Synthesis of 1-piperideine-6-carboxylic acid produced by L-lysine-epsilon-aminotransferase from the Streptomyces clavuligerus gene expressed in Escherichia coli

The gene (lat) encoding L-lysine epsilon-aminotransferase (LAT) in Streptomyces clavuligerus was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of lat predicted a single open reading frame (ORF) of 1371 bp, encoding a polypeptide of 457 amino acids with calculated molecular mass of 49.89 kDa. S. clavuligerus LAT was grouped into aminotransferase subfamily II of alpha family on the basis of sequence homology. A model system composed of the recombinant LAT in phosphate buffer was set up to study the biosynthesis of 2-acetyltetrahydropyridine. Lysine was found to be transformed to 1-piperideine-6-carboxylic acid. 2-Acetyltetrahydropyridine was characterized from the mixture of 1-piperideine-6-carboxylic acid and methylglyoxal. For the first time, we demonstrated that the L-lysine epsilon-aminotransferase is responsible for the formation of 1-piperideine-6-carboxylic acid, which may react with methylglyoxal to generate the acylated N-heterocyclic odorant 2-acetyltetrahydropyridine.     

重组甘蔗ACC氧化酶cDNA原核表达的纯化复性研究

重组甘蔗ACC氧化酶基因在大肠杆菌中表达,其产物以不溶性包涵体存在。用N i2 -NTA亲和层析柱对其进行纯化,结果显示,在层析柱上直接复性及纯化的方法比在变性条件下N i2 -NTA纯化然后稀释透析进行复性的方法简捷,效果好,获得的目的蛋白质纯度大于98%,活性为132.58 nm o l C2H4/(m g.h)。     

玫瑰黄链霉菌几丁质酶基因的克隆及拼接表达

应用PCR扩增克隆到玫瑰黄链霉菌(Streptomyces roseoflavus)的几丁质酶基因chiC的整个催化域部分,将其与包含信号肽区、纤维素结合区、几丁质结合区三个结构功能域的链霉菌(S.lividans)chiC基因片段相连接,插入pET23b( )质粒上,构建表达载体pLCH,用来转化大肠杆菌(Escherichia coli)JM109(DE3),转化子进行破壁之后在胶体几丁质酶培养基上表现活性。     

orfX基因整合表达在阿维链霉菌工业育种中的应用

阿维链霉菌（Streptomyces avermitilis）基因组中含有orfX基因，通过增加orfX基因剂量可以提高阿维菌素的产量。本研究以pIJ8600为出发质粒，构建了含红霉素启动子（ermEp*）和orfX结构基因DNA片段的整合型表达载体pWJ827。通过接合转移将pWJ827导入阿维菌素的工业菌株BIB9903中，得到稳定高产的重组菌株BIB0827。高效液相（HPLC）分析结果表明，与出发菌株BIB9903相比，BIB0827发酵产物阿维菌素产量提高了1.5倍，达到3350μg/mL。传代稳定性实验证明，BIB0827传代10代后依然稳定，在工业生产上有较大的应用价值。     

实时荧光定量PCR方法检测大肠杆菌O157:H7

针对大肠杆菌(Escherichia coli )O157:H7的致病基因eae设计了特异性引物，并用荧光染料SYBR GreenⅠ 进行了实时定量聚合酶链式反应（Real-time PCR）。熔解曲线显示产物特异性较强，无非目的条带和二聚体产生。并对反应程序进行了优化，确定了最佳的反应程序，最终在合适的模板浓度内得出了标准曲线。结果显示Real-time PCR 比普通PCR灵敏1 000倍。     

Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay

Humic acids are ubiquitous and abundant in terrestrial environments; therefore, they are often co-extracted with nucleic acids and interfere with quantitative PCR (qPCR) assays. In this study a recently developed NanoGene assay that is resistant to interference by humic acids was evaluated for gene detection in soil samples. The NanoGene assay utilizes a combination of magnetic beads, dual quantum dots labels, and DNA hybridization in solution. Seven soil samples containing different amounts of organic matter were tested to compare NanoGene and qPCR assays for their respective ability to detect a bacterial pathogen. We spiked the soils with Escherichia coli O157:H7, extracted genomic DNA, and conducted NanoGene and qPCR assays targeting the E. coli O157:H7-specific eaeA gene. To prevent the inhibition of PCR that is common when using DNA extracted from soils, we used a range of template DNA concentrations and BSA addition in the qPCR assay. Compared to the qPCR assay the NanoGene assay was significantly more resistant to the inhibitory effect of humic acids, successfully quantifying the eaeA gene within a linear (R² = 0.99) range of 10⁵ through 10⁸ CFU/g soil for all seven soil samples tested. In contrast, the qPCR assay was significantly inhibited using the same template DNA isolated from soils containing a range of organic content (2.0%–12%). Interestingly, the qPCR assay was still inhibited despite additional purification steps, suggesting that humic acids were still associated with DNA at a level that was inhibitory to qPCR. This study demonstrated that the NanoGene assay is suitable for quantitative gene detection in diverse soil types and is not susceptible to inhibition by humic acids and other organic compounds that commonly lead to false negative results in qPCR assays.     

Response of Escherichia coli O157:H7 survival to pH of cultivated soils

Purpose

The Escherichia coli (E. coli) O157:H7 survival dynamics in original and pH-modified agricultural soils were investigated to determinate how E. coli O157:H7 survival responded to the pH values of different soils, identify the relationships between E. coli O157:H7 survival time (t _d ) and soil properties, and assess the potential pathogen contamination after soil pH changed.

Materials and methods

The six soil samples were collected from different provinces of China, and 18 pH-modified soil samples were obtained from original soils by treating the original soils with direct electric current. The E. coli O157:H7 cells were inoculated into 24 soils and incubated at soil moisture of ?33 kPa and 25 °C. The soils were sampled for determining the numbers of E. coli O157:H7 at given time intervals over the incubation. The effects of soil pH change and other properties on the t _d values were analyzed.

Results and discussion

The t _d values in the test soils were between 7.1—24.7 days. Results indicate that soil pH, texture, and free Fe₂O₃ (Fe_d) were the most important factors impacting the t _d values in the test soils. Further, the response of E. coli O157:H7 survival to pH change varied with different soils. In the acidic soils (shorter t _d values), the t _d values decreased as the pH decreased and Fe_d increased, while in the neutral or alkaline soils (pH?≥?6.45, longer t _d values), the t _d values did not change significantly with pH.

Conclusions

The changes of amorphous and free sesquioxides induced by pH change might strengthen the response of E. coli O157:H7 survival to soil pH. Closer attention should be paid to E. coli O157:H7 long survival in soils and its potential environmental contamination risk.     

Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli

The plasmid pHG contains a cyclodextrin glycosyltransferase (CGTase) gene (cgt) derived from Bacillus macerans. Two transformants, Bacillus subtilis (pHG) and Escherichia coli (pHG), were found to produce CGTases with the same primary structure as the enzyme from B. macerans. However, the beta-cyclodextrin coupling activity of the CGTase from E. coli (pHG) was 14-fold higher than that of the enzymes from the other strains. By contrast, no differences in alpha-cyclodextrin coupling activities were observed among these CGTases. CGTase from E. coli (pHG) was found to be less thermostable than the other CGTases. When the CGTase produced by B. subtilis was treated with increasing urea concentrations (10-1000 mM) to promote increasing degrees of protein unfolding, a bell-shaped beta-cyclodextrin coupling activity profile was obtained. Subtle differences in the conformation of the CGTase produced by E. coli are therefore proposed to be responsible for the markedly increased beta-cyclodextrin coupling activity of this enzyme.     

Volatile compounds from Escherichia coli O157:H7 and their absorption by strawberry fruit

Volatile compounds emitted by cultures of two strains of the pathogenic bacterium Escherichia coliO157:H7 and a nonpathogenic strain of E. coli were trapped on Super-Q porous polymer and identified by GC-MS. The predominant compound produced by all three strains was indole with lesser amounts of other components including methyl ketones, 2-heptanone, 2-nonanone, 2-undecanone, and 2-tridecanone. The vapor-phase profiles of these strains were similar for most chemicals identified but differed with regard to ketones. Strawberry fruit was shown to be a suitable host for E. coli O157:H7 with the population of the bacterium either increasing or remaining stable after 3 days depending on inoculation level. Headspace analysis of the volatile compounds from inoculated fruit yielded no detectable quantity of indole. Strawberry fruit readily absorbed indole and other volatile compounds produced by the bacteria and in some cases metabolized the compounds to new volatile products. Thus, headspace "marker" compounds indicating possible bacterial contamination of fruit were largely removed from the vapor phase by the strawberries.     

Inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium in manure-amended soils studied in outdoor lysimeters

The inactivation of Escherichia coli O157:H7 (CCUG 44857) and Salmonella enterica serovar Typhimurium was investigated in two agricultural soils (sandy loam and silty clay) amended with poultry manure, cattle manure slurry or human urine. The study was performed in soil lysimeters placed outdoors, and was repeated over two consecutive years. The amendments, inoculated with E. coli O157:H7 and Salmonella Typhimurium, were mixed with soil on the top of the lysimeters. Samples were collected from the top 5-cm layer of each lysimeter at regular intervals, and the inactivation was monitored over 6 months, by the plate spread method and by enrichment. The inactivation was modelled by fitting a non-linear model to the data, and pathogen reduction times were calculated (90 and 99% reduction). The results showed that the inactivation of E. coli O157:H7 and Salmonella Typhimurium varied depending on the manure type used and its carbon content. The longest inactivation time occurred in samples amended with poultry manure, in which both E. coli O157:H7 and Salmonella Typhimurium were detected up to day 90 with the spread plate method. The most rapid inactivation for both pathogens occurred in soil amended with urine. However, low amounts of culturable E. coli O157:H7 and Salmonella Typhimurium were detected by enrichment throughout the study period (180 days), regardless of manure type.     

Inactivation of Escherichia coli in soil by solarization

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g⁻¹ dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces.     

Inactivation of Escherichia coli in soil by solarization

Abstract

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g^?1 dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces.     

胸腺素家族多肽在大肠杆菌中的表达

为提高胸腺素(又名胸腺肽,thymosin)的体外表达效率,本研究采用设计融合标签的方法对胸腺素α1和β4(Tα1,Tβ4)进行体外重组表达,通过超高效液相色谱-飞行时间质谱联用(LC/MS)法,对融合蛋白的完整分子量进行验证,利用反相超高效液相色谱法对不同表达载体的单位表达量进行测定.结果 表明,A 18-KEKE、...     

Fate of chlorate present in cattle wastes and its impact on Salmonella Typhimurium and Escherichia coli O157:H7

Chlorate salts are being developed as a feed additive to reduce the numbers of pathogens in feedlot cattle. A series of studies was conducted to determine whether chlorate, at concentrations expected to be excreted in urine of dosed cattle, would also reduce the populations of pathogens in cattle wastes (a mixture of urine and feces) and to determine the fate of chlorate in cattle wastes. Chlorate salts present in a urine-manure-soil mixture at 0, 17, 33, and 67 ppm had no significant effect on the rates of Escherichia coli O157:H7 or Salmonella Typhimurium inactivation from batch cultures. Chlorate was rapidly degraded when incubated at 20 and 30 degrees C with half-lives of 0.1 to 4 days. Chlorate degradation in batch cultures was slowest at 5 degrees C with half-lives of 2.9 to 30 days. The half-life of 100 ppm chlorate in an artificial lagoon system charged with slurry from a feedlot lagoon was 88 h. From an environmental standpoint, chlorate use in feedlot cattle would likely have minimal impacts because any chlorate that escaped degradation on the feedlot floor would be degraded in lagoon systems. Collectively, these results suggest that chlorate administered to cattle and excreted in wastes would have no significant secondary effects on pathogens present in mixed wastes on pen floors. Lack of chlorate efficacy was likely due to low chlorate concentrations in mixed wastes relative to chlorate levels shown to be active in live animals, and the rapid degradation of chlorate to chloride at temperatures of 20 degrees C and above.     

Glycopeptide derived from hen egg ovomucin has the ability to bind enterohemorrhagic Escherichia coli O157:H7

Ovomucin glycopeptide (OGP) was prepared by size exclusion chromatography after Pronase digestion of hen egg ovomucin, and the binding of OGP to foodborne pathogens (Bacillus cereus,Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, and Staphylococcus aureus) was investigaed. Binding assays with biotinylated bacteria as probes in microtiter plates showed that OGP bound to only E. coli O157:H7 among these foodborne pathogens. Periodate treatment markedly reduced the binding ability, indicating that E. coli O157:H7 bound to carbohydrate moieties of OGP. Lectin blot analysis with Maackia amurensis (MAA) and Sambucus nigra (SNA), which are specific for oligosaccharides containing sialic acid, revealed their binding sites in OGP were similar to the E. coli O157:H7 binding sites that were probed with biotinylated E. coli O157:H7 after Western blotting of OGP. Sialydase treatment of OGP abolished its ability to bind E. coli O157:H7, demonstrating that sialic acid played an important role in the binding. These results suggest that OGP has E. coli O157:H7-specific binding sites that consist of sialic acid. On the basis of these properties, OGP has the potential to be an ingredient with a protective effect against E. coli O157:H7 infection and to be a novel probe for the detection of E. coli O157:H7 in the food hygiene field.     

Fate of Escherichia coli and Escherichia coli O157 in soils and drainage water following cattle slurry application at 3 sites in southern Scotland

Abstract. Slurry from farm animals may contaminate water supplies, rivers and bathing waters with faecal coliforms, such as Escherichia coli . Where animals harbour the O157 strain the hazard to human health is particularly high, but both the hazard level, and the low incidence and sporadic nature of the excretion of E. coli O157 make it difficult to study this strain under field conditions. The survival of total E. coli and of E. coli O157 were compared in the laboratory for two soils under controlled temperature and moisture. E. coli O157 die-off rate was the same as or quicker than for total E. coli . This result meant that field experiments studying the fate of total E. coli should give a satisfactory evaluation of the risk of water contamination by the O157 strain. In four field experiments at three sites, slurry containing total E. coli numbers of 2.2 × 10⁴ to 5.7 × 10⁵ colony forming units per mL (c.f.u. mL^–1) was applied to drained field plots. Field die-off was faster than expected from laboratory experiments, especially in one experiment where two weeks dry weather followed application. In all but this experiment, the first drain flow events after slurry application led to very high E. coli concentrations in the drains (10³ to 10⁴ c.f.u. mL^–1). E. coli O157 was present in the slurry used for two of the experiments (33 c.f.u. per 100 mL in each case). However the proportion of E.coli O157 was very low (about 1 in 10⁵) and it was not detected in the drainage water. After the first week E. coli drainage water numbers decreased rapidly but they were 1–10 c.f.u. mL^–1 for much of the sampling period after slurry application (1–3 months).     

重组犬IFN-γ在大肠杆菌中的高效表达

提取Concavadin A诱导培养的犬脾细胞总RNA，经RT-PCR扩增出犬IFN-γ，克隆到pMD18-T载体并测序鉴定，然后把IFN-γ基因克隆到原核表达载体PJLA605，构建pRL-Ca／IFN-γ表达质粒；应用M9培养基通过摇瓶发酵，确定诱导时机和诱导表达时间。结果表明，工程菌pRL-CaIFN-γ在30℃培养至OD600为1．5于42℃诱导4h，菌体收获量湿重达20．0gm，目标蛋白表达量约占菌体总蛋白的32％，外源基因在该基因工程菌中得到了高效表达。     

Environmental Controls on the Fate of Escherichia coli in Soil

An improved understanding of factors that influence the survival and/or growth of Escherichia coli (E. coli) in soil is essential to allow the formation of land management practices to control the spread of the pathogenic strains of the bacteria, whose transmission to fresh produce is a threat to food safety. Persistence of E. coli in soils held at different water potentials and with carbon additions then subjected to post-freezing incubation temperatures and in the presence of Klebsiella terrigena (K. terrigena) were investigated. Soil samples adjusted to different water potentials (?0.03, ?0.1 and ?1.5 MPa) were inoculated with a multi-antibiotic resistant strain of E. coli (E. coli 2+), which allowed recovery of the organism from soil samples. In addition to manipulation of water content, different C levels were added and samples were frozen for varying lengths of time, thawed and incubated. In freezing studies, initial soil moisture content significantly affected E. coil 2+ survival in soils following thawing, resulting in lower survival rate (k) at water potential of ?0.03 than at ?0.1 and ?1.5 MPa. The effect of length of freezing time was significant only at ?0.03 MPa. Glucose addition at 1.25 mg C g^?1 improved survival rate versus glucose at 0.125. The low level glucose increased die-off rate versus no addition, suggesting that unless amendments provide C above a certain threshold level, they might facilitate the death of the bacteria. E. coli 2+ survival improved in the presence of K. terrigena at 6°C but not at 23°C. Persistence of E. coli under the interactive influence of various environmental factors highlights the urgency and importance of understanding its potential for transmission to fresh produce and water bodies.     

小鼠表皮生长因子在Escherichia coli中重组表达

以大肠杆菌（Escherichia coli）BL21为表达宿主，重组表达小鼠表皮生长因子。以pET30a为出发载体，构建了包含mEGF编码序列的重组表达载体pETmEGF，采用低温诱导表达和无破胞程序的冻融法进行纯化，得到了浓度为17．24μg/mL和纯度为57％的mEGF融合蛋白，其活性相当于标准小鼠表皮生长因子的29．16％。     

pfl-act基因在大肠杆菌中的高效表达及其纯化

丙酮酸甲酸裂解酶是肠道细菌在厌氧代谢中十分关键的酶,丙酮酸甲酸裂解酶激活因子(pyruvateform ate lyase activator,PFL-A)在功能上具有重要的作用。为进一步研究PFL-A的激活机理,以大肠杆菌K-12的基因组为模板,通过G enB ank上公布的序列设计引物,扩增出目的基因,克隆到pM D 18-T载体,经测序,所扩增出的基因与p f l-act基因具有99%的同源性。将p f l-act连接到高效表达载体pET-22b( )中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,结果发现,p f l-act以包涵体形式表达。改变诱导剂、诱导剂量或培养温度,对包涵体的形成均没有明显的影响。