynthesized Silver Nanoparticles from Extract of <Emphasis Type="Italic">Nitzschia palea</Emphasis> Against Curli-Mediated Biofilm of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Extended spectrum beta lactamase (ESBL) are emerging beta-lactamases in Gram-negative pathogens, causing serious problems in hospitalized patients worldwide. Biofilm mode of virulence has decreased the efficiency of antibiotics used for treatment against ESBL pathogens. Therefore, there is an urgent need for alternative agents such as nanoparticles that can prevent and inhibit the biofilm formation. The aim of the present study was to inhibit the biofilm formed by ESBL-producing Escherichia coli using silver nanoparticles (AgNPs) synthesized with fresh water diatom (Nitzschia palea). AgNPs were characterized using UV-Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscope (FESEM), energy-dispersive X-ray spectroscopy (EDX), and XRD. AgNPs at their biofilm inhibitory concentration (BIC) of 300 ng ml^?1 significantly reduced the biofilm formed by E. coli. Interestingly, Congo red assay revealed the reduction of curli, essential for biofilm formation in the presence of AgNPs. Light and CLSM examination of the biofilm images also validated that in the presence of AgNPs, the biofilm architecture was disintegrated and the thickness was significantly reduced. Overall, the present study exemplifies the use of AgNPs as a plausible alternative for conventional coating agents on implant devices to prevent and control biofilm-associated urinary tract infections.