Evidence for demand-regulation of ribosome accumulation in E coli

We have determined the relative concentrations of ribosomes accumulated under different growth conditions for a number of translational mutants as well as for some natural isolates of Escherichia coli. The mutants are a tRNA modification mutant (miaA), a streptomycin resistant (SmR) and a streptomycin pseudodependent (SmP) mutant as well as two ribosome ambiguity (ram) mutants. The natural isolates used in this study are known to function with submaximal ribosome kinetics. The data show that for all the ribosome mutants the concentration of ribosomes relative to that in wild type bacteria increases when the growth rate decreases. A small increase is also seen in the natural isolates. In contrast, the miaA mutant shows no increase in ribosome concentration under the same slow growth conditions. The results suggest that bacteria with kinetically impaired ribosomes can to some extent increase the number of ribosomes accumulated under poor growth conditions in order to compensate for their slower function. We use this observation to explain in part how bacteria growing in natural environments can escape the strong selection for maximized growth rates and for optimized ribosomes that are characteristic of laboratory strains.     

Specific stimulation of ribosomal RNA synthesis in E. coli by a protein factor

Summary Ribosomal RNA synthesis in a purified system is stimulated by a crude protein fraction prepared from E. coli. The positive effector which is not associated with RNA polymerase, nor is the sigma factor, increases the initiation frequency on a rRNA operon. The additional rRNA synthesis is inhibited by ppGpp to the same extent as the basal one.The evidence presented points to the existence of a positive control element for rRNA synthesis, which activity depends upon the physiological state of the cell.     

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     

Mapping and characterization of an E. coli mutant defective in IS1-mediated deletion formation

Summary A mutant of E. coli has been isolated in which the frequency of IS1-mediated deletion formation is reduced as much as 100 fold. The mutation causing this reduction, designated del, was mapped to a position near 61 min, in the vicinity of lysA and galR. Strains carrying a deletion of the del gene were constructed, and these exhibit a significant reduction in the frequency of IS1 excision in addition to impairment of deletion formation. A bacteriophage capable of transducing lysA and del was also isolated.     

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.     

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.     

Inducible, error-free DNA repair in tsl recA mutants of E. coli

Summary Host cell reactivation and UV reactivation and mutagenesis of UV-irradiated phage were measured in tsl recA ⁺ and tsl recA host mutants. Host cell reactivation was slightly more efficient in the tsl recA strain compared to the tsl ⁺ recA strain. Phage was UV-reactivated in the tsl recA strain with about one-half the efficiency of that in the wild type strain, but there was no corresponding mutagenesis of phage. UV-reactivation was also slightly lower and mutagenesis several-fold lower than normal in the tsl recA ⁺ strain. To account for these observations, we propose that there is an inducible, error-free pathway of DNA repair in E. coli that competes with error-prone repair for repair of phage lesions.     

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.     

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.     

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.     

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。     

肥胖基因的分离及其在大肠杆菌中的表达

利用PCR技术自外周血白细胞染色体DNA中扩增获取了肥胖基因(ob基因)的外显子2和3序列.经过拼接,获得了全长的ob基因编码序列. 测序结果表明,获得的序列与文献报道完全一致.利用PCR技术扩增出成熟蛋白的编码序列,克隆至表达载体pBV220中获得了表达菌株,并对表达产物进行了初步纯化,为进一步研究ob基因产物的功能与应用奠定了基础.     

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。