The tyrosyl-tRNA synthetase from Escherichia coli: Complete nucleotide sequence of the structural gene

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on M_r of 47 403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.     

Construction and Characterisation of a Genetically Engineered Escherichia coli Strain for the Epoxide Hydrolase-catalysed Kinetic Resolution of Epoxides

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.     

Syntheses of novel myxopyronin B analogs as potential inhibitors of bacterial RNA polymerase

Based upon observations from our initial findings, additional myxopyronin B analogs have been prepared and tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against Escherichia coli and Staphylococcus aureus.     

Organometallic flavonoid derivatives as spectroscopic probes

Derivatives of naringenin have been synthesized with organometalcarbonyl reporting groups for IR spectroscopy attached at C-2, C-3′, or C-6, and the products have been tested for the induction of nod gene expression using a Rhizobium leguminosarum strain which contains the Escherichia coli lacZ (β-galactosidase) gene fused to nodABC. Derivatives with an OMe substituent within the reporting group moiety showed residual gene induction activity.     

酵母合成2'-岩藻糖基乳糖的研究进展*

2'-岩藻糖基乳糖(2'-fucosyllactose,2'-FL)是人乳寡糖中含量最高的岩藻糖基化寡糖,具有促进双歧杆菌增殖和调节肠道菌群等作用,已被广泛用于婴幼儿食品行业。目前,2'-FL已经成功在大肠杆菌、酿酒酵母、解脂耶氏酵母、枯草芽孢杆菌和谷氨酸棒杆菌中实现从头合成。考虑到食品安全、消费者认知和接受程度,酵母作为GRAS(generally recognized as safe)菌株,在2'-FL的生物合成中更具工业应用前景和商业价值。概述2'-FL的生物合成路径和现状,对2'-FL生物合成常用的底盘细胞进行比较,从底物转运、前体GDP-L-岩藻糖供应、α-1, 2-岩藻糖基转移酶、产物外排和拓宽底物谱等方面综述酵母合成2'-FL的工程化策略,提出酵母合成2'-FL的限制性因素,并对未来研究进行展望。     

A bacteriophage lambda vector for cloning with BamHI and Sau3A

A phage lambda cloning vector has been constructed which contains a single site for the restriction endonuclease BamHI. Since Sau3A and BglII produce the same cohesive ends as BamHI, this vector can also be used to clone DNA fragments generated with either of these enzymes. We have used this vector to construct an Escherichia coli library using partial digestion with Sau3A. This vector will be most useful for applications requiring genetic analysis of cloned E. coli genes.     

Sensitive and specific detection of Xanthomonas campestris pv campestris by PCR using species-specific primers based on hrpF gene sequences

A sensitive and specific assay was developed to detect bacterial black rot of crucifers caused by Xanthomonas campestris pv. campestris (X. c. pv. campestris), in cabbage seed and plant. Primers XCF and XCR from hrpF homologous to nolX, host recognition protein, were used to amplify a 525 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally infected seed and plant of cabbage. The PCR product was only produced from X. c. pv. campestris among 40 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference bacteria.     

Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

Expression of hepatitis B virus surface antigen gene in Escherichia coli

Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10⁵ polypeptide molecules per bacterial cell.     

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNA^Phe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNA^Phe/(U)₉/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319–322).     

Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).     

The oxygen reaction of the cytochrome d-terminated respiratory chain of Escherichia coli at sub-zero temperatures: Kinetic resolution by EPR spectroscopy of two high-spin cytochromes

The oxygen reaction of the fully reduced respiratory chain in membranes from oxygen-limited Escherichia coli was studied at sub-zero temperatures using EPR spectroscopy. Laser photolysis of CO-liganded cytochrome oxidase d precedes oxidation of at least 2 kinetically separable high-spin cytochromes. At −120 to −100°C, a rhombic signal appears, attributable to cytochrome d, followed at above −100°C, by appearance of a second, axial signal near g = 6, here assigned to cytochrome(s) b, and changes in the redox state of iron-sulphur clusters. The data kinetically resolve the 2 high-spin signals attributed to the oxidase complex and suggest schemes for electron flow to oxygen.

Cytochrome oxidase Escherichia coli Cytochrome b-d complex Bacterial electron transport Low-temperature technique     

分子生物学方法提高电活性微生物胞外电子传递效率的研究进展*

微生物细胞与电极之间的胞外电子传递效率是限制微生物电化学技术发展的关键因素,而分子生物学的发展为提高胞外电子传递效率带来了光明前景。从四种具有代表性的纯培养电活性微生物(奥奈达希瓦氏菌、铜绿假单胞菌、硫还原地杆菌和工程大肠杆菌)和混合培养电活性微生物出发,综述了利用分子生物学手段改造几种电活性微生物的研究成果,阐明了针对特异的电活性微生物,如何采取相应的分子生物学手段提高其胞外电子传递的效率,并展望了未来的研究方向。     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.     

Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.     

Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus

The gene for Escherichia coli translational initiation factor 3 (infC) has been inserted into an overexpression plasmid under the control of the bacteriophage T7 promoter. The infC plasmid was then used to transform a host with a chromosomal T7 RNA polymerase gene controlled by the lacUVS promoter. Induction of T7 RNA polymerase expression in the host cells resulted in a 200-fold overexpression of infC mRNA and a 100-fold overproduction of initiation factor 3. Rapid batch purification of biologically active IF3 yielded predominantly the long form of IF3, implying that the short form is an artifact of purification by traditional methods.     

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.