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E. Coli Strains (e + coli_strain)
Kinds of E. Coli Strains Selected AbstractsEnhancement of the NAD(P)(H) Pool in Escherichia coli for BiotransformationENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007F. Heuser Abstract In pyridine nucleotide-dependent, reductive whole cell biotransformation with resting cells of Escherichia coli, the availability of intracellular NAD(P)(H) is a pivotal point for an efficient and highly productive substrate conversion. The question whether an increase of the intracellular NAD(P)(H) concentration could increase the productivity was discussed controversially in the past. This is the first report on an E. coli strain with an increased NAD(P)(H) pool which was tested in a reductive biotransformation system for an increased productivity. Biotransformation was performed with a strain overexpressing a gene encoding an (R)-specific alcohol dehydrogenase for the stereospecific, NADPH-dependent reduction of methyl acetoacetate (MAA) to (R)-methyl-3-hydroxybutanoate (MHB). Cofactor regeneration was implemented via glucose oxidation by coexpression of a gene encoding glucose dehydrogenase. The specific MHB productivity (mmol mg,1 cell dry weight,1h,1) enabled a comparison between the E. coli,BL21(DE3) wild-type and a genetically modified strain. The enhancement of the NAD(P)(H) pool was achieved by genetic manipulation of the NAD(H) biosynthetic pathways. After simultaneous overexpression of the pncB and nadE genes, encoding nicotinic acid phosphoribosyltransferase and NAD synthetase, measurements of the total NAD(P)(H) pool, sizes showed a 7-fold and 2-fold increased intracellular concentration of NAD(H) and NADP(H), respectively. However, the implementation of an E.,coli strain carrying a genomically integrated pncB gene with an upstream T7,promoter for biotransformation did not result in reproducible increased specific cell productivity. [source] Novel DNA repair alkyltransferase from Caenorhabditis elegansENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2001Sreenivas Kanugula Abstract O6 -Alkylguanine DNA-alkyltransferase (AGT) is a widely distributed DNA repair protein that protects living organisms from endogenous and exogenous alkylation damage to DNA at the O6 -position of guanine. The search of the C. elegans genome database for an AGT protein revealed the presence of a protein (cAGT-2) with some similarity to known AGTs in addition to the easily recognized cAGT-1 protein. The predicted protein sequence of cAGT-2 contains the amino acid sequence ,ProCysHisPro, at the presumed active site of the protein, whereas all other known AGTs have ,ProCysHisArg,. A truncated version of the cAGT-2 protein was expressed in E. coli. This purified recombinant protein was able to repair O6 -methylguanine and O4 -methylthymine adducts in DNA in vitro and also reacted with the bulky benzyl adduct in O6 -benzylguanine. This fragment of cAGT-2 (104 amino acids) is the smallest protein possessing AGT activity yet described. The full-length cAGT-2 protein (274 amino acids) totally lacks the N-terminal domain present in all other known AGTs but has a long C-terminal extension that has significant homology to histone 1C. Expression of cAGT-2 in an E. coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N -methyl- N,-nitro- N -nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase. Environ. Mol. Mutagen. 38:235,243, 2001. © 2001 Wiley-Liss, Inc. [source] Theoretical study of lipid biosynthesis in wild-type Escherichia coli and in a protoplast-type L-form using elementary flux mode analysisFEBS JOURNAL, Issue 4 2010Dimitar Kenanov In the present study, we investigated lipid biosynthesis in the bacterium Escherichia coli by mathematical modeling. In particular, we studied the interaction between the subsystems producing unsaturated and saturated fatty acids, phospholipids, lipid A, and cardiolipin. The present analysis was carried out both for the wild-type and for several in silico knockout mutants, using the concept of elementary flux modes. Our results confirm that, in the wild type, there are four main products: L1-phosphatidylethanolamine, lipid A, lipid A (cold-adapted), and cardiolipin. We found that each of these compounds is produced on several different routes, indicating a high redundancy of the system under study. By analysis of the elementary flux modes remaining after the knockout of genes of lipid biosynthesis, and comparison with publicly available data on single-gene knockouts in vivo, we were able to determine the metabolites essential for the survival of the cell. Furthermore, we analyzed a set of mutations that occur in a cell wall-free mutant of Escherichia coli W1655F+. We postulate that the mutant is not capable of producing both forms of lipid A, when the combination of mutations is considered to make a nonfunctional pathway. This is in contrast to gene essentiality data showing that lipid A synthesis is indispensable for the survival of the cell. The loss of the outer membrane in the cell wall-free mutant, however, shows that lipid A is dispensable as the main component of the outer surface structure in this particular E. coli strain. [source] Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coliFEBS JOURNAL, Issue 7 2003Christian Schwöppe UhpC is a membrane-bound sensor protein in Escherichia coli required for recognizing external glucose-6-phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His-UhpC protein in a UhpC-deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild-type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing. [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] Palindromic AT-rich repeat in the NF1 gene is hypervariable in humans and evolutionarily conserved in primates,HUMAN MUTATION, Issue 4 2005Hidehito Inagaki Abstract Palindromic sequences are dispersed in the human genome and may cause chromosomal translocations in humans. They constitute unsequenced gaps in the human genome because of their resistance to PCR amplification, cloning into vectors, and sequencing. We have overcome these difficulties by using a combination of optimized PCR conditions, cloning in a recombination-deficient E. coli strain, and RNA polymerases in sequencing. Using these methods, we analyzed a palindromic AT-rich repeat (PATRR) in the neurofibromatosis type 1 (NF1) gene on chromosome 17 (17PATRR). The 17PATRR manifests a size polymorphism due to a highly variable length of (AT)n dinucleotide repeats within the PATRR. 17PATRRs can be categorized into two types: a longer one that comprises a nearly or completely perfect palindrome, and a shorter one that represents its deleted asymmetric derivative. In vitro analysis shows that the longer 17PATRR is more likely to form a cruciform structure than the shorter one. Two reported t(17;22)(q11;q11) patients with NF1, whose breakpoints were identified within the 17PATRR, have translocations that are derived from perfect or nearly perfect palindromic alleles. This implies that the symmetric structure of a PATRR can induce a translocation. We identified conserved PATRRs within the NF1 gene in great apes and similar inverted repeats in two Old World monkeys, but not in New World monkeys or other mammals. This indicates that the palindromic region appeared approximately 25 million years ago and elongated during primate evolution. Although such palindromic regions are usually unstable and disappear rapidly due to deletion, the 17PATRR in the NF1 gene was stably conserved during evolution for reasons that are still unknown. Hum Mutat 26(4), 332,342, 2005. © 2005 Wiley-Liss, Inc. [source] Fate of inoculated Escherichia coli in hayJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007Z.G. Weinberg Abstract Aims:, To monitor the fate of inoculated Escherichia coli in dry and moist hay of various types, under laboratory conditions. Methods and Results:, Wheat, vetch and clover hay were used as received or wetted to 250,300 g kg,1 moisture. The hay was inoculated at about 106 CFU g,1 with a kanamycin-resistant E. coli strain that expresses the green fluorescence protein, and was stored in small open glass jars that were covered with aluminium foil. Three jars per treatment were sampled on days 1 and 3, or 4 and 7, or 8, 20 and 50, respectively, after the initiation of the experiments, and the numbers of E. coli in the hay were determined. The results indicated that E. coli disappeared from both dry and moist hay by 7,8 days after inoculation. However, in a few cases colonies that were presumed to be E. coli developed after incubation in Luria broth medium. Conclusions:, The tagged E. coli strain usually disappeared rapidly from both the dry and the moist hay, in spite of the high level of inocula used. However, in some cases a few, possibly injured E. coli might have persisted, and could be detected after incubation in a rich growth medium. Significance and Impact of the Study:, This study is part of a risk assessment associated with sewage irrigation of forage crops in Israel. The results indicate that E. coli added to the hay is not likely to pose a health risk to cattle or to humans. Nevertheless, more research with natural strains of E. coli and other enteric pathogens that might be more adapted to forage conditions is warranted in order to ensure the safety of sewage-irrigated crops. [source] Molecular characterization of polyphosphate (PolyP) operon from Serratia marcescensJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2006Seung-Jin Lee The polyphosphate (polyP) operon was cloned from a genomic library of Serratia marcescens KCTC 2172 by Southern hybridization using E. coli ppk gene as a probe. The polyP operon was composed of a polyphosphate promoter, polyphosphate kinase (ppk ) and exopolyphosphatase (ppx ). A potential CRP binding site and pho box sequence were found in the region upstream of the putative promoter in the regulatory region. The ppk gene comprises 2,063 nucleotides and encodes 686 amino acids yielding a protein with a molecular mass of 70 kDa. The ppx gene contains 1611 nucleotides and encodes 536 amino acids with a molecular 58 kDa. An E. coli strain transformed with the ppk gene had a 16-fold increased in polyphosphate kinase activity, while introduction of the ppx gene produced a 25-fold increase in polyphosphatase activity. E. coli strains transformed with ppk and ppx genes also displayed increased accumulation of polyphosphate. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] ACID TOLERANCE OF ESCHERICHIA COLI FOLLOWING COLD SHOCK TREATMENTJOURNAL OF FOOD SAFETY, Issue 2 2003GREG BLANK ABSTRACT The effect of an initial cold shock treatment (2 h at 10C), following an abrupt downshift in temperature from 37 to 10C, on the subsequent growth and survival of Escherichia coli strains O157:H7 and MY20 (Biotype 1) in acidified Trypticase soy broth (TSB) and fruit juices (orange, apple) was investigated. Overall, no difference in growth at 37C was observed between each cold shocked and noncold shocked E. coli strain when cultured in TSB adjusted with either acetic acid (pH 6.0)or malic, citric and tartaric acid (each adjusted to: pH 4.5, 5.0, 5.5, 6.0). However, significant (P ± 0.05) differences in survival were observed between cold shocked and noncold shocked populations in TSB acidified with acetic acid (pH 5.0) or citric, malic and tartaric acid (pH 4.0). For strain MY20, survivor levels for cold shocked cells in TSB acidified with acetic acid citric, malic and tartaric acid at 8C were significantly (P ± 0.05) higher than in noncold shocked populations. Also, at 37C survival levels for cold shocked cells were significantly (P ± 0.05) higher than noncold shocked cells in TSB acidified with either malic or tartaric acid (pH 4.0). For the O157:H7 strain, survivor levels were higher (P ± 0.05) for cold shocked cells when maintained in TSB at 37C regardless of acid type. At 8C, cold shock treatment only increased (P ± 0.05) the survival of the O157:H7 strain in TSB adjusted with acetic acid (pH 6.0). Acid cross protection induced by cold shocking, as evidenced by enhanced survival, was not apparent for either E. coil strain in apple (pH 3.5) or orange juice (pH 3.8) maintained at 8C. [source] Consequences of RNase E scarcity in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2002Chaitanya Jain Summary The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10,20% of normal and that the feedback mecha-nism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein. [source] Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone SkpPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23-24 2008Svenja Jarchow Abstract The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep,tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep,Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm. [source] Inhibition of bacterial translocation by chronic ethanol consumption in the rat,APMIS, Issue 12 2001VALERIA BENDER BRAULIO Chronic ethanol ingestion has been associated with small intestine morphological changes, disrupted host mucosal defenses and bacterial overgrowth. Since bacterial translocation (BT) may result from such alterations, we have investigated the potential effect of chronic ethanol consumption on BT. For this purpose, male Wistar rats were fed a liquid diet containing 5% v/v ethanol for 4 weeks (EG, n=16), and a pair-fed group received equal daily amounts of calories in a similar diet without ethanol (PFG, n=16). On experimental day 29, distal ileum ligature and small intestine inoculation of a tetracycline-resistant E. coli strain (Tc®E. coli R6) followed by duodenal ligature was performed. After 1 or 5 h post inoculation, mesenteric lymph nodes, liver, spleen and kidney were excised. Unexpectedly, rats of the EG presented markedly less BT to the mesenteric lymph nodes (p<0.001) and to the other organs examined compared to rats of the PFG. This BT inhibition was observed at 1 and 5 h after bacterial inoculation, and may be attributed exclusively to chronic ethanol ingestion. Since alcoholism is well known to decrease host immunity, these results suggest that other factors, independent of the immune function, may be involved in the BT inhibition observed in this study. [source] Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activityBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Xinkai Xie Abstract Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed ,50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes. Biotechnol. Bioeng. 2009;102: 20,28. © 2008 Wiley Periodicals, Inc. [source] Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphatesBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Jie Bao Abstract The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi,Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis,Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use. © 2005 Wiley Periodicals, inc. [source] Design of Metabolic Engineering Strategies for Maximizing l -(,)-Carnitine Production by Escherichia coli.BIOTECHNOLOGY PROGRESS, Issue 2 2005Bioreactor Levels, Integration of the Metabolic In this work metabolic engineering strategies for maximizing l -(,)-carnitine production by Escherichia coli based on the Biochemical System Theory (1,3) and the Indirect Optimization Method are presented (4). The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of l -(,)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in l -(,)-carnitine production (74% increase in production rate), in close agreement with the modelapos;s predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the l -(,)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value. [source] Understanding 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] Novel Hemoglobins to Enhance Microaerobic Growth and Substrate Utilization in Escherichiacoli,BIOTECHNOLOGY PROGRESS, Issue 5 2001Christian J. T. Bollinger Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiellapneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmpgenes from P. aeruginosa, S. typhi, C. jejuni, K.pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A600 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A600 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D.radiodurans hemoprotein-expressing E. colirelative to the E. coli control carrying the parental plasmid without any hemoglobin gene. [source] Production of Lewis x Tetrasaccharides by Metabolically Engineered Escherichia coliCHEMBIOCHEM, Issue 2 2006Claire Dumon Dr. Abstract Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram-scale by high-cell-density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for ,-3 fucosyltransferase and the Neisseria meningitidis lgtB gene for ,-4 galactosyltransferase. The first compound Gal,-4(Fuc,-3)GlcNAc,-4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin-synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Gal,-4(Fuc,-3)GlcNAc,-3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for ,-3 N-acetylglucosaminyltransferase. [source] Emergence of ciprofloxacin resistance in Escherichia coli isolates from outpatient urine samplesCLINICAL MICROBIOLOGY AND INFECTION, Issue 3 2007C. Gagliotti Abstract This study investigated the association between prescription of fluoroquinolones and emergence of ciprofloxacin resistance among Escherichia coli isolates in the urine of outpatients from whom a ciprofloxacin-sensitive E. coli strain had been isolated previously. Patients were identified and followed using the healthcare databases of Emilia-Romagna Region, Italy. The outcome of interest was the first isolation from urine of an E. coli strain resistant to ciprofloxacin. Prescription of fluoroquinolones during the previous 6 months was associated independently with the emergence of ciprofloxacin resistance; the strength of the association varied according to individual fluoroquinolone agents. [source] Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separationsELECTROPHORESIS, Issue 7-8 2004Yi Zhu Abstract Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains. [source] Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity testsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2002M. Vijayaraj Reddy Abstract 3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the 32P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts. Environ. Mol. Mutagen. 40:1,17, 2002. © 2002 Wiley-Liss, Inc. [source] Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex methodENVIRONMENTAL MICROBIOLOGY, Issue 10 2008David M. Gordon Summary It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85,90% of E. coli strains can be assigned to a phylo-group and that 80,85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group. [source] Phenotypic and genotypic characterization of encapsulated Escherichia coli isolated from blooms in two Australian lakesENVIRONMENTAL MICROBIOLOGY, Issue 5 2005Michelle L. Power Summary Escherichia coli has long been used as an indicator organism for water quality assessment. Recently there has been an accumulation of evidence that suggests some strains of this organism are able to proliferate in the environment, a characteristic that would detract from its utility as an indicator of faecal pollution. Phenotypic and genotypic characterization of E. coli isolated from blooms in two Australian lakes, separated by a distance of approximately 200 km, identified that the blooms were dominated by three E. coli strains. A major phenotypic similarity among the three bloom strains was the presence of a group 1 capsule. Genetic characterization of a conserved region of the cps gene cluster, which encodes group 1 capsules, identified a high degree of genetic variation within the bloom isolates. This differs from previously described encapsulated E. coli strains which are highly conserved at the cps locus. The phenotypic or genotypic profiles of the bloom strains were not identified in 435 E. coli strains isolated from vertebrates. The occurrence of these encapsulated strains suggests that some E. coli have evolved a free-living lifestyle and do not require a host in order to proliferate. The presence of the same three strains in bloom events in different geographical regions of a temperate climate, and at different times, indicates that free-living E. coli strains are able to persist in these water reservoirs. This study provides further evidence of circumstances where caution is required in using E. coli as an indicator organism for water quality. [source] Usage of Aplysia lectin interactions with T antigen and poly- N -acetyllactosamine for screening of E. coli strains which bear glycoforms cross-reacting with cancer-associated antigensFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2001Nechama Gilboa-Garber Abstract Aplysia gonad lectin (AGL), which strongly agglutinates cancer cells, was found, in the present study, to bind to erythrocyte T antigen, in addition to its affinity to Ii system antigens. These antigens were reported to be overexpressed and to contribute to tumor progression and invasion. In healthy human sera, there are antibodies against them, stimulated by the normal intestinal microflora, which bear similar glycoforms. Since the levels of these antibodies were reported to be lower in most cancer patients' sera, we have examined the applicability of AGL to isolation of enteric commensal Escherichia coli strains which bear glycoforms cross-reacting with the cancer-associated antigens. Among 30 E. coli isolates examined, two were agglutinated by AGL. One of them was also agglutinated by certain related galactophilic lectins, which bind to the T and Tn antigens. The agglutination of the two bacteria by healthy human sera, as a group, was stronger than that displayed by the cancer patients' sera. These results indicate that AGL might be useful for identification of the desired bacteria, which could potentially serve for cancer diagnosis and therapy. [source] Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strainsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Tecilli Cabellos-Avelar Abstract The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22 000 transposon-induced and 26 000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process. [source] Metabolism of phenylpropionic acid in enteropathogenic Escherichia coli belonging to serogroup O111 and its application for diagnosisFEMS MICROBIOLOGY LETTERS, Issue 1 2001Kinue Irino Abstract We evaluated a biochemical assay based on the ability to metabolise ,-phenylpropionic acid (PPA) as a diagnostic aid in the identification of typical enteropathogenic Escherichia coli (EPEC) strains. A total of 1061 E. coli strains of serogroups O55, O111, and O119 were initially characterised regarding their H types (serotypes) and the presence of EPEC DNA sequences, eae, EAF, and bfpA. In case of the serogroup O111 strains, 84.6% carried the typical EPEC markers, and the great majority of those (98.1%) were PPA-positive. In contrast, only 0.9% of the serogroups O55 and O119 strains carrying the typical EPEC markers (53.6% and 75.4%, respectively) were PPA-positive. We conclude that the PPA test is a useful method to detect typical EPEC strains only among strains of the O111 serogroup. [source] Proposed mechanism of inactivating Escherichia coli O157:H7 by ultra-high pressure in combination with tert -butylhydroquinoneJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2008A.S. Malone Abstract Aims:, Investigating mechanisms of lethality enhancement when Escherichia coli O157:H7, and selected E. coli mutants, were exposed to tert -butylhydroquinone (TBHQ) during ultra-high pressure (UHP) treatment. Methods and Results:,Escherichia coli O157:H7 EDL-933, and 14 E. coli K12 strains with mutations in selected genes, were treated with dimethyl sulfoxide solution of TBHQ (15,30 ppm), and processed with UHP (400 MPa, 23 ± 2°C for 5 min). Treatment of wild-type E. coli strains with UHP alone inactivated 2·4,3·7 log CFU ml,1, whereas presence of TBHQ increased UHP lethality by 1·1,6·2 log CFU ml,1; TBHQ without pressure was minimally lethal (0,0·6 log reduction). Response of E. coli K12 mutants to these treatments suggests that iron,sulfur cluster-containing proteins ([Fe,S]-proteins), particularly those related to the sulfur mobilization (SUF system), nitrate metabolism, and intracellular redox potential, are critical to the UHP,TBHQ synergy against E. coli. Mutations in genes maintaining redox homeostasis and anaerobic metabolism were associated with UHP,TBHQ resistance. Conclusions:, The redox cycling activity of cellular [Fe,S]-proteins may oxidize TBHQ, potentially leading to the generation of bactericidal reactive oxygen species. Significance and Impact of the Study:, A mechanism is proposed for the enhanced lethality of UHP by TBHQ against E. coli O157:H7. The results may benefit food processors using UHP,based preservation, and biologists interested in piezophilic micro-organisms. [source] High prevalence of antimicrobial-resistant genes and integrons in Escherichia coli isolates from Black-headed Gulls in the Czech RepublicJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2007M. Dolejska Abstract Aims:, To carry out an assessment of the occurrence of resistance to antimicrobials in Escherichia coli that has been isolated from young Black-headed Gulls in three nesting colonies. Methods and Results:, A total of 257 isolates were tested for sensitivity to eight antibacterial substances by disk diffusion method. The polymerase chain reaction was used for detecting specific genes of antibacterial resistance and class 1 integrons in resistant E. coli isolates. A total 75 (29·9%) of 257 isolates were resistant to one or more antimicrobial agents. The dominant type of resistance was to tetracycline, detected in 49 (19·1%) isolates. Resistance to ampicillin was detected in 30 (11·7%), cephalothin in 11 (4·3%), streptomycin in 24 (9·3%), sulphonamides in 20 (7·8%) and chloramphenicol in 5 (1·9%) isolates. Nine isolates carrying integrons were detected. Conclusions:, The study suggests that young Black-headed Gulls are an important host reservoir of resistant E. coli strains, probably reflecting the presence of such strains in their sources of food and/or water. Significance and Impact of the Study:, Although Black-headed Gulls do not naturally come into contact with antibiotics, these birds can be infected with resistant E. coli and potentially serve as their reservoirs, vectors and bioindicators in the environment. [source] Formation of nonculturable Escherichia coli in drinking waterJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2005L.A. Bjergbæk Abstract Aims:, To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. Methods and Results:, Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp -tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20,60 days in nondisinfected drinking water resulted in detection of only 0·7,5% of the initial E. coli population using standard cultivation methods, whereas 1,20% could be resuscitated to a culturable state, and 17,49% could be clearly detected using cultivation-independent methods. Conclusions:, Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10,20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. Significance and Impact of the Study:, The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water. [source] Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strainsJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2005G. P. Schamberger Abstract Aims:, To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. Methods and Results:,Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. Conclusions:,Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. Significance and Impact of the Study:, The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used. [source] | |||||||||||||||||||||||||||||||||||||