Temperature-Sensitive Nonsense Mutations in Essential Genes of Escherichia coli

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4(ts) gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.     

大肠杆菌棉子糖操纵子

大肠杆菌(Escherichiacoli)棉子糖(raf)操纵子位于质粒,它编码能够使大肠杆菌吸收和利用三糖棉子糖的蛋白,即一个主动运输系统(Raf透性酶),α半乳糖苷酶和蔗糖水解酶。raf操纵子包括启动子rafP,调节基因rafR,操纵基因rafO以及rafA,rafB,rafD三个结构基因。这个操纵子由一个阻遏蛋白RafR负控制,同时以环腺苷代谢降解物基因激活蛋白(cAMPCAP)为中介的正调控也参与调节。     

利用Red重组工程技术解决外源基因在大肠杆菌染色体lac操纵子中的表达

为了应用Red重组工程技术实现外源基因在大肠杆菌染色体上的表达, 寻找染色体上外源蛋白的稳定高效表达位点, 使用Red重组工程系统和kan/sacB无痕迹修饰技术, 将易于定量分析的荧光素酶报告基因替换DY330染色体lac操纵子中的lacZ基因。检测该位点的表达效率结果显示: 大肠杆菌染色体上lac操纵子能够高效稳定表达外源基因, 初步证明了染色体可以作为外源蛋白或抗原的表达载体, 不会影响细菌的生长繁殖。     

利用Red重组工程技术解决外源基因在大肠杆菌染色体lac操纵子中的表达

为了应用Red重组工程技术实现外源基因在大肠杆菌染色体上的表达, 寻找染色体上外源蛋白的稳定高效表达位点, 使用Red重组工程系统和kan/sacB无痕迹修饰技术, 将易于定量分析的荧光素酶报告基因替换DY330染色体lac操纵子中的lacZ基因。检测该位点的表达效率结果显示: 大肠杆菌染色体上lac操纵子能够高效稳定表达外源基因, 初步证明了染色体可以作为外源蛋白或抗原的表达载体, 不会影响细菌的生长繁殖。     

Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K_m values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K_m for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.A wide variety of fermentation products can be made using sugars from lignocellulosic biomass as a substrate (9, 13, 16, 39). Prior to fermentation, however, the carbohydrate polymers cellulose and hemicellulose must be converted to soluble sugars, using a combination of chemical and enzymatic processes (40, 42, 43). Chemical processes are accompanied by side reactions that produce a mixture of minor products such as alcohols, acids, and aldehydes that have a negative effect on the metabolism of microbial biocatalysts. Alcohols (catechol, syringol, etc.) have been shown to act by permeabilizing the cell membrane and toxicity correlated well with the hydrophobicity of the molecule (47). Organic acids (acetate, formate, etc.) are thought to cross the membrane in neutral form and ionize within the cytoplasm, inhibiting growth by acidifying the cytoplasm and collapsing the proton motive force (32, 48). The inhibitory mechanisms of aldehydes are more complex. Aldehydes can react to form products with many cellular constituents, in addition to direct physical and metabolic effects (27, 36). In aggregate, these side products from chemical pretreatments can retard cell growth and slow the fermentation of biomass-derived sugars (10, 31).Furfural (a dehydration product of pentose sugars) is of particular importance. Furfural content in dilute acid hydrolysates of hemicellulose has been correlated with toxicity (23). Removal of furfural by lime addition (pH 10) rendered hydrolysates readily fermentable while readdition of furfural restored toxicity (22). Furfural has also been shown to potentiate the toxicity of other compounds known to be present in acid hydrolysates of hemicellulose (47-49). Furfural has been reported to alter DNA structure and sequence (3, 17), inhibit glycolytic enzymes (6), and slow sugar metabolism (11).The ability of fermenting organisms to function in the presence of these inhibitors has been researched extensively. Encapsulation of Saccharomyces cerevisiae in alginate has been shown to be protective and improve fermentation in acid hydrolysates of hemicellulose (38). Strains of S. cerevisiae have been previously described with improved resistance to hydrolysate inhibitors (1, 20, 29). Escherichia coli (7), S. cerevisiae (2, 19), and other microorganisms (4) have been shown to contain enzymes that catalyze the reduction of furfural to the less toxic product, furfuryl alcohol (49). In E. coli, furfural reductase activity appears to be NADPH-dependent (7). An NADPH-dependent furfural reductase was purified from E. coli although others may also be present. Two NADPH-dependent enzymes capable of reducing 5-hydroxymethyl furfural (a dehydration product of hexose sugars) have been characterized in S. cerevisiae and identified as the ADH6 gene product (2, 34) and a mutant form of ADH1 (2).In this paper, we describe the isolation of a furfural-resistant E. coli mutant (EMFR9) in which furfural reductase activity is lower than that of the parent (LY180) due to decreased expression of yqhD and dkgA. The reduction of furfural by these two NADPH-dependent oxidoreductases is proposed to inhibit growth by depleting the NADPH needed for biosynthesis.     

大肠埃希菌trp operon的克隆与表达

色氨酸操纵子所表达酶的高效表达和酶活性的提高,从而构建高产色氨酸菌株.利用PCR的方法从大肠埃希菌基因组中直接克隆色氨酸操纵子,并将其连接到原核表达载体pBV220中,得到重组质粒pBV220-trp operon,转化大肠埃希菌DH5α,温度诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性.通过凝胶电泳观察PCR扩增产物大小约为7 kb.SDS-PAGE鉴定目的蛋白得到了高效表达,邻氨基苯甲酸合成酶和色氨酸合成酶的活性分别比对照提高了3.4倍和2.5倍.成功构建了重组质粒pBV220-trp operon,邻氨基苯甲酸合成酶和色氨酸合成酶的表达量和表达活性在大肠埃希菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础.     

rpoS Function Is Essential for bgl Silencing Caused by C-Terminally Truncated H-NS in Escherichia coli

From evolutionary and physiological viewpoints, the Escherichia coli bgl operon is intriguing because its expression is silent (Bgl(-) phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing. However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl(+) phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10cam transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be leuO, hns, and bglJ, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively. The former encodes the stationary-phase-specific sigma factor, sigma(S), and the latter encodes a LysR-like DNA-binding protein. It was found that sigma(S) is defective in both types of mutant cells. These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in the hns60 background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.     

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.     

Effects of Dimethylsulfoxide on the Lactose Operon in Escherichia coli

Fowler, Audree V. (University of California, Los Angeles), and Irving Zabin. Effects of dimethylsulfoxide on the lactose operon in Escherichia coli. J. Bacteriol. 92:353-357. 1966.-Dimethylsulfoxide (DMSO) at a concentration of 5% (v/v) in the culture medium inhibits the growth of Escherichia coli to only a slight extent, and does not affect the differential rate of synthesis of beta-galactosidase. Resting cells remain viable after shaking in the presence of 20% DMSO for 3 hr at 37 C. Both beta-galactosidase and thiogalactoside transacetylase retain almost all activity after incubation in even higher concentrations of the solvent for many hours. DMSO decreases the permeability barrier. The rate of hydrolysis of o-nitrophenyl-beta-d-galactoside (ONPG) in whole cells containing beta-galactosidase but lacking permease is increased in cells treated with 5% DMSO. Several permeaseless strains preinduced for beta-galactosidase will grow on lactose in the presence, but not in the absence, of 5% DMSO. When permeaseless strains are grown on tetrazolium-lactose-agar, the presence of 5% DMSO causes a definite but not marked shift toward the lactose-positive character.     

Use of the tna Operon as a New Molecular Target for Escherichia coli Detection

A quantitative real-time PCR targeting the tnaA gene was studied to detect Escherichia coli and distinguish E. coli from Shigella spp. These microorganisms revealed high similarity in the molecular organization of the tna operon.     

Production of Active Chimeric Pediocin AcH in Escherichia coli in the Absence of Processing and Secretion Genes from the Pediococcus pap Operon

Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys⁺¹ to Cys⁺⁴⁴) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH₂ terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys⁺¹-Tyr-Tyr-Gly-Asn-Gly-Val⁺⁷-sequence at the NH₂ terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.     

Mechanism of 3-Methylanthranilic Acid Derepression of the Tryptophan Operon in Escherichia coli

3-Methylanthranilic acid (3MA) inhibits growth and causes derepression of the tryptophan biosynthetic enzymes in wild-type strains of Escherichia coli. Previous reports attributed this effect to an inhibition of the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate to indole-3-glycerol phosphate and a consequent reduction in the concentration of endogenous tryptophan. Our studies have shown that 3MA-resistant mutants linked to the tryptophan operon have a feedback-resistant anthranilate synthetase; mutants with an altered indole-3-glycerol phosphate synthetase were not found. 3MA or 7-methylindole can be metabolized to 7-methyltryptophan, and 3MA, 7-methylindole, and 7-methyltryptophan lead to derepression of the tryptophan operon. Furthermore, 3MA-resistant mutants are also resistant to 7-methylindole derepression. These results strongly suggest that the primary cause of derepression by 3MA is through its conversion to 7-methyltryptophan, which can inhibit anthranilate synthetase, thereby decreasing the concentration of endogenous tryptophan. Unlike 5- or 6-methyltryptophan, 7-methyltryptophan does not appear to function as an active corepressor.