大肠杆菌苏氨酸合成途径动力学模型的构建与分析

动力学模型分析有利于理解生物系统的调控机制,从而为高效细胞工厂的理性设计提供指导。基于以往发表的相关途径动力学模型和测量的酶动力学数据,开发了大肠杆菌苏氨酸合成途径的动力学模型。模型包含从天冬氨酸至苏氨酸的合成途径及葡萄糖开始的为合成途径提供前体以及能量的代谢途径。与以往模型不同的是新模型中考虑了能量和还原力的平衡,从而使模型模拟的系统自身成为一个不需要从外界提供能量和还原力的自洽系统。模型稳态分析的结果表明PTS、G6PDH和HDH等反应对苏氨酸合成反应的通量控制系数较大,通过过表达这些反应的酶可以有效增加苏氨酸合成反应的通量。     

产多聚唾液酸的菌种筛选及产酸条件

通过对40株大肠杆菌进行产多聚唾液酸的筛选,得到一株高产多聚唾液酸菌株C-8,对该菌的一系列培养条件进行了研究。最佳培养基为：山梨醇2.5％,硫酸铵0.5％,磷酸氢二钾90mmol／L,胰蛋白陈1.5％,硫酸镁0.04％,pH7．8。在37℃,250r／min摇床培养65h,可使菌体在每毫升培养液中产多聚唾液酸1200μg。     

温度对大肠杆菌L-色氨酸发酵过程的影响

目的：研究变温控制对大肠杆菌TRTH L-色氨酸补料分批发酵过程中生物量、色氨酸产量、比生长速率及质粒稳定性的影响。方法：利用5L自控发酵罐对L-色氨酸补料分批发酵过程进行温度控制,对不同温度下相关参数进行分析比较,确定优化的温度控制方案。结果：以30-36％顺序升温的工艺进行发酵得到理想结果,与单一温度控制策略相比,L-色氨酸产量提高了15．4％;色氨酸的比合成速率提高了21．6％;质粒稳定性增加,未出现质粒丢失现象,质粒拷贝数保持在恒定水平。结论：温度对大肠杆菌L-色氨酸发酵有重要影响。     

重组大肠杆菌生产谷胱甘肽发酵条件的研究

研究了重组Ｅ．Ｃｏｌｉ产ＧＳＨ的发酵条件,重点考察了添加酵母膏、前体氨基酸和ＡＴＰ的影响。结果发现,前体氨基酸和ＡＴＰ均能促进胞内ＧＳＨ的积累,若在发酵０ｈ和１２ｈ分别加入２０ｇ／ＬＡＴＰ和９ｍｍｏｌ／Ｌ前体氨基酸,则细胞干重和胞内ＧＳＨ含量可分别比对照提高２４％和１４倍。应用正交试验得出的针对细胞干重和ＧＳＨ总量的最佳组合,最大细胞干重和ＧＳＨ总量比原试验中的最好结果分别提高了１０％和２６％。在分析了该菌对葡萄糖利用情况的基础上,对该菌进行了指数流加培养,２５ｈ细胞干重与发酵液内ＧＳＨ总量分别达到８０ｇ／Ｌ和８８０ｍｇ／Ｌ,比摇瓶最好结果分别提高了８３和４６倍。     

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs

Abstract We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA ( E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA⁺ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA⁻ or eaeA⁺ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and efficacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli .     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

禽源致病性大肠埃希菌安徽分离株I型菌毛pilA基因的序列测定与分析

目的探明大肠埃希菌I型菌毛pilA基因在不同宿主来源菌株间的同源性,为利用Ⅰ型菌毛基因诊断和防治大肠埃希菌病提供理论基础。方法以禽源致病性大肠埃希菌安徽分离株基因组DNA为模板,采用PCR方法扩增Ⅰ型菌毛pilA基因,并进行序列测定与分析。结果 13株不同血清型禽源致病性大肠埃希菌安徽分离株均携带pilA基因,彼此间该基因的核苷酸序列和氨基酸序列同源性分别介于84.9%～99.7%和86.2%～99.2%。pilA基因核苷酸序列在安徽分离株与鸡源参考株、猪源参考株以及人源参考株之间的同源性分别为85.9%～99.7%、85.9%～93.9%和87.5%～100%,氨基酸序列同源性分别为83.1%～99.2%、88.5%～93.8%和90%～100%。系统发育进化树分析显示JD16、JD34、JD11、JD24、YD2、JD8和YD1禽源安徽分离株与人源参考株sPH2的亲缘关系较近,在进化树的同一分支上;GD3、YD5、GD2、YD3、YD5和YD7禽源安徽分离株与鸡源参考株PDI-386和猪源参考株107/86的亲缘关系较近,在进化树中属于同一分支;GD1禽源安徽分离株与鸡源参考株HY1-2和MS2-1的亲缘关系较近,在进化树中属于另一分支。结论大肠埃希菌I型菌毛pilA基因在不同宿主来源菌株及不同血清型菌株之间高度保守,可作为大肠埃希菌病的诊断基因和疫苗候选基因。     

Effect of growth conditions on the rate of loss of the plasmid pAT153 from continuous cultures of Escherichia coli HB101

Abstract The plasmid pAT153 was lost less rapidly from carbon, nitrogen, phosphorous or sulphur-limited continuous cultures of Escherichia coli HB101 as the dilution rate increased. At a fixed dilution rate of 0.3 h⁻¹, the plasmid was maintained longer as the growth-limiting nutrient was changed from glucose to casamino acids (nitrogen-limited), phosphate or sulphate. These differences in the stability of maintenance were not due to parallel changes in the plasmid copy number. We propose that the rate of loss of pAT153 from E. coli HB101 is determined primarily by the ratio of growth rates of plasmid-containing bacteria and plasmid-free bacteria. This ratio increases with increasing growth rate and depends markedly on the growth-limiting nutrient, sulphate-limited growth being particularly suitable for the maintenance of this host-plasmid combination.     

Use of glucose starvation to limit growth and induce protein production in Escherichia coli

The use of glucose starvation to uncouple the production of recombinant beta-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control beta-galactosidase synthesis. beta-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of beta-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombiant beta-galactosidase occurred continuously throughout the experiment. The cst-1 controlled beta-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.