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1.
Diversification of Escherichia coli genomes: are bacteriophages the major contributors? 总被引:10,自引:0,他引:10
Determination of the genome sequence of enterohemorrhagic Escherichia coli O157 Sakai and genomic comparison with the laboratory strain K-12 has revealed that the two strains share a highly conserved 4.1-Mb sequence and that each also contains a large amount of strain-specific sequence. The analysis also revealed the presence of a surprisingly large number of prophages in O157, most of which are lambda-like phages that resemble each other. Based on these results, we discuss how the E. coli strains have diverged from a common ancestral strain, and how bacteriophages contributed to this process. We also describe possible mechanisms by which O157 acquired many closely related phages, and raise the possibility that such bacteria might function as 'phage factories', releasing a variety of chimeric or mosaic phages into the environment. 相似文献
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A key element in eukaryotic immune defenses against invading microbes is the production of reactive oxygen and nitrogen species.
One of the main targets of these species are the iron–sulfur clusters, which are essential prosthetic groups that confer to
proteins the ability to perform crucial roles in biological processes. Microbes have developed sophisticated systems to eliminate
nitrosative and oxidative species and promote the repair of the damages inflicted. The Ric (Repair of Iron Centers) proteins
constitute a novel family of microbial di-iron proteins with a widespread distribution among microbes, including Gram-positive
and Gram-negative bacteria, protozoa and fungi. The Ric proteins are encoded by genes that are up-regulated by nitric oxide
and hydrogen peroxide. Recent studies have shown that the active di-iron center is involved in the restoration of Fe–S clusters
damaged by exposure to nitric oxide and hydrogen peroxide. 相似文献
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B.-Y. Chen 《Biotechnology Techniques》1997,11(10):709-712
Temperature induction of bacteriophage in Escherichia coli depends on bacterial population density. The lowest rate of viability loss at the temperature threshold results in maximal gene expression of . -Infection causes bacterial cells to lose cell viability and thus decrease temperature induction efficiency. In addition, shifting-up in temperature increases the probability of progeny ; thus, the mortality of bacterial hosts increases and the expression of recombinant proteins by naked significantly decrease. 相似文献
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Rotanova T. V. Mel'nikov E. E. Tsirulnikov K. B. 《Russian Journal of Bioorganic Chemistry》2003,29(1):85-87
Two subfamilies of Lon proteases that differ in the structure of fragments containing the catalytically active Ser residue were revealed by the comparison of more than sixty sequences of Lon proteases from various sources. The absence of the classic catalytic triad in the active site of Lon proteases was confirmed. The catalytic site of Lon proteases was shown to be represented by the Ser–Lys dyad. 相似文献
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Nobuo Nakamura Tatsuo Fujio Masao Tanaka 《Bioscience, biotechnology, and biochemistry》2013,77(12):2251-2253
Oxidation of grayanotoxin (GTX) II with lead (IV) acetate in methanol gave a new derivative, the 1(R)-spiro-3,6(S),14,16-tetra-hydroxy-5-keto derivative. Treatment of GTX-II tetraacetate in acetic acid by using Pb(IV) acetate as an oxidizing agent gave a novel 1,5-seco-GTX derivative, Δ1(10)-1,5-seco-GTX-pentaacetate, together with the 1,5-seco-GTX-1(R) derivative. Oxidation of GTX-II-tetraacetate with Tl(III) acetate in acetic acid or benzene gave the 1,5-seco-GTX-1(S) derivative. 相似文献
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Two highly contrasted images depict genomes: at first sight, genes appear to be distributed randomly along the chromosome. In contrast, their organisation into operons (or pathogenicity islands) suggests that, at least locally, related functions are in physical proximity. Analysis of the codon usage bias in orthologous genes in the genome of bacteria which diverged a long time ago suggested that some physical (architectural) selection pressure organised the distribution of genes along the chromosome. The metabolism of highly reactive species such as sulphur-containing molecules must be compartmentalised to escape the deleterious actions of diffusible reagents such as gases or radicals. We analysed the distribution of sulphur metabolism genes in the genome of Escherichia coli and found a number of them to be clustered into statistically significant islands. Another interesting feature of these genes is that the proteins they encode are significantly deprived of cysteine and methionine residues, as compared to the bulk proteins. We speculate that this clustering is associated to the organisation of sulphur metabolism proteins into islands where the sensitive sulphur-containing molecules are protected from reacting with elements in the environment such as dioxygen, nitric oxide or radicals. 相似文献
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Tabakov V. Yu. Emelyanova L. K. Antonova S. V. Voeikova T. A. 《Russian Journal of Genetics》2001,37(3):332-334
The bacterial hemoglobin vhbgene was cloned from sliding bacterium Vitreoscillasp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhbgene was transferred fromEscherichia colito some Streptomycesstrains, producers of antibiotics, by the method of intergeneric conjugation using conjugative–integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhbgene on processes of conjugation and antibiotic production in a number of examined strains was shown. 相似文献
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《亚洲两栖爬行动物研究(英文版)》2017,(4)
The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin(CATH) PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low(3195.88 and 2838.34 Da, respectively). Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid p ET-32 a. This reconstructed plasmid was then transfected into the expression vector E. coliBL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The antimicrobial activity assay using Staphylococcus aureus(Song) and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method. 相似文献
12.
Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained
were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in
fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and
high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified
free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence
of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05
M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following
incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small
amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable
activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan. 相似文献
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Gao Y Liu B Strum S Schutzbach JS Druzhinina TN Utkina NS Torgov VI Danilov LL Veselovsky VV Vlahakis JZ Szarek WA Wang L Brockhausen I 《Glycobiology》2012,22(8):1092-1102
The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis. 相似文献
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Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL–ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL–GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL–ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL–ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. 相似文献
19.
Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis. 相似文献
20.
Panagiotis Fotakis Andreas K. Kateifides Christina Gkolfinopoulou Dimitra Georgiadou Melissa Beck Katharina Gründler Angeliki Chroni Efstratios Stratikos Dimitris Kardassis Vassilis I. Zannis 《Journal of lipid research》2013,54(12):3281-3292
We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I−/− × apoE−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype. 相似文献