Virulence Factors of Enteric Pathogenic Escherichia coli: A Review

Escherichia coli are remarkably versatile microorganisms and important members of the normal intestinal microbiota of humans and animals. This harmless commensal organism can acquire a mixture of comprehensive mobile genetic elements that contain genes encoding virulence factors, becoming an emerging human pathogen capable of causing a broad spectrum of intestinal and extraintestinal diseases. Nine definite enteric E. coli pathotypes have been well characterized, causing diseases ranging from various gastrointestinal disorders to urinary tract infections. These pathotypes employ many virulence factors and effectors subverting the functions of host cells to mediate their virulence and pathogenesis. This review summarizes new developments in our understanding of diverse virulence factors associated with encoding genes used by different pathotypes of enteric pathogenic E. coli to cause intestinal and extraintestinal diseases in humans.     

Proof of Concept for the Detection with Custom Printed Electrodes of Enterobactin as a Marker of Escherichia coli

The rapid and decentralized detection of bacteria from biomedical, environmental, and food samples has the capacity to improve the conventional protocols and to change a predictable outcome. Identifying new markers and analysis methods represents an attractive strategy for the indirect but simpler and safer detection of pathogens that could replace existing methods. Enterobactin (Ent), a siderophore produced by Escherichia coli or other Gram-negative bacteria, was studied on different electrode materials to reveal its electrochemical fingerprint—very useful information towards the detection of the bacteria based on this analyte. The molecule was successfully identified in culture media samples and a future goal is the development of a rapid antibiogram. The presence of Ent was also assessed in wastewater and treated water samples collected from the municipal sewage treatment plant, groundwater, and tap water. Moreover, a custom configuration printed on a medical glove was employed to detect the target in the presence of another bacterial marker, namely pyocyanin (PyoC), that being a metabolite specific of another pathogen bacterium, namely Pseudomonas aeruginosa. Such new mobile and wearable platforms offer considerable promise for rapid low-cost on-site screening of bacterial contamination.     

牛源大肠杆菌F41菌毛蛋白的原核表达和多克隆抗体的制备

目的原核表达及纯化牛源产肠毒素性大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)F41菌毛蛋白,并制备F41菌毛蛋白的多克隆抗体。方法以ETEC基因组为模板,采用PCR法扩增F41菌毛基因,克隆入表达载体pQE-30,转化大肠杆菌XL1-Blue,IPTG诱导表达,并进行SDS-PAGE分析,表达产物经切胶纯化,免疫新西兰大白兔,制备多克隆抗体,采用间接ELISA法测定抗血清效价,Western blot分析F41菌毛蛋白与多抗的反应原性。结果重组表达质粒pQE-30-F41经双酶切及测序证实构建正确;F41菌毛重组蛋白以包涵体形式表达,相对分子质量约为32 000;重组蛋白的表达量约占菌体总蛋白的35%,纯化的重组蛋白纯度可达92%;制备的抗血清效价达1∶2.56×106以上,Western blot结果表明F41菌毛蛋白与制备的多抗具有良好的反应原性。结论已成功原核表达并纯化了F41菌毛重组蛋白,且制备了高效价的多克隆抗体,为单克隆抗体制备及其免疫检测方法的建立奠定了基础。     

The isolation of poly(3‐hydroxybutyrate) from recombinant Escherichia coli XL1‐blue using the digestion method

This study examines the isolation of poly(3‐hydroxybutyrate) (PHB) from recombinant Escherichia coli XL1‐blue pBHB2 OLD‐2 harbouring the PHB biosynthesis gene. Six types of commercial surfactants (Emal 10P, Emal AD‐25, Emal S PASTE, Neopelex S‐S, Triton X‐100 and cetyl trimethyl ammonium bromide (CTAB)) were screened for PHB isolation by solubilising non‐polymer cellular material (NPCM) in the cell. Emal 10P, Emal AD‐25, Emal S PASTE and Triton X‐100 are suitable surfactants for PHB isolation. Factors such as the reaction temperature, reaction time, ratio of NPCM/surfactant and pH were investigated to achieve optimum conditions. For 80% of the PHB content in dry cells, the purity and recovery of the obtained PHB was 98% and 99% when 0.67 of NPCM/Emal S PASTE was used at 70°C for 30 min. The cost of the used surfactant is below 0.5 USD/kg (PHB). The pretreatment and multistage digestion method must be combined when the PHB content is lower than 80%. © 2011 Canadian Society for Chemical Engineering     

外源蛋白在大肠杆菌中的可溶性表达策略

长期以来,大肠杆菌一直是表达外源蛋白的首选表达系统. 但由于外源蛋白在表达过程中容易被宿主细胞蛋白酶降解或者形成包涵体,其应用受到了限制. 本文综述了在大肠杆菌中表达可溶外源蛋白的策略和进展,以期提高具有生物活性的外源基因的表达水平.     

Optimization of 1,3‐propanediol production by novel recombinant Escherichia coli using response surface methodology

Response surface methodology was used to optimize 1,3-propanediol production by a novel recombinant Escherichia coli JM109 (pHsh-dhaB-yqhD). The optimal fermentation parameters for enhanced 1,3-propanediol yield were found to be: glycerol 61.8 g L⁻¹, yeast extract 6.2 g L⁻¹, Vitamin B₁₂ 0.049 g L⁻¹ and fermentation time 30 h. Subsequent experimental trials confirmed the validity of the model. These optimal fermentation conditions in the cultivation flask culture led to a 1,3-propanediol concentration of 43.1 g L⁻¹ and a conversion rate of 69.7% (g g⁻¹). A maximum 1,3-propanediol concentration of 41.1 g L⁻¹ was achieved in a 5 L fermenter using the optimized parameters. Copyright © 2006 Society of Chemical Industry     

大肠杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种重要的化工原料,具有广阔的市场. 微生物发酵法生产琥珀酸可以解决常规化学合成法对石油的依赖. 代谢工程的兴起使重组大肠杆菌生产琥珀酸变为可能,也取得了一定的成效,但是其发酵强度还不够高,且过程中伴随着其他有机酸的积累,因此还不适于工业化生产. 代谢工程以系统生物学为基础,为重组大肠杆菌的进一步改造提供了更合理的依据. 本工作以大肠杆菌生产琥珀酸所涉及的关键酶、代谢途径及其改造为对象,系统综述了大肠杆菌生产琥珀酸所涉及的代谢工程技术及其最新研究进展,并探讨了将来的发展前景.     

大肠杆菌偏利共培养系统合成大豆苷元

大豆苷元是一种植物雌激素,具有多种生物活性,但在大肠杆菌中的生物全合成还未见报道。基于大豆苷元合成途径的三个模块（对香豆酸、甘草素和大豆苷元模块）,构建大肠杆菌共培养系统从头合成大豆苷元。将对香豆酸和甘草素模块分配到两株大肠杆菌中构建双菌共培养系统,合成甘草素。在此基础上,探索了三种共培养模式合成大豆苷元,结果显示,三菌共培养系统比其他两种双菌共培养系统的产量更高,达到27.8 mg/L。共培养菌株间通过苯丙氨酸的单向流动形成了偏利共生的关系,有助于平衡代谢途径,提高大豆苷元产量。该共培养系统在大肠杆菌中实现大豆苷元的从头合成,为其他黄酮类化合物的生物合成提供了即插即用的平台。     

大肠杆菌偏利共培养系统合成大豆苷元

大豆苷元是一种植物雌激素,具有多种生物活性,但在大肠杆菌中的生物全合成还未见报道。基于大豆苷元合成途径的三个模块（对香豆酸、甘草素和大豆苷元模块）,构建大肠杆菌共培养系统从头合成大豆苷元。将对香豆酸和甘草素模块分配到两株大肠杆菌中构建双菌共培养系统,合成甘草素。在此基础上,探索了三种共培养模式合成大豆苷元,结果显示,三菌共培养系统比其他两种双菌共培养系统的产量更高,达到27.8 mg/L。共培养菌株间通过苯丙氨酸的单向流动形成了偏利共生的关系,有助于平衡代谢途径,提高大豆苷元产量。该共培养系统在大肠杆菌中实现大豆苷元的从头合成,为其他黄酮类化合物的生物合成提供了即插即用的平台。     

Characterization of promoters in Escherichia coli and application for xylitol synthesis

Promoters are the most important tools to control and regulate the gene expression in synthetic biology and met-abolic engineering. The expression of target genes in Escherichia coli is usually control...     

代谢工程改造蛋氨酸代谢途径构建高产L-蛋氨酸大肠杆菌

以Escherichia coli BL21(DE3)为出发菌株,敲除其蛋氨酸合成途径中关键酶的阻遏基因metJ,使菌株积累蛋氨酸达22 mg/L. 在此基础上,利用紫外诱变筛选育出一株抗蛋氨酸结构类似物的蛋氨酸高产菌株YB12,使蛋氨酸产量提高到60 mg/L. 通过过量表达蛋氨酸合成途径中的metA和cysE基因及编码蛋氨酸转运蛋白的yeaS基因,YB12菌株积累蛋氨酸量提高到251 mg/L. 结果表明,蛋氨酸在胞外的积累受多个基因、多种代谢途径调控,单独敲除某个基因或改造某个途径不能使蛋氨酸大量合成和积累,对多个代谢途径共同改造是构建蛋氨酸工程菌的最有效方法.     

Application of a Recombinant Escherichia coli Whole‐Cell Catalyst Expressing Hydroxynitrile Lyase and Nitrilase Activities in Ionic Liquids for the Production of (S)‐Mandelic Acid and (S)‐Mandeloamide

The conversion of benzaldehyde and cyanide into mandelic acid and mandeloamide by a recombinant Escherichia coli strain which simultaneously expressed an (S)‐hydroxynitrile lyase (oxynitrilase) from cassava (Manihot esculenta) and an arylacetonitrilase from Pseudomonas fluorescens EBC191 was studied. Benzaldehyde exhibited a pronounced inhibitory effect on the nitrilase activity in concentrations ≥25 mM. Therefore, it was tested if two‐phase systems consisting of a buffered aqueous phase and the ionic liquid 1‐butyl‐1‐pyrrolidinium bis(trifluoromethanesulfonyl)imide (BMpl NTf₂) or 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMim PF₆) could be used for the intended biotransformation. The distribution coefficients of the substrates, intermediates and products of the reaction were determined and it was found that BMpl NTf₂ and BMim PF₆ were highly efficient as substrate reservoirs for benzaldehyde. The recombinant E. coli strain was active in the presence of BMpl NTf₂ or BMim PF₆ phases and converted benzaldehyde and cyanide into mandelic acid and mandeloamide. The two‐phase systems allowed the conversion of benzaldehyde dissolved in the ionic liquids to a concentration of 700 mM with product yields (=sum of mandelic acid and mandeloamide) of 87–100%. The cells were slightly more effective in the presence of BMpl NTf₂ than in the presence of BMim PF₆. In both two‐phase systems benzaldehyde and cyanide were converted into (S)‐mandeloamide and (S)‐mandelic acid with enantiomeric excesses ≥94%. The recombinant E. coli cells formed, in the two‐phase systems with ionic liquids and increased substrate concentrations, higher relative amounts of mandeloamide than in a purely aqueous system with lower substrate concentrations.     

Nonlinear Predictive Control of Fed‐Batch Cultures of Escherichia coli

A strategy for controlling a fed‐batch Escherichia coli culture is described to maintain the culture at the boundary between oxidative and oxido‐fermentative regimes. A nonlinear predictive controller is designed to regulate the acetate concentration, constraining the feed rate to follow an optimal reference profile which maximizes the biomass growth. For the sake of simplicity and efficiency, the original problem is converted into an unconstrained nonlinear programming problem, solved by control vector parameterization techniques. The robustness of the structure is further improved by explicitly including the difference between system and model prediction. A robustness study based on a Monte Carlo approach is used to evaluate the performance of the proposed controller. This control law is finally compared to the generic model control strategy.     

基因重组大肠杆菌生产类人胶原蛋白补氮策略优化研究

优化重组大肠杆菌高密度发酵的调控工艺.利用重组大肠杆菌高密度发酵生产类人胶原蛋白,考察了连续流加和分阶段式两种不同补氮策略对细胞生长和类人胶原蛋白产量的影响.不同补氮方式显著影响类人胶原蛋白的产量.分阶段式补氮方式优于连续流加补氮方式,有利于细胞生长和类人胶原蛋白的表达,最终细胞浓度为76.01 g·L-1(DCW),类人胶原蛋白浓度为16.75 g·L-1,蛋白表达量为22.1%.     

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.     

The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Crosstalks with the Divisome

The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli, it consists of a transmembrane β-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane.     

Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cellbound glutamate decarboxylase(GAD) activity of recombinant Escherichia coli(BL21(DE3)-p ET28a-gad B) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-p ET28a-gad B, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 μg·ml-1. Using this concentration,the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-p ET28a-gad B and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21(DE3)-p ET28a-gad B cells with 5 μg·ml-1ampicillin resulted in structural changes to the cell envelope,but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol(PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid(GABA) at the 10 th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after30 days of storage at 4 °C.