大肠杆菌的蛋白分泌机制

大肠杆菌的分泌蛋白定位于内膜、外膜、周质空间和胞外环境,它们在N端或C端带有一定的结构包含着分泌信号,这两类分泌蛋白在各自特定的一组蛋白因子的协助下跨越内膜,再通过目前尚不清楚的方式实现其最终定位.N端带有信号肽的分子在跨越内膜时得到Sec家族蛋白因子协助,信号肽在跨膜过程中可能被切除,该过程由ATP和电化学势提供能量.C端带分泌信号的分子主要受到Hly家族分子协助,一次穿过内膜和外膜而不经过周质空间.     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

Cloned truncated recA genes in E. coli

Summary Plasmids pMH1 and pDR1461, possessing the control region and 22% or 73% of the E. coli recA gene, conferred UV sensitivity to wild-type uvrA, and umuC bacteria. Sensitization was less in recA441 (tif-1) mutants and absent in lexA cells. Radiosensitization correlated with inhibition of recombinational repair, even through induced recA protein synthesis and recombination in Hfr matings were normal. Plasmids pMH1 and pDR1461 also prevented induction of some, but not all, SOS functions. Mutagenic reversion to tryptophan prototrophy and induced reactivation of UV-irradiated phage were eliminated, and the efficiency of lysogenic induction reduced. However, naladixic acid induced filamentous growth, mitomycin-C induced uvrA gene expression and post UV-irradiation DNA degradation control were little changed. Explanations of these effects are discussed which involve the presence of either truncated recA protein or multiple copies of the recA gene control sequence.A preliminary account of this work is presented in Chromosome Damage and Repair, edited by E. Seeberg and K. Klepper, to be published by Plenum Press     

大肠杆菌碱性磷酸酶的体外定向进化研究

大肠杆菌碱性磷酸酶（E.coli alkaline phosphatase, EAP, EC 3.1.3.1）是一个非特异性二聚体磷酸单酯酶. 采用易错聚合酶链反应(error prone PCR)的方法,在原有高活力突变株的基础上,对EAP远离活性中心催化三联体的区域进行定向进化,经两轮error prone PCR,获得催化活力较亲本D101S突变株提高3倍、较野生型酶提高35倍的进化酶4-186,并对该酶的催化动力学特征进行了分析. 进化酶基因的DNA测序表明4-186含两个有义氨基酸置换：K167R和S374C,二者既不位于底物结合位点,也不位于酶的金属离子结合位点.     

氧化葡萄糖酸杆菌中一种潜在的双组分系统蛋白的功能研究

氧化葡萄糖酸杆菌(Gluconobacteroxydans)基因组编码的蛋白质中,有相当数量的传感器激酶和反应调控蛋白组成了细菌的多个双组分信号转导系统(two-componentsignaltransduction systems, TCSs),这些系统能够介导细菌对外界环境变化做出反应。但目前对G. oxydans中潜在的双组分系统成员蛋白质结构和功能缺少必要的研究【。目的】研究菌株G. oxydans 621H中GOX0645基因序列所编码蛋白质的自磷酸化活性,探究其与细菌趋化性运动的关联,揭示其是否作为一种双组分系统成员蛋白在细胞内发挥作用。【方法】以菌株G. oxydans 621H基因组中一段可能编码双组分系统蛋白质的基因GOX0645为基础,通过生物信息学分析其保守结构域;采用体外化学发光实验证明其编码蛋白的自磷酸化活性;利用基因定点突变筛选出与自磷酸化活性相关的氨基酸位点;通过差速离心法寻找双组分蛋白的亚细胞定位;最后运用体内双分子荧光互补和体外生物大分子相互作用实验印证其与下游鞭毛马达蛋白之间的相互作用。【结果】生物信息学分析发现GOX0645编码蛋白同时具有组氨酸激...     

The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli

Background  

The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed.     

Replication of E. coli duplex DNA in vitro

Summary An E. coli lysate after being gently washed to remove soluble components, supports replicative DNA synthesis, if soluble proteins and the deoxyribonucleotide triphosphates are added. This DNA synthesis is dependent on ATP and on the presence of the gene products of the dnaB, dnaG, and polC (DNA polymerase III) genes. It continues at the replication forks preformed in vivo and Okazaki fragments are intermediate products of the reaction.Two different methods were used to prepare the washed DNA containing fraction. The one method involves washing of a cell lysate situated on a dialysis membrane. The other method involves DNAase treatment of a lysate and sedimentation of the degraded DNA through a glycerol gradient. Both washed preparations contain not only the DNA and the replication forks but also functional amounts of DNA polymerase III and of the dnaB gene product. Other factors, that are essential for replicative DNA synthesis, including the dnaG gene product, are washed out of the DNA containing preparations and the system is reconstituted by readdition of the soluble proteins.     

真菌双组分信号转导系统及其抑制剂研究进展

双组分信号转导系统存在于包括真菌在内的大部分低等真核生物、原核生物及一些植物中。真菌双组分信号转导蛋白在细胞新陈代谢、毒力以及致病性等方面具有重要作用,且目前在人类细胞中尚未发现双组分信号转导系统。因此,探明真菌双组分信号转导系统的机制,可为抑制剂的设计和寻找提供多个"靶点",从而研制出能够抗致病性真菌而不对宿主细胞造成损伤的新型抗真菌药物。本文就近年来真菌双组分信号转导系统及其潜在抑制剂进行综述。     

基于卷积神经网络的大肠杆菌启动子预测

目的 基于位点特异性打分矩阵（position-specific scoring matrices,PSSM）的预测模型已经取得了良好的效果,基于PSSM的各种优化方法也在不断发展,但准确率相对较低,为了进一步提高预测准确率,本文基于卷积神经网络（convolutional neural networks,CNN）算法做了进一步研究。方法 采用PSSM将启动子序列处理成数值矩阵,通过CNN算法进行分类。大肠杆菌K-12（Escherichia coli K-12,E.coli K-12,下文简称大肠杆菌）的Sigma38、Sigma54和Sigma70 3种启动子序列被作为正集,编码（Coding）区和非编码（Non-coding）区的序列为负集。结果 在预测大肠杆菌启动子的二分类中,准确率达到99%,启动子预测的成功率接近100%;在对Sigma38、Sigma54、Sigma70 3种启动子的三分类中,预测准确率为98%,并且针对每一种序列的预测准确率均可以达到98%以上。最后,本文以Sigma38、Sigma54、Sigma70 3种启动子分别和Coding区或者Non-coding区序列做四分类,预测得到的准确性为0.98,对3种Sigma启动子均衡样本的十交叉检验预测精度均可以达到0.95以上,海明距离为0.016,Kappa系数为0.97。结论 相较于支持向量机（support vector machine,SVM）等其他分类算法,CNN分类算法更具优势,并且基于CNN的分类优势,编码方式亦可以得到简化。     

Rcs双组分调节系统对细菌环境应答的分子调控研究进展

荚膜异多糖酸合成调节(Regulator of Capsule Synthesis,Rcs)系统是存在于许多肠杆菌科细菌中非典型的双组分调节系统,由3种核心蛋白(跨膜感应激酶RcsC、跨膜蛋白RcsD和响应调节剂RcsB)及多种辅助蛋白共同构成.Rcs系统能整合环境信号、调节基因表达并改变细菌的生理行为.近年来,对细菌...     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

On the nature of sbcA mutations in E. coli K12

Summary We have recently shown (Kaiser and Murray 1979) that many E. coli K12 strains carry a defective prophage (Rac) located a few minutes clockwise of the trp operon on the genetic map. The Rac genome contains recE, the determinant for the ATP-independent exonuclease, ExoVIII. E. coli K12 strains which carry sbcA mutations express recE constitutively. This paper describes an investigation of several such strains. We show that the SbcA phenotype may arise from more than one type of mutational change. The most readily explained SbcA phenotype is that of sbcA8 strains in which a large section of the Rac genome (including one hybrid attachment site and probably the prophage repressor gene) is deleted. Three sbcA ^- strains carry multiple (and probably tandemly repeated) copies of the Rac genome while two others carry a single Rac prophage that is indistinguishable in its hybridisation behaviour from that carried by sbcA ⁺ strains.     

产NDM-5大肠埃希菌的鉴定及特征分析

对分离自血液标本的1株碳青霉烯类耐药大肠埃希菌(Escherichia coli)SCNJ06进行特征分析,以期为临床耐药菌株感染的防治提供理论参考。采用全基因组测序以及生物信息学分析,该菌株属于序列型167(ST167),含有11种耐药基因,分别是rmtB、aph(3″)-Ib、aph(6)-Id、bla_(NDM-5)、bla_(TEM-1B)、bla_(CTX-M-55)、fosA3、floR、sul2、tet(A)和mdf(A)。其中,bla_(NDM-5)位于IncX3型质粒pNDM5_SCNJ06上,mdf(A)位于染色体上,其余耐药基因位于IncFII型质粒prmtB_SCNJ06上。接合试验显示,pNDM5_SCNJ06和prmtB_SCNJ06均能够发生接合转移。应加强抗菌药物临床应用管理和医院感染防控措施,重视细菌耐药监测工作。     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

大肠杆菌细胞DNA复制、修复和重组途径的衔接

以大肠杆菌为例围绕相关领域的研究动态进行分析和总结.DNA复制、损伤修复和重组过程的相互作用关系研究是当今生命科学研究的前沿和热点之一.越来越多的研究表明,在分子水平上,DNA复制、损伤修复和重组过程既彼此独立,又相互依存.上述途径可以通过许多关键蛋白质之间的相互作用加以协调和整合,并籍此使遗传物质DNA得到有效的维护和忠实的传递.需要指出的是,基于许多细胞内关键蛋白及其功能在生物界中普遍保守性的事实,相信来自大肠杆菌有关DNA复制、修复和重组之间的研究成果也会对相关真核生物的研究提供借鉴.     

Genetic locations of uvrD and pol genes of E. coli

Summary UvrD maps between metE and ilv genes, and pol maps near metE, between metE and rha genes.Abbreviations used Hcr Host cell reactivation - UVr Ultraviolet light reactivation This is the fifth of the series Studies on radiation-sensitive mutants of E. coli. The fourth of which is A. Miura and J. Tomizawa, Proc. nat. Acad. Sci. (Wash.), in press.