Ecological restoration of arsenic contaminated soil by Arundo donax L

The possible arsenic tolerance mechanisms were explored in Arundo donax L. under various supplied arsenic concentrations. The treatments included control (no metal) and five doses of arsenic trioxide i.e., 0, 50, 100, 300, 600 and 1000 μg L⁻¹ As to A. donax. The phytoextraction ability of A. donax L. plants was assessed using both the translocation and bioaccumulation factors. The transpirates were collected to analyze the arsenic concentration volatilized along-with study of anatomical characteristics of the plant parts. In general, the arsenite and arsenate accumulation linearly increased in roots, shoot and leaves with the increasing supplied arsenic levels i.e., from 2.348, 2.775 and 3.25 μg g⁻¹ at 50 μg L⁻¹ to 50, 53.125 and 64.25 μg g⁻¹ arsenite, at 1000 μg L⁻¹, from 4.075, 5.425 and 13.56 μg g⁻¹ at 50 μg L⁻¹ to 71, 62.02 and 436.219 μg g⁻¹ arsenate at 1000 μg L⁻¹, respectively. The order of arsenic accumulation in A. donax L. was: solution As(III) < Root As(III) < Shoot As(III) < Leaf As(III) < Solution As(V) < Root As(V) < Shoot As(V) < Leaf As(V). The range of arsenic volatilization by A. donax L. was 7.2–22% at higher supplied arsenic (300–1000 μg L⁻¹). Volatilization was an important mechanism to avoid toxic effects of arsenic by A. donax L. in addition to bioaccumulation.     

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.     

Production of a fusion protein of sweet potato sporamin from recombinant E. coli XL1 Blue by fed-batch fermentations

Glucose-stat and pH-stat control strategies were employed in order to culture a recombinant E. coli XL1 Blue to produce a fusion protein of sweet potato sporamin (SPA) and glutathione S-transferase (GST) from the recombinant E. coli XL1 Blue. Cell densities up to 25 g l^–1 and 28.9 mg fusion protein (GST-SPA) g^–1 cell dry weight (CDW) was achieved from a fed-batch fermentation controlled by glucose-stat strategy. A pH-stat control fermentation using glycerol as a carbon source gave E. coli up to 27 g l^–1 and 31.5 mg GST-SPA g^–1 CDW. Additionally, a pH-stat control strategy using glucose as a carbon source gave E. coli up to 15 g l^–1 and about 22.7 mg g^–1 CDW of GST-SPA.     

Overexpression of a Single Membrane Component from the Bacillus mer Operon Enhanced Mercury Resistance in an Escherichia coli Host

Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 μM Hg²⁺, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.     

Cephalexin-Induced Morphological Alterations in the Surface Structures of Staphylococcus aureus and Escherichia coli Demonstrated by Scanning Electron Microscopy

The scanning electron microscope was used to investigate the alterations in surface morphology of Staphylococcus aureus 209P and Escherichia coli NIH induced by the action of cephalexin known to interfere with cell-wall synthesis. Exposure to cephalexin produced a series of changes on the surface morphology in proportion to the concentrations of cephalexin added. Untreated S. aureus cells had smooth contours. Exposure to 1 μg/ml of cephalexin during the logarithmic phase of growth in S. aureus did not produce any detectable changes. Upon exposure of S. aureus to 5 μ/ml or 10 μg/ml, some cells were larger than normal and showed abnormal cell division-like structures in part. When S. aureus was exposed to 50 μg/ml, cell division was completely inhibited, and no formation of grape-like clusters was observed. Untreated E. coli cells appeared to have smooth and regular contours. E. coli propagated almost normally upon exposure of the organisms to 1 μg/ml of cephalexin. Filamentous structures were observed with the exposure of E. coli to 12.5 μg/ml or 25 μg/ml, but spheroplast-like structures were not observed. Exposure to 100 μg/ml of cephalexin resulted in the formation of marked filamentous cells and spheroplast-like structures having multiple small saccular outpouchings. Scanning electron microscope demonstrated more completely the morphological abnormalities induced by cephalexin.     

Purification of Recombinant HIV-1 Protease

Abstract

A method is described to purify recombinant HIV-1 protease from soluble extracts of Escherichia coli. The isolation involves QAE-Sepharose anion exchange chromatography, hexyl agarose hydrophobic interaction chromatography, MonoS cation exchange chromatography, and Superose 6 size exclusion chromatography. Approximately 100 μg of protease was obtained from 18 g E. coli paste. The protein was judged to be homogeneous due to the presence of a single band on a silver-stained SDS polyacrylamide gel.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.     

Simple monitoring of cell leakiness and viability in Escherichia coli bioprocesses—A case study

In a recently published study, we developed a simple methodology to monitor Escherichia coli cell integrity and lysis during bioreactor cultivations, where we intentionally triggered leakiness. In this follow‐up study, we used this methodology, comprising the measurement of extracellular alkaline phosphatase to monitor leakiness and flow cytometry to follow viability, to investigate the effect of process parameters on a recombinant E. coli strain producing the highly valuable vascular endothelial growth factor A165 (VEGF‐A165) in the periplasm. Since the amount of soluble product was very little (<500 μg/g dry cell weight), we directly linked the effect of the three process parameters temperature, specific uptake rate of the inducer arabinose and specific growth rate (μ) to cell integrity and viability. We found that a low temperature and a high μ were beneficial for cell integrity and that an elevated temperature resulted in reduced viability. We concluded that the recombinant E. coli cells producing VEGF‐A165 in the periplasm should be cultivated at low temperature and high μ to reduce leakiness and guarantee high viability. Summarizing, in this follow‐up study we demonstrate the usefulness of our simple methodology to monitor leakiness and viability of recombinant E. coli cells during bioreactor cultivations.     

Arsenic species uptake and translocation in Elodea canadensis

Abstract

The capacity of Elodea canadensis to phytofiltrate arsenic species from water was evaluated. Plants were adapted to tap water and supplemented with 15 and 250?µg L^?1 of As. Inorganic arsenic species (As III, As V), and organic arsenic compounds: monomethylarsonate (MMA) and dimethylarsinate (DMA) were analyzed. Sampling was carried out at different times after exposure in culture water and plant organs. Plants exposed to 15?µg L^?1 of As concentration showed no significant difference on As concentration (95% confidence level) in their organs compared to controls. When plants were exposed to 250?µg L^?1 of As concentration, a significant increase of As concentration in plant organs was observed. After 1?h exposure, plants reduce 63.16% the As concentration in the culture water, with a bioaccumulation factor (BF) of 4.3. Under these conditions, E. canadensis accumulate As V in roots and do not translocate it to stems (transfer factor <1). MMA was determined in stems and leaves. E. canadensis effectively phytofiltrate As from tap water of a city located in an arsenic endemic area from concentrations of 36?µg L^?1 to undetectable levels (10?ng L^?1).     

The characteristics of Escherichia coli adsorption of arsenic(III) from aqueous solution

The potential of using Escherichia coli (E. coli) as adsorbent for the adsorption of As(III) from aqueous solution was assessed. Various parameters like pH, initial concentration and temperature have been investigated. It is found that the adsorption of As(III) is pH dependent and the optimum value was 2.0. Kinetics studies revealed that the equilibrium of the adsorption of As(III) on to E. coli was obtained within 30 min and the process was followed with the pseudo-2nd-order kinetic model. The equilibrium adsorption data obtained at different temperatures were fitted better with Langmuir than Freundich isotherm. The adsorption capacity of E. coli for As(III) is increased with the increasing of temperature and the maximum adsorption capacity is 1.111 mg/g which obtained at 40°C. Potentiometric titration and Fourier transform infra-red spectroscopy were applied to reveal the mechanisms of the As(III) binding. Amino, amide, amine group and C–H of the alkane are determined to be involved in As(III) binding based on the results.     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1 -fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.     

Renal bioaccumulation of trace elements in urban and rural Sri Lankan populations: A preliminary study based on post mortem tissue analysis

IntroductionEnvironmental pollution, especially by toxic trace elements, is a global health concern. Heavy metals such as Cadmium (Cd), Arsenic (As) and Lead (Pb) are associated with numerous disorders and are considered by some as an aetiological factor for the Chronic Kidney Disease (CKDu¹) epidemic in Sri Lanka. This study explores patterns of bioaccumulation of six trace elements in kidneys obtained during forensic autopsies from urban and rural regions in Sri Lanka.MethodsKidney samples obtained from one urban district (n = 13) and three rural districts (n = 18) were lyophilized, microwave digested and profiled by ICP-MS techniques.Results and DiscussionThe mean age of the sampled population was 47.9 ± 11.3 yrs. Median (IQR) for Cd, As, Pb, Cr, Zn and Se were, 14.67(8.04–22.47) μg/g, 0.44(0.29–0.56) μg/g, 0.11(0.07–0.30) μg/g, 0.15(0.1096–0.3274), 25.55(17.24–39.35) μg/g and 0.52(0.37−0.84) μg/g, respectively. Cd, Zn and Se levels were significantly higher (p < 0.05) among the urban samples compared to that of the rural group. Zn and Se levels were higher among younger age groups. As, Pb and Cr did not show any significant differences between the two cohorts nor any correlations with age.ConclusionThis population-specific baseline study provides an insight into the differences in exposure to toxic trace elements and essential elements between urban and rural populations. Residents in CKDu affected rural districts did not appear to be at risk of toxic heavy metal exposure, however their renal bioaccumulation of nephroprotective essential elements was lower than urban residents.     

Expression of a soluble flavone synthase allows the biosynthesis of phytoestrogen derivatives in Escherichia coli

Flavones are plant secondary metabolites with potent pharmacological properties. We report the functional expression of FSI, a flavonoid 2-oxoglutarate-dependent dioxygenase-encoding flavone synthase from parsley in Escherichia coli. This expression allows the biosynthesis of various flavones from phenylpropanoid acids in recombinant E. coli strains simultaneously expressing five plant-specific flavone biosynthetic genes. The gene ensemble consists of 4CL-2 (4-coumarate:CoA ligase) and FSI (flavone synthase I) from parsley, chsA (chalcone synthase) and chiA (chalcone isomerase) from Petunia hybrida, and OMT1A (7-O-methyltransferase) from peppermint. After a 24-h cultivation, the recombinant E. coli produces significant amounts of apigenin (415 μg/l), luteolin (10 μg/l), and genkwanin (208 μg/l). The majority of the flavone products are excreted in the culture media; however, 25% is contained within the cells. The metabolic engineering strategy presented demonstrates that plant-specific flavones are successfully produced in E. coli for the first time by incorporating a soluble flavone synthase confined only in Apiaceae.     

Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence

New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation. Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

Development of a full‐length human protein production pipeline

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in Escherichia coli and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip^® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.     

The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P < 0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts.