DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Influence of DNA supercoiling on the loss of culturability of Escherichia coli cells incubated in seawater

The relationship between the loss of culturability of Escherichia coli cells in seawater and the DNA supercoiling level of a reporter plasmid (pUC8) have been studied under different experimental conditions. Transfer to seawater of cells grown at low osmolarity decreased their ability to grow without apparent modification of the plasmid supercoiling. We found that E. coli cells could be protected against seawater-induced loss of culturability by increasing their DNA-negative supercoiling in response to environmental factors: either a growth at high osmolarity before the transfer to seawater, or addition of organic matter (50-mg/l peptone) in seawater. We further found conditions where a DNA-induced relaxation was accompanied by an increase in seawater sensitivity. Indeed, inactivation of either one of the subunits A and B of DNA gyrase, which leads to important DNA relaxation, was accompanied in both cases by an increased loss of culturability of conditional mutants after transfer to seawater which could not be explained uniquely by the increase in the temperature required to inactivate the gyrase. Similarly, a strain harbouring a mutation in topoisomerase I, compensated by another mutation in subunit B of the gyrase, was more sensitive to seawater than the isogenic wild-type cell and this greater sensitivity was correlated to a relaxation of plasmid DNA. Again, in these different cases, a previous growth at high osmolarity protected against this seawater sensitivity. We thus propose that the ability of E. coli cells to survive in seawater and maintain their ability to grow on culture media could be linked, at least in part, to the topological state of their DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potassium ions and changes in bacterial DNA supercoiling under osmotic stress

Escherichia coli transiently increases both the [ATP]/[ADP] ratio and the negative supercoiling of plasmid DNA when it is shifted to high osmolarity. Here we report that a mutant lacking all saturable K+ transport systems increases the negative supercoiling of the plasmid DNA under upshock but cannot further relax DNA. The mutant dnaK756 behaves like the K+ transport mutant.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

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.     

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。     

Heterologous protein expression using a novel stress-responsive protein of E. coli RpoA as fusion expression partner

E. coli proteome response to the stressor 2-HEDS was analyzed through two-dimensional gel electrophoresis (2-DE), and we identified DNA-directed RNA polymerase -subunit (RpoA) as stress-responsive protein. Even under stress situation where the total number of soluble proteins decreased by 9.8%, the synthesis level of RpoA was increased 1.5-fold. As a fusion expression partner as well as solubility enhancer, RpoA facilitated the folding and increased significantly the solubility of many aggregation-prone heterologous proteins (human minipro-insulin, human epidermal growth factor, human prepro-ghrelin, human interleukin-2, human activation induced cytidine deaminase, human glutamate decarboxylase, Pseudomonas putida cutinase, human ferritin light chain, human granulocyte colony-stimulating factor, and cold inflammatory syndrome1 protein Nacht domain) in E. coli cytoplasm. Due probably to intrinsic high folding efficiency and/or chaperone-like activity, RpoA was very effective in shielding interactive surfaces of heterologous proteins that are associated with non-specific protein–protein interaction leading to the formation of inclusion bodies. RpoA was also well suited for the production of biologically active fusion mutant of Pseudomonas putida cutinase that is of much biotechnological and commercial interest.     

大肠杆菌严谨型RNA聚合酶的筛选及体内转录活性测定

利用选择性培养基筛选大肠杆菌自然突变菌株,经噬菌体P1转导和蛋白质互补试验,发现一株突变体(LCH001)的突变基因发生在编码RNA聚合酶β′亚基的rpoC基因上,经DNA序列分析,发现突变位点发生在第3406个碱基上,由G变成了T,导致编码的氨基酸由甘氨酸(GGT)变成半胱氨酸(TGT)。体内转录试验表明该突变RNA聚合酶转录严谨型启动子控制基因的活性显著降低,其β-半乳糖苷酶的活性是野生型菌株的18%,而转录非严谨型启动子控制基因的活性显著提高,其β-半乳糖苷酶的活性约是野生型菌株的5倍。研究结果对探讨RNA聚合酶结构与功能的关系以及RNA聚合酶在细菌严谨反应过程中的作用具有重要意义。     

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.     

A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability

A method of λ-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)₆ tag, into the chromosome of a strain lacking tufB. Received: 15 July 1998 / Accepted: 13 October 1998     

Nucleotide excision by E. coli DNA polymerase I in proofreading and non-proofreading modes

Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms. When it binds to a template, the proofreading activity is usually switched off. As the enzyme progresses along the template, it becomes more and more competent for excision. This phenomenon introduces a link between fidelity and processivity. Processivity is best studied when the chain-length distributions of synthesized polymers are stationary. Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme. When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed. It seems that the polymerase starts by hydrolyzing the primer down to a length of 7–10 nucleotides and only then starts to add nucleotides. It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading. The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3′ → 5′ exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.     

Torque-induced deformations of charged elastic DNA rods: thin helices,loops, and precursors of DNA supercoiling

We study the deformations of charged elastic rods under applied end forces and torques. For neutral filaments, we analyze the energetics of initial helical deformations and loop formation. We supplement this elastic approach with electrostatic energies of bent filaments and find critical conditions for buckling depending on the ionic strength of the solution. We also study force-induced loop opening, for parameters relevant for DNA. Finally, some applications of this nano-mechanical DNA model to salt-dependent onset of the DNA supercoiling are discussed.