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Coli Cells (coli + cell)
Kinds of Coli Cells Selected AbstractsUnderstanding and Improving NADPH-Dependent Reactions by Nongrowing Escherichia coli CellsBIOTECHNOLOGY PROGRESS, Issue 2 2004Adam Z. Walton We have shown that whole Escherichia coli cells overexpressing NADPH-dependent cyclohexanone monooxygenase carry out a model Baeyer-Villiger oxidation with high volumetric productivity (0.79 g ,-caprolactone/L·h ) under nongrowing conditions (Walton, A. Z.; Stewart, J. D. Biotechnol. Prog.2002, 18, 262,268). This is approximately 20-fold higher than the space-time yield for reactions that used growing cells of the same strain. Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively. Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E. coli cells could support even more efficient NADPH-dependent bioconversions if a more suitable enzyme-substrate pair were identified. This was demonstrated by reducing ethyl acetoacetate with whole cells of an E. coli strain that overexpressed an NADPH-dependent, short-chain dehydrogenase from bakerapos;s yeast ( Saccharomyces cerevisiae). Under glucose-fed, nongrowing conditions, this reduction proceeded with a space-time yield of 2.0 g/L·h and a final product titer of 15.8 g/L using a biocatalyst:substrate ratio (g/g) of only 0.37. These values are significantly higher than those obtained previously. Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 ± 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions. This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway. [source] Biosynthesis of the Vitamin E Compound ,-Tocotrienol in Recombinant Escherichia coli CellsCHEMBIOCHEM, Issue 15 2008Christoph Albermann Dr. Abstract The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol-cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mgL,1 homogentisate and 84 ,gL,1 geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac-promoter resulted in the production of methyl-6-geranylgeranyl-benzoquinol (67.9 ,g,g,1). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound,,-tocotrienol (15 ,g,g,1),in E. coli. [source] Cell architecture comes to phage biologyMOLECULAR MICROBIOLOGY, Issue 5 2008Roger Hendrix Summary Nothing is more iconic of the early days of molecular biology than the image of a bacteriophage infecting an Escherichia coli cell. It is perhaps surprising therefore that more than 50 years later, it is still possible to learn something entirely new and unexpected about how phage infection works, as we see in the paper by Edgar et al. of this issue of Molecular Microbiology. The results give fundamental new insight into the way these viruses infect their hosts and promise to open new windows on the virus,host interactions that have shaped the evolution of both. [source] Facilitating the hyphenation of CIEF and MALDI-MS for two-dimensional separation of proteinsELECTROPHORESIS, Issue 15 2010Chang Cheng Abstract Both CIEF and MALDI-MS are frequently used in protein analysis, but hyphenation of the two has not been investigated proportionally. One of the major reasons is that the additives (such as carrier ampholytes and detergent) in CIEF severely suppress the MALDI-MS signal, which hampers the hyphenation of the two. In this paper, we develop a simple means to alleviate the above signal-suppressing effect. We first deposit 1,,L of water onto a MALDI-MS target, deliver a fraction of CIEF-separated protein (,0.1,,L) to the water droplet, evaporate the solvent, add 0.5,,L of MALDI matrix to the sample spot, dry the matrix and move the target plate to a MALDI-TOF-MS for mass spectrum measurement. We optimize the droplet volume and the laser-ablation region. Under the optimized conditions, we improve the S/N by two- to tenfold. We also apply this method for 2-D separations of standard proteins and apolipoprotein A,I, a membrane protein expressed in Escherichia coli cells. [source] Single-step purification of the recombinant green fluorescent protein from intact Escherichia coli cells using preparative PAGEELECTROPHORESIS, Issue 17 2009Few Ne Chew Abstract Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%. [source] Development of a CE-MS method to analyze components of the potential biomarker vascular endothelial growth factor 165ELECTROPHORESIS, Issue 13 2009Angel Puerta Abstract The vascular endothelial growth factor 165 (VEGF165) is the predominant form of the complex VEGF-A family. Its angiogenic effect is involved in many physiological and pathological events. For this reason, its roles as a potential biomarker and as a therapeutic drug have been considered. Nevertheless, very little is known about the existence of different forms of VEGF165 arising from glycosylation and potentially from other PTMs. This aspect is important because different forms may differ in biological activity (therapeutic drug application) and the pattern of the different forms can vary with pathological changes (biomarker application). In this work a CE-MS method to separate up to seven peaks containing, at least, 19 isoforms of intact VEGF165 is described. Comparison between human VEGF165 expressed in a glycosylating system, i.e. insect cells, and in a non-glycosylating system, i.e. E. coli cells, has been carried out. The method developed provides structural information (mass fingerprint) about the different forms of VEGF165 and after the deconvolution and the analysis of the MS spectra, PTMs pattern of VEGF165 including glycosylation and loss of amino acids at the N- and C-terminus was identified. Glycans involved in PTMs promoting different glycoforms observed in the CE-MS fingerprint were confirmed by MALDI-MS after deglycosylation with peptide N-glycosidase F. This approach is a starting point to study the role of VEGF165 as a potential biomarker and to perform quality control of the drug during manufacturing. To our knowledge this is the first time that a CE-MS method for the analysis of VEGF165 has been developed. [source] On-line concentration of proteins by SDS-CGE with LIF detectionELECTROPHORESIS, Issue 2 2008Cheng-Ju Yu Abstract We present a simple approach for on-line concentration of SDS-protein complexes by using poly(vinyl alcohol) (PVA) solution in CGE. In comparison to the coated capillary, the presence of EOF in CGE omitted the need to fill the capillaries with polymer solutions prior to the analysis. More importantly, we found that highly reproducible separation of eight proteins by 3.5% PVA was achieved between runs and without the regeneration of high bulk EOF; the RSD of migration times was less than 0.7%. To further improve the concentration sensitivity, neutral PVA was introduced into the capillary with the help of EOF to act as sieving matrix. The occurrence of stacking at the boundary between the PVA and the sample zone is mainly due to the retardation of proteins by PVA. As a result, the LODs at an S/N of 3 for SDS,protein complexes are of the order of sub-nM to several nM. For example, the LOD for BSA is 0.78 nM, which is a 91-fold sensitivity enhancement over the normal injection. In addition, our stacking method has been applied to the analyses of proteins in Escherichia coli cells. The peak for ,-galactosidase (E. coli) was observed after 0.1 ,M ,-galactosidase was spiked into the E. coli samples. [source] A novel microstep device for the size separation of cellsELECTROPHORESIS, Issue 10-11 2004Sarah Vankrunkelsven Abstract We report on a series of preliminary experiments investigating the applicability of a novel method for the size separation of nano- and microsized particles and cells. The working principle is based on the application of a shear-driven flow through stepwise tapered micro- or nanochannels. Size separations of mixtures of 0.5 and 1.0 ,m carboxylated polystyrene beads as well as of binary mixtures of Staphylococcus aureus and Saccharomycescerevisiae cells and of S. cerevisiae and Escherichia coli cells are demonstrated. [source] Amifostine protection against induced DNA damage in ,-irradiated Escherichia coli cells depend on recN DNA repair gene product activityENVIRONMENTAL TOXICOLOGY, Issue 2 2010Eliseo Almeida Abstract Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against ,-rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild-type genotypes and in uvr, recF, recB, recB-recC-recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against ,-radiation-induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double-strand breaks. The results are discussed in relation to amifostine's chemopreventive potential. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010. [source] Immunocompetence of bivalve hemocytes as evaluated by a miniaturized phagocytosis assayENVIRONMENTAL TOXICOLOGY, Issue 3 2002C. Blaise Abstract Immune function in bivalves can be adversely affected by long-term exposure to environmental contaminants. Investigating alterations in immunity can therefore yield relevant information about the relationship between exposure to environmental contaminants and susceptibility to infectious diseases. We have developed a rapid, cost-effective, and miniaturized immunocompetence assay to evaluate the phagocytic activity, viability, and concentration of hemocytes in freshwater and marine bivalves. Preliminary experiments were performed to optimize various aspects of the assay including 1) the time required for adherence of hemocytes to polystyrene microplate wells, 2) the time required for internalization of fluorescent bacteria, 3) the ratio of hemocytes to fluorescent bacteria in relation to phagocytosis, 4) hemolymph plasma requirements, and 5) the elimination of fluorescence from (noninternalized) bacteria adhering to the external surface of hemocytes. The results of these experiments showed the optimal adherence time for hemocytes in microplate wells to be 1 h, that phagocytosis required at least 2 h of contact with fluorescently labeled E. coli cells, that the number of fluorescent E. coli cells had a positive effect on phagocytic activity, that at least 2.5 million cells/mL were required to measure a significant intake, and that a linear increase in uptake of bacteria (R = 0.91; p < 0.01) could be obtained with concentrations of up to 1.3 × 106 hemocytes/mL. Afterward, the assay was used in two field studies to identify sites having the potential to affect the immunocompetence of bivalves. The first study was conducted on Mya arenaria clams collected at selected contaminated sites in the Saguenay River (Quebec, Canada), and the second examined Elliptio complanata freshwater bivalves that had been exposed to a municipal effluent plume in the St. Lawrence River (Quebec, Canada). In the Saguenay River field study a significant increase in phagocytosis was observed at sites closest to polluted areas. Phagocytotic activity varied over time and was highest during the warmest months (June, July, and August), closely paralleling the spawning period of Mya arenaria clams. In contrast, a drop in phagocytic activity was observed in Elliptio complanata mussels exposed to surface water 4 km downstream of a major municipal effluent plume, with a concomitant increase in the number of hemocytes in the hemolymph. It appears that both immunosuppressive and immunostimulative effects are likely to occur in the field and that responses will be influenced by the type and intensity of contaminants at play. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 160,169, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10047 [source] Biological measurement of estrogenic activity in urine and bile conjugates with the in vitro ER-CALUX reporter gene assayENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2002Juliette Legler Abstract Although estrogens are excreted as biologically inactive conjugates, they can be reconverted to an active form, possibly by bacteria. A simple method was developed to deconjugate estrogen metabolites present in human urine and fish bile back to active estrogens by enzymatic hydrolysis with ,-glucuronidase or live Escherichia coli cells. Deconjugated extracts were tested for estrogenic activity in the in vitro stable estrogen receptor,mediated chemical-activated luciferase gene expression (ER-CALUX) assay. Estrogen glucuronides in urine obtained from human males and females were effectively converted to active forms after incubation with ,-glucuronidase or E. coli. The highest estrogenic activity was found in deconjugated metabolites from urine of a pregnant woman, in which levels up to 3,000 nmol estradiol equivalents per liter of urine were found after overnight incubation of urine with E. coli. Bile sampled from male bream and flounder from various freshwater and marine locations was also deconjugated and a good correlation was found between high biliary estrogenic activity and elevated levels of xenoestrogenic activity in surface water as well as in plasma vitellogenin. Therefore, the measurement of deconjugated bile could form a useful (indirect) biomarker for internal dose of xenoestrogens in male fish. [source] Microbial Baeyer,Villiger Oxidation of Prochiral Polysubstituted Cyclohexanones by Recombinant Whole-Cells Expressing Two Bacterial MonooxygenasesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 5 2005Marko D. Mihovilovic Abstract The microbial Baeyer,Villiger oxidation of prochiral 3,5-dimethylcyclohexanones bearing various functionalities with recombinant E. coli cells overexpressing cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 and cyclopentanone monooxygenase from Comamonas sp. NCIMB 9872 has been investigated. A distinct difference in substrate specificity and stereoselectivity of the two enzymes was observed, and enantiocomplementary products were obtained in some cases. The biocatalytic systems enabled access to chiral lactones as valuable intermediates for the total synthesis of various natural compounds. Substituents with varying lipophilicity and hybridization have been prepared by a diastereoselective synthetic route and subsequently bio-transformed for the investigation of conformational and electronic effects on the biooxidation,. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3FEBS JOURNAL, Issue 18 2009Xiao Luo Insulin-like peptide 3 (INSL3), which is primarily expressed in the Leydig cells of the testes, is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate descent of the testes of the male fetus via interaction with its G protein-coupled receptor, RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombination. To establish an alternative approach to the preparation of human INSL3, we designed and recombinantly expressed a single-chain INSL3 precursor in Escherichia coli cells. The precursor was solubilized from the inclusion body, purified almost to homogeneity by immobilized metal-ion affinity chromatography and refolded efficiently in vitro. The refolded precursor was subsequently converted to mature human INSL3 by sequential endoproteinase Lys-C and carboxypeptidase B treatment. CD spectroscopic analysis and peptide mapping showed that the refolded INSL3 possessed an insulin-like fold with the expected disulfide linkages. Recombinant human INSL3 demonstrated full activity in stimulating cAMP activity in RXFP2-expressing cells. Interestingly, the activity of the single-chain precursor was comparable with that of the mature two-chain INSL3, suggesting that the receptor-binding region within the mid- to C-terminal of B-chain is maintained in an active conformation in the precursor. This study not only provides an efficient approach for mature INSL3 preparation, but also resulted in the acquisition of a useful single-chain template for additional structural and functional studies of the peptide. [source] Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricinFEBS JOURNAL, Issue 17 2002Goran Kragol Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure,antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2,10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin. [source] Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylaseFEBS JOURNAL, Issue 23 2000Evidence that GDP-mannose, GDP-Glc pyrophosphorylases are different proteins GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1- P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an , subunit (43 kDa) and a , subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1- P and GTP) and GDP-Man (from Man-1- P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the , subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the , subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native , subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1- P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1- P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the , subunit is the GMPP, whereas the , subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity. [source] Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24FEBS JOURNAL, Issue 20 2000Toshiyuki Sakaki Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1,,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1,,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43,48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3 -26,23-lactol to 25(OH)D3 -26,23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1,,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1,,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. [source] The archaeal Hjm helicase has recQ-like functions, and may be involved in repair of stalled replication forkGENES TO CELLS, Issue 2 2006Ryosuke Fujikane The archaeal Hjm is a structure-specific DNA helicase, which was originally identified in the hyperthermophilic archaeon, Pyrococcus furiosus, by in vitro screening for Holliday junction migration activity. Further biochemical analyses of the Hjm protein from P. furiosus showed that this protein preferably binds to fork-related Y-structured DNAs and unwinds their double-stranded regions in vitro, just like the E. coli RecQ protein. Furthermore, genetic analyses showed that Hjm produced in E. coli cells partially complemented the defect of functions of RecQ in a recQ mutant E. coli strain. These results suggest that Hjm may be a functional counterpart of RecQ in Archaea, in which it is necessary for the maintenance of genome integrity, although the amino acid sequences are not conserved. The functional interaction of Hjm with PCNA for its helicase activity further suggests that the Hjm works at stalled replication forks, as a member of the reconstituted replisomes to restart replication. [source] A cryptic lysis gene near the start of the Q, replicase gene in the +1 frameGENES TO CELLS, Issue 10 2004Tohru Nishihara The maturation/lysis (A2) protein encoded by the group B single-stranded RNA bacteriophage Q, mediates lysis of host Escherichia coli cells. We found a frameshift mutation in the replicase (,-subunit) gene of Q, cDNA causes cell lysis. The mutant has a single base deletion 73 nucleotides (nt) 3, from the start of the replicase gene with consequent translation termination at a stop codon 129,131 nt further 3,. The 43-amino acid C-terminal part of the 67-amino acid product encoded by what in WT (wild-type) is the +1 frame, is rich in basic amino acids This 67-aa protein can mediate cell lysis whose characteristics indicate that the protein may cause lysis by a different mechanism and via a different target, than that caused by the A2 maturation/lysis protein. Synthesis of a counterpart of the newly discovered lysis product in wild-type phage infection would require a hypothetical ribosomal frameshifting event. The lysis gene of group A RNA phages is also short, 75 codons in MS2, and partially overlaps the first part of their equivalently located replicase gene, raising significant evolutionary implications for the present finding. [source] SsrA-mediated protein tagging in the presence of miscoding drugs and its physiological role in Escherichia coliGENES TO CELLS, Issue 7 2002Tatsuhiko Abo Background: We have shown recently that read-through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho-independent terminator leads to SsrA-mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read-through of stop codons and SsrA-mediated protein tagging. Results: Miscoding antibiotics caused translational read-through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA-mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read-through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3, end of the mRNA defined by the terminator hairpin. The SsrA-defective cells were more sensitive to the miscoding antibiotics compared to the wild-type cells. Conclusion: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics. [source] Bidirectional migration of SeqA-bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coliGENES TO CELLS, Issue 5 2000Sota Hiraga Background We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions. Results The present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 °C. Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division. Conclusions A single cluster of SeqA-bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies. [source] Bacterial cell death induced by human pro-apoptotic Bax is blocked by an RNase E mutant that functions in an anti-oxidant pathwayGENES TO CELLS, Issue 3 2000Rika Nanbu-Wakao Background Bax is a member of the Bcl-2 family and induces apoptosis of mammalian cells. We have shown that a trace amount of human Bax induces the cell death of Escherichia coli, accompanied by damage to DNA, and that the region of Bax which is lethal to E. coli is also responsible for apoptosis-inducing activity in the mammalian cells. Results We isolated a Bax-resistant mutant from E. coli cells that survive in the presence of paraquat, a generator of superoxide, by screening a library constructed from the random insertion of a transposon. Psb1 (paraquat-resistant, suppressor of Bax-1) mutant had a Tn 10 transposon inserted in the rne gene of E. coli, splitting the RNase E gene (rne) into N- and C-terminal halves. The introduction of the truncated 5, end of rne specifically enhanced resistance to paraquat, prevented cell death induced by Bax and decreased the intracellular H2O2 concentration. The region responsible for the paraquat- and Bax-resistance was not the catalytic site for the endoribonuclease activity of RNase E. Conclusions The N-terminal region of the RNase E protein inhibits bacterial death induced by human Bax as well as paraquat through a unique mechanism that is distinct from RNA digestion. This study implies that the protection of bacterial death induced by Bax is associated with an anti-oxidant pathway and that a mutant RNase E has a novel function as an anti-oxidant. [source] Development and Evaluation of a DNA Vaccine Based on Helicobacter pylori urease B: Failure to Prevent Experimental Infection in the Mouse ModelHELICOBACTER, Issue 6 2006Livania Zavala-Spinetti Abstract Background:, The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). Methods:, The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 µg each) and intranasal (16 µg) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. Results:, Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. Conclusion:, A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations. [source] The OsDHODH1 Gene is Involved in Salt and Drought Tolerance in RiceJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2009Wen-Ying Liu Abstract In the present paper, we identified and cloned OsDHODH1 encoding a putative cytosolic dihydroorotate dehydrogenase (DHODH) in rice. Expression analysis indicated that OsDHODH1 is upregulated by salt, drought and exogenous abscisic acid (ABA), but not by cold. By prokaryotic expression, we determined the enzymatic activity of OsDHODH1 and found that overproduction of OsDHODH1 significantly improved the tolerance of Escherichia coli cells to salt and osmotic stresses. Overexpression of the OsDHODH1 gene in rice increased the DHODH activity and enhanced plant tolerance to salt and drought stresses as compared with wild type and OsDHODH1 -antisense transgenic plants. Our findings reveal, for the first time, that cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought. [source] Atomic force microscopy study of the role of LPS O-antigen on adhesion of E. coliJOURNAL OF MOLECULAR RECOGNITION, Issue 5 2009Joshua Strauss Abstract The O-antigen is a highly variable component of the lipopolysaccharide (LPS) among Escherichia coli strains and is useful for strain identification and assessing virulence. While the O-antigen has been chemically well characterized in terms of sugar composition, physical properties such as O-antigen length of E. coli LPS have not been well studied, even though LPS length is important for determining binding of bacteria to biomolecules and epithelial cells. Atomic force microscopy (AFM) was used to characterize the physicochemical properties of the LPS of eight E. coli strains. Steric repulsion between the AFM tip (silicon nitride) and the E. coli cells was measured and modeled, to determine LPS lengths for three O157 and two O113 E. coli strains, and three control (K12) strains that do not express the O-antigen. For strains with an O-antigen, the LPS lengths ranged from 17,±,10 to 37,±,9,nm, and LPS length was positively correlated with the force of adhesion (Fadh). Longer lengths of LPS may have allowed for more hydrogen bonding between the O-antigen and silanol groups of the AFM silicon nitride tip, which controlled the magnitude of Fadh. For control strains, LPS lengths ranged from 3,±,2 to 5,±,3,nm, and there was no relationship between LPS length and adhesion force between the bacterium and the silicon nitride tip. In the absence of the O-antigen, we attributed Fadh to electrostatic interactions with lipids in the bacterial membrane. Copyright © 2009 John Wiley & Sons, Ltd. [source] Monolithic Ceramic Foams for Ultrafast Photocatalytic Inactivation of BacteriaJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 8 2009Pinggui Wu Palladium-modified nitrogen-doped titanium dioxide (TiON/PdO) foams were synthesized by a sol,gel process on a polyurethane foam template. The TiON/PdO foam was tested for microbial killing using Escherichia coli cells as a target. Under visible-light illumination, the TiON/PdO foam displayed a strong antimicrobial effect on the bacteria cells in water. The antimicrobial effect was found to be dependent on the palladium content and the calcination temperature. In a flow-through dynamic photoreactor, the new photocatalyst efficiently inactivated E. coli within a short contact time (<1 min), the shortest ever reported for the photocatalytic killing of bacteria. The strong antimicrobial functions of the TiON/PdO foam were related to charge trapping by PdO and the high contact efficiency of the foam structure. [source] Application of a microfluidic device for counting of bacteriaLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2006K.-I. Inatomi Abstract Aims:, To develop a miniaturized analytical system for counting of bacteria. Methods and Results:,Escherichia coli cells were used throughout the experiments. The system consists of a microfluidic chamber, a fluorescence microscope with a charge-coupled device (CCD) camera and syringe pumps. The chamber was made of a silicone rubber (30 × 30 mm and 4 mm high). The E. coli cells were flowed from a micro-nozzle fabricated in the chamber and detected with the CCD camera. The individual cells were indicated as signal peaks on a computer. The cell counts showed a good correlation compared with that of a conventional plate counting method, and results of the simultaneous detection of live and dead cells were also presented. Conclusions, Significance and Impact of the Study:, The system having a small disposable nozzle has the advantages for low cost and safe medical or environmental analysis, when compared with a conventional flow cytometer. This is the first step of the development of a one-chip microbe analyzer. [source] Analysis of a dextran-binding domain of the dextranase of Streptococcus mutansLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2002H. Morisaki Aims: To examine the dextran-binding domain of the dextranase (Dex) of Streptococcus mutans. Methods and Results: Deletion mutants of the Dex gene of Strep. mutans were prepared by polymerase chain reaction and expressed in Escherichia coli cells. Binding of the truncated Dexs to dextran was measured with a Sephadex G-150 gel. Although the Dexs which lacked the N-terminal variable region lost enzyme activity, they still retained dextran-binding ability. In addition, further deletion into the conserved region from the N-terminal did not influence the dextran-binding ability. However, the Dex which carried a deletion in the C-terminus still possessed both enzyme activity and dextran-binding ability. Further deletion into the conserved region from the C-terminal resulted in complete disappearance of both enzyme and dextran-binding activities. Conclusions: Deletion analysis of the Dex gene of Strep. mutans showed that the C-terminal side (about 120 amino acid residues) of the conserved region of the Dex was essential for dextran-binding ability. Significance and Impact of the Study: The dextran-binding domain was present in a different area from the catalytic site in the conserved region of the Dex molecule. The amino acid sequence of the dextran-binding domain of the Dex differed from those of glucan-binding regions of other glucan-binding proteins reported. [source] Protection of red sea bream Pagrus major against red sea bream iridovirus infection by vaccination with a recombinant viral proteinMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2010Hajime Shimmoto ABSTRACT Megalocytivirus infections cause serious mass mortality in marine fish in East and Southeast Asian countries. In this study the immunogenicity of crude subunit vaccines against infection by the Megalocytivirus RSIV was investigated. Three capsid proteins, 18R, 351R and a major capsid protein, were selected for use as crude subunit vaccines. High homology among Megalocytivirus types was found in the initial sequence examined, the 351R region. Red sea bream (Pagrus major) juveniles were vaccinated by intraperitoneal injection of recombinant formalin-killed Escherichia coli cells expressing these three capsid proteins. After challenge infection with RSIV, fish vaccinated with the 351R-recombinant bacteria showed significantly greater survival than those vaccinated with control bacteria. The 351R protein was co-expressed with GAPDH from the bacterium Edwardsiella tarda in E. coli; this also protected against viral challenge. A remarkable accumulation of RSIV was observed in the blood of vaccinated fish, with less accumulation in the gills and spleen tissues. Thus, the 351R-GAPDH fusion protein is a potential vaccine against Megalocytivirus infection in red sea bream. [source] Damped oscillations in the adaptive response of the iron homeostasis network of E. coliMOLECULAR MICROBIOLOGY, Issue 2 2010Amnon Amir Summary Living organisms often have to adapt to sudden environmental changes and reach homeostasis. To achieve adaptation, cells deploy motifs such as feedback in their genetic networks, endowing the cellular response with desirable properties. We studied the iron homeostasis network of E. coli, which employs feedback loops to regulate iron usage and uptake, while maintaining intracellular iron at non-toxic levels. Using fluorescence reporters for iron-dependent promoters in bulk and microfluidics-based, single-cell experiments, we show that E. coli cells exhibit damped oscillations in gene expression, following sudden reductions in external iron levels. The oscillations, lasting for several generations, are independent of position along the cell cycle. Experiments with mutants in network components demonstrate the involvement of iron uptake in the oscillations. Our findings suggest that the response is driven by intracellular iron oscillations large enough to induce nearly full network activation/deactivation. We propose a mathematical model based on a negative feedback loop closed by rapid iron uptake, and including iron usage and storage, which captures the main features of the observed behaviour. Taken together, our results shed light on the control of iron metabolism in bacteria and suggest that the oscillations represent a compromise between the requirements of stability and speed of response. [source] RecA-mediated excision repair: a novel mechanism for repairing DNA lesions at sites of arrested DNA synthesisMOLECULAR MICROBIOLOGY, Issue 1 2007Marc Bichara Summary In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism , RecA-mediated excision repair , in which RecA-dependent pairing of the mono-modified single-stranded template with a complementary sequence allows its repair by the UvrABC excinuclease. [source] | |||||||||||||||||||||||||||||||