Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.

In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.

Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.     

Effects of Cystine and Hydrogen Peroxide on Glutathione Status and Expression of Antioxidant Genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cysteine or cystine was earlier shown to multiply enhance the toxic effect of hydrogen peroxide on Escherichia coli cells. In the present work, the treatment of E. coli with H₂O₂ in the presence of cystine increased fivefold the level of extracellular oxidized glutathione (GSSG_out) and decreased fivefold the GSH/GSSG_out ratio (from 16.8 to 3.6). The same treatment of cells with deficiency in glutathione oxidoreductase (GOR) resulted in even more severe oxidation of GSH_out, so that the level of oxidized glutathione exceeded that of reduced glutathione and the GSH/GSSG_out ratio decreased to 0.4. Addition of cystine to the GOR deficient cells resulted in significant oxidation of extracellular glutathione even in the absence of oxidant and in tenfold increase in intracellular oxidized glutathione along with a decrease in the GSH/GSSG_out ratio from 282 to 26. However, in the cytoplasm of wild type cells, the level of oxidized glutathione (GSSG_in) was changed insignificantly and the GSH/GSSG_in ratio increased by 26% (from 330 to 415). Data on glutathione status and cystine reduction in the E. coli gsh and gor mutants suggested that exogenous cystine at first should be reduced with extracellular GSH outside the cells and then imported into them. The high toxicity of H₂O₂ in the presence of cystine resulted in disorders of membrane functions and inhibition of the expression of genes including those responsible for neutralization of oxidants and DNA repair.__________Translated from Biokhimiya, Vol. 70, No. 8, 2005, pp. 1119–1129.Original Russian Text Copyright © 2005 by Smirnova, Muzyka, Oktyabrsky.     

大肠杆菌基因组减小研究进展

大肠杆菌是遗传重组领域广泛应用的宿主之一,用于生产重组蛋白、氨基酸和其他化学品。基因组减小可以减少代谢调节网络中的冗余,提高其预测性和可控性。我们介绍了最小基因组的研究策略、应用无痕敲除技术减小大肠杆菌基因组的方法,以及基因组减小后对菌体生长特性、附加体稳定性、重组蛋白表达和代谢的影响。     

原核系统可溶性表达策略

获得大量目的蛋白的最简单最经济的方法是利用原核表达系统表达外源基因.但由于原核系统的自身特点,使所表达的蛋白常常形成无活性的包涵体.多年来世界各国的研究为解决这一问题尝试了多种方法.本简单介绍原核表达系统的特点及提高蛋白可溶性表达的常用方法.     

志贺菌基因组进化研究进展

志贺菌属(Shigella)是引起全球范围内细菌性痢疾的重要病原菌.近年来,多重耐药型和新血清型志贺菌的不断出现给志贺菌的监测和防控带来了新的挑战.基因组学的快速发展为深入了解志贺菌的进化来源、变异机制及传播规律等提供了极大的帮助,对控制细菌性痢疾的蔓延具有重要的科学意义.本文首先从遗传来源角度探讨志贺菌与大肠杆菌的进化关系及其可能的分子机制,随后对福氏、宋内和1型痢疾志贺菌的基因组进化进展进行了总结,详细描述了它们的时空分布特点以及耐药基因变异在进化中所发挥的作用,以期为志贺菌的研究和防控提供参考.     

Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis

Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species. A region of the S. avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli. Production of the brown pigment was attained in E. coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG. The cloned S. avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E. coli vector. The gene involved in pigment production could be used as a tool to analyse gene expression in S. avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.     

人Hepcidin融合表达载体的构建及在大肠杆菌中的表达

为了在大肠杆菌中表达生产hepcidin,根据大肠杆菌密码子偏好性,化学合成了人hepcidin的基因序列,并构建了hepcidin的融合表达载体pET -hpc。pET- hpc在大肠杆菌BL2 1 (DE3)中表达的hepcidin融合蛋白以包涵体形式存在,其N端带有 6个组氨酸。通过优化诱导表达条件,该融合蛋白表达水平显著提高,占总蛋白的 2 5 . 2 %。表达的包涵体经 1 %TritonX 1 0 0洗涤后溶于8mol L尿素,在变性条件下采用金属螯合层析进行纯化,所得融合蛋白纯度大于 95 %。     

大肠杆菌不同菌株基因组DNA的多态性分析

从大肠杆菌K12菌株JM109基因组克隆了两段DNA重复序列,长度为0.9和0.6kb,分别命名为ECR-1和ECR-6。以ECR-1和ECR-6重复序列作DNA多态性分析的探针,可以鉴别大肠杆菌非常相近的菌株。表明ECR-1和ECR-6 DNA序列可用于大肠杆菌菌株的分类、流行病学和微生态学研究以及大肠杆菌各种致病菌株的临床诊断。     

利用Tn5转座子介导突变提高大肠杆菌丁醇生产水平

目前,对于构建高产丁醇大肠杆菌工程菌株的工作,主要是对丁醇通路和相关途径的基因进行理性改造。为进一步提升菌株的丁醇生产能力,需要发掘基因组上可影响丁醇生产能力的基因,但这很难通过已有认识或计算机模型进行预测。本工作以一株实验室前期构建的产丁醇大肠杆菌工程菌株为研究对象,利用Tn5转座子构建了一个含有1 196个菌株的突变文库。丙酮酸是丁醇的前体,并且在发酵终产物中,副产物丙酮酸的含量与丁醇的含量呈反相关,因此,可以利用丙酮酸的含量来间接反映丁醇的含量,而丙酮酸可用二硝基苯肼显色法进行快速测定,基于此,建立了96孔板——酶标仪快速筛选方法。利用该方法成功筛选到了比对照菌株丁醇产量提高了29%、49%、56%的3个突变体菌株。利用反向PCR及测序的方法,确定了其转座子插入位置分别为:pyk A、tdk、cad C基因。这些基因可以作为进一步提高菌株丁醇产量的靶点,同时这种利用Tn5转座子筛选基因靶标的策略也为构建其他微生物细胞工厂提供了新思路。     

A 718-kb DNA Sequence of the Escherichia coli K-12 Genome Corresponding to the 12.7-28.0 min Region on the Linkage Map

The 718,122 base pair (bp) sequence of the Escherichia coliK-12 genome corresponding to the region from 12.7 to 28.0 minuteson the genetic map is described. This region contains at least682 potential open reading frames, of which 278 (41%) have beenpreviously identified, 147 (22%) were homologous to other knowngenes, 138 (20%) are identical or similar to the hypotheticalgenes registered in databases, and the remaining 119 (17%) didnot show a significant similarity to any other gene. In thisregion, we assigned a cluster of cit genes encoding multienzymecitrate lyase, two clusters of fimbrial genes and a set of lysogenicphage genes encoding integrase, excisionase and repressor inthe e14 genetic element. In addition, a new valine tRNA gene,designated valZ, and a family of long directly repeated sequences,LDR-A, -B and -C, were found.     

整合子与多重耐药大肠埃希菌相关性研究

目的探讨整合子在多重耐药大肠埃希菌耐药性中的作用。方法对临床分离的93株多重耐药大肠埃希菌的I、Ⅱ型整合酶基因进行检测,并分析药敏结果。结果多重耐药临床分离株中Ⅰ型整合子阳性率为60.2%。所检出整合子共有3种长度即1000、1600和2000 bp;主要携带aadA和dfrA类基因盒;未检出Ⅱ型整合子。结论整合子形成是细菌产生多重耐药的重要原因。     

产超广谱β-内酰胺酶大肠埃希菌的耐药性分析

目的了解杭州市第一人民医院产ESBLs大肠埃希菌的发生比例及对临床上常用的24种抗菌药物的耐药率变化.方法收集2003 2004年该院各类临床标本中分离的大肠埃希菌,采用NCCLS推荐的表型确证试验方法检测ESBLs菌株;药敏试验采用纸片扩散法.结果2003年与2004年,产ESBLs的大肠埃希菌分离率分别为46.11%（184/399）、57.44%（386/672）（P=0.0003）;2年来,ESBLs阳性菌对临床常用的24种药物表现出较高的耐药性,耐药率上升非常显著（P=0.0005）;非产ESBLs大肠埃希菌对大多数抗菌药物仍保持较高的敏感率;但2004年ESBLs阴性的大肠埃希菌的耐药性,比2003年显著上升（χ^2=37.785,P=0.0005）.结论尽早开展产ESBLs菌的监测,合理使用抗菌药物,对于有效控制产ESBLs菌的播散与流行是一项重要措施.     

A 460-kb DNA Sequence of the Escherichia coli K-12 Genome Corresponding to the 40.1-50.0 min Region on the Linkage Map

The 465,813 base pair sequence corresponding to the 40.1–50.0min region on the genetic map of Escherichia coli K-12 (W3110)was determined. Analysis of the sequence revealed that thisregion contained at least 466 potential open reading frames,of which 187 (40%) were previously reported, 105 (23%) werehomologous to other known genes, 103 (22%) were identical orsimilar to hypothetical genes registered in databases, and theremaining 71 (15%) did not show a significant similarity toany other gene. At the 45.2–46.0 min region, we founda very large cluster of about 30 genes, whose functions areinvolved in the biosynthesis of polysaccharides as the componentsof outer membranes. In addition, we identified anew asn-tRNAgene, designated asnW, between the asnT and asnU genes and anew lysogenic phage attachment site as the cis-element.     

人血红蛋白α,β基因在大肠杆菌中的表达

从人外周血细胞中提取出ＲＮＡ,通过逆转录聚合酶链反应（ＲＴＰＣＲ）得到人血红蛋白的α和β基因片段,与ｐＴ７Ｂｌｕｅ质粒连接,然后将α、β基因克隆进ｐＢＶ２２０原核表达载体中,采用Ｓａｎｇｅｒ双脱氧终止法测序的结果与文献报道一致。宿主菌经热诱导,在１．６×１０５处有特异的蛋白带表达,表达量占总菌体总蛋白的２０％。     

多表位恶性疟疾疫苗M.RCAg-1在大肠杆菌表达的条件优化和大规模培养

通过以培养基配方、IPTG浓度、金属离子复合液浓度、镁离子浓度、表达时间、接种量、诱导时间点等发酵的重要条件对重组蛋白表达量影响的研究,确定多表位恶性疟疾疫苗M.RCAg-1蛋白最佳表达条件为以改良TB培养基培养、最优Mg2+,诱导剂IPTG和金属离子复合液浓度分别为10mmol/L,0.5mmol/L,6μl/ml,接种量为10%,表达时间为4.5h,将优化后的参数用于50L发酵罐进行连续3批中试规模的发酵,最终收获菌体湿重平均为31.8±1.78g/L,目的蛋白表达量可占菌体总蛋白的50%左右,试验确定了恶性疟疾多表位随机组合蛋白M.RCAg-1在大肠杆菌中的最优表达条件,该条件能够适合大规模培养需要。     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

纳豆激酶基因在大肠杆菌中活性表达的比较研究

实现纳豆激酶基因 (nattokinasegene)在大肠杆菌中高活性表达 ,并说明前肽 ( pro序列 )对纳豆激酶的活性表达必不可少。以纳豆芽孢杆菌基因组DNA为模板 ,采用PCR方法分别扩增编码信号肽、前肽及成熟肽的序列 ( pre pro NK)和编码前肽、成熟肽的序列 (pro NK) ,构建了大肠杆菌表达质粒 pTYB1 0 1 ,pTYB1 0 2 ,转化大肠杆菌ER2 5 66。在IPTG诱导下 ,分别在 1 5℃ ( 1 4h) ,3 0℃ ( 3h)和 3 7℃ ( 2h)培养。结果可见 ,pTYB1 0 2能表达有活性的纳豆激酶。SDS PAGE表明 ,1 5℃表达杂蛋白更少。薄层扫描显示表达的纳豆激酶占菌体总蛋白的 3 0 %以上。成功制备了表达纳豆激酶的工程菌。