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Sample records for escherichia coli bl21de3

  1. Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains

    PubMed Central

    Wurm, David J.; Hausjell, Johanna; Ulonska, Sophia; Herwig, Christoph; Spadiut, Oliver

    2017-01-01

    When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (qs,glu) and the corresponding maximum specific lactose uptake rate (qs,lac,max) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv). We showed the effect of qs,lac,max on productivity and product location underlining its importance for recombinant protein production. In the present study we investigated the mechanistic qs,glu/qs,lac,max correlation for four pET-based E. coli BL21(DE3) strains producing different recombinant products and thereby proved the mechanistic model to be platform knowledge for E. coli BL21(DE3). However, we found that the model parameters strongly depended on the recombinant product. Driven by this observation we tested different dynamic bioprocess strategies to allow a faster investigation of this mechanistic correlation. In fact, we succeeded and propose an experimental strategy comprising only one batch cultivation, one fed-batch cultivation as well as one dynamic experiment, to reliably determine the mechanistic model for qs,glu/qs,lac,max and get trustworthy model parameters for pET-based E. coli BL21(DE3) strains which are the basis for bioprocess development. PMID:28332595

  2. Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3)

    PubMed Central

    Austin, Brian P.; Waugh, David S.

    2011-01-01

    The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25 mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5 mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4 °C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration. PMID:22197595

  3. Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

    PubMed

    Robichon, Carine; Luo, Jianying; Causey, Thomas B; Benner, Jack S; Samuelson, James C

    2011-07-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

  4. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity

    PubMed Central

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M.; Park, Jin-Byung

    2016-01-01

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass. PMID:27681369

  5. Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity.

    PubMed

    Woo, Ji-Min; Kim, Ji-Won; Song, Ji-Won; Blank, Lars M; Park, Jin-Byung

    The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

  6. Development of a Method To Produce Hemoglobin in a Bioreactor Culture of Escherichia coli BL21(DE3) Transformed with a Plasmid Containing Plesiomonas shigelloides Heme Transport Genes and Modified Human Hemoglobin Genes ▿

    PubMed Central

    Smith, B. J. Z.; Gutierrez, P.; Guerrero, E.; Brewer, C. J.; Henderson, D. P.

    2011-01-01

    We describe a method for production of recombinant human hemoglobin by Escherichia coli grown in a bioreactor. E. coli BL21(DE3) transformed with a plasmid containing hemoglobin genes and Plesiomonas shigelloides heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates. PMID:21803893

  7. Identification of riboflavin: revealing different metabolic characteristics between Escherichia coli BL21(DE3) and MG1655.

    PubMed

    Wang, Xinran; Wang, Qian; Qi, Qingsheng

    2015-06-01

    There are many physiological differences between Escherichia coli B and K-12 strains, owing to their different origins. Deeper insight into the metabolic and regulative mechanisms of these strains will inform improved usage of these industrial workhorses. In the present study, we observed that BL21 fermentation broth gradually turned yellow during cultivation. By spectral analysis and liquid chromatography-mass spectrometry identification, we confirmed for the first time that the yellow substance accumulated in the fermentation broth is riboflavin. Comparing the enzyme sequences involved in riboflavin metabolism between BL21 and MG1655, we identified a site mutation on the 115 residue of bifunctional riboflavin kinase/FMN adenylyltransferase (RibF) in BL21. This His115Leu mutation was found to reduce enzyme activity to 55% of that of MG1655, which is probably one reason for riboflavin accumulation in BL21. Quantitative PCR analysis showed that genes of the entire branch of the riboflavin and FAD biosynthesis pathways in BL21 were up-regulated. Several physiological and metabolic characteristics of BL21 and MG1655 were found to be different, and may also be related to the riboflavin accumulation.

  8. Tracing ancestors and relatives of Escherichia coli B, and the derivation of B strains REL606 and BL21(DE3).

    PubMed

    Daegelen, Patrick; Studier, F William; Lenski, Richard E; Cure, Susan; Kim, Jihyun F

    2009-12-11

    Antecedents of Escherichia coli B have been traced through publications, inferences, and personal communication to a strain from the Institut Pasteur in Paris used by d'Herelle in his studies of bacteriophages as early as 1918 (a strain not in the current collection). This strain appears to have passed from d'Herelle to Bordet in 1920, and from Bordet to at least three other laboratories by 1925. The strain that Gratia received from Bordet was apparently passed to Bronfenbrenner by 1924 and from him to Luria around 1941. Delbrück and Luria published the first paper calling this strain B in 1942. Its choice as the common host for phages T1-T7 by the phage group that developed around Delbrück, Luria, and Hershey in the 1940s led to widespread use of B along with E. coli K-12, chosen about the same time for biochemical and genetic studies by Tatum and Lederberg. Not all currently available strains related to B are descended from the B of Delbrück and Luria; at least three strains with somewhat different characteristics were derived independently by Hershey directly from the Bronfenbrenner strain, and a strain that appears to have passed from Bordet to Wollman is in the current Collection of the Institut Pasteur. The succession of manipulations and strains that led from the B of Delbrück and Luria to REL606 and BL21(DE3) is given, established in part through evidence from their recently determined complete genome sequences.

  9. Enhancing functional expression of codon-optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons.

    PubMed

    Zhong, Chao; Wei, Ping; Zhang, Yi-Heng Percival

    2017-05-01

    Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon-optimized (i.e., rare codon-free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha-helix, beta-strand, or linker in the first half of rare codon-free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta-strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi-SRC-containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP-folding reporter when it was fused to the 3' end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six-fold enhancement in soluble protein expression level as compared to the wild-type and rare codon-free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co-translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054-1064. © 2016 Wiley Periodicals, Inc.

  10. Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction

    PubMed Central

    Kwon, Soon-Kyeong; Kim, Seong Keun; Lee, Dae-Hee; Kim, Jihyun F.

    2015-01-01

    Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. To accomplish successful overexpression of membrane proteins in a workhorse organism such as E. coli, defined and rational optimization strategies based on an understanding of the genetic background of the toxicity-escape mechanism are desirable. To this end, we sequenced the genomes of E. coli C41(DE3) and its derivative C43(DE3), which were developed for membrane protein production. Comparative analysis of their genomes with those of their ancestral strain E. coli BL21(DE3) revealed various genetic changes in both strains. A series of E. coli variants that are able to tolerate transformation with or overexpression of membrane proteins were generated by in vitro evolution. Targeted sequencing of the evolved strains revealed the mutational hotspots among the acquired genetic changes. By these combinatorial approaches, we found non-synonymous changes in the lac repressor gene of the lac operon as well as nucleotide substitutions in the lacUV5 promoter of the DE3 region, by which the toxic effect to the host caused by overexpression of membrane proteins could be relieved. A mutation in lacI was demonstrated to be crucial for conferring tolerance to membrane protein overexpression. PMID:26531007

  11. Comparative genomics and experimental evolution of Escherichia coli BL21(DE3) strains reveal the landscape of toxicity escape from membrane protein overproduction.

    PubMed

    Kwon, Soon-Kyeong; Kim, Seong Keun; Lee, Dae-Hee; Kim, Jihyun F

    2015-11-04

    Achieving sufficient yields of proteins in their functional form represents the first bottleneck in contemporary bioscience and biotechnology. To accomplish successful overexpression of membrane proteins in a workhorse organism such as E. coli, defined and rational optimization strategies based on an understanding of the genetic background of the toxicity-escape mechanism are desirable. To this end, we sequenced the genomes of E. coli C41(DE3) and its derivative C43(DE3), which were developed for membrane protein production. Comparative analysis of their genomes with those of their ancestral strain E. coli BL21(DE3) revealed various genetic changes in both strains. A series of E. coli variants that are able to tolerate transformation with or overexpression of membrane proteins were generated by in vitro evolution. Targeted sequencing of the evolved strains revealed the mutational hotspots among the acquired genetic changes. By these combinatorial approaches, we found non-synonymous changes in the lac repressor gene of the lac operon as well as nucleotide substitutions in the lacUV5 promoter of the DE3 region, by which the toxic effect to the host caused by overexpression of membrane proteins could be relieved. A mutation in lacI was demonstrated to be crucial for conferring tolerance to membrane protein overexpression.

  12. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3), and comparison of the closely related E. coli B and K-12 genomes

    SciTech Connect

    Studier, F.W.; Daegelen, P.; Lenski, R. E.; Maslov, S.; Kim, J. F.

    2009-12-01

    Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations ({approx} 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions. P1 transductions from K-12 into the two B lineages produced 317 single-base-pair differences and 9 insertions or deletions, reflecting differences between B DNA in BL21(DE3) and integrated restriction fragments of K-12 DNA inherited by REL606. Two sites showed selective enrichment of spontaneous mutations. No unselected spontaneous single-base-pair mutations were evident. The genome sequences revealed that a progenitor of REL606 had been misidentified, explaining initially perplexing differences. Limited sequencing of other B strains defined characteristic properties of B and allowed assembly of the inferred genome of the ancestral B of Delbrueck and Luria. Comparison of the B and K-12 genomes shows that more than half of the 3793 proteins of their basic genomes are predicted to be identical, although {approx} 310 appear to be functional in either B or K-12 but not in both. The ancestral basic genome appears to have had {approx} 4039 coding sequences occupying {approx} 4.0 Mbp. Repeated horizontal transfer from diverged Escherichia coli genomes and homologous recombination may explain the observed variable distribution of single-base-pair differences. Fifteen sites are occupied by phage-related elements, but only six by comparable elements at the same site. More than 50 sites are occupied by IS elements in both B and K, 16 in common, and likely founding IS elements are identified. A signature of widespread cryptic phage P4-type mobile elements was

  13. Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)

    PubMed Central

    2011-01-01

    Background Gene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3). Results The deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ΔarcAΔiclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ΔiclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ΔarcA ΔiclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a

  14. Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome.

    PubMed

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Kountourakis, Nikos; Koukaki, Marina; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Tsolis, Konstantinos C; Karamanou, Spyridoula; Economou, Anastassios

    2016-01-01

    Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.

  15. Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level.

    PubMed

    Bartlow, Patrick; Uechi, Guy T; Cardamone, John J; Sultana, Tamanna; Fruchtl, McKinzie; Beitle, Robert R; Ataai, Mohammad M

    2011-08-01

    Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.

  16. Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.

    PubMed

    Wang, Hengwei; Wang, Fengqing; Wang, Wei; Yao, Xueling; Wei, Dongzhi; Cheng, Hairong; Deng, Zixin

    2014-01-01

    Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

  17. Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

    PubMed

    Kumar, Sandeep; Jain, Kavish Kumar; Singh, Anupam; Panda, Amulya K; Kuhad, Ramesh Chander

    2015-06-01

    Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

  18. Cloning and construction of recombinant palI gene from Klebsiella oxytoca on pET-32b into E. coli BL21 (DE3) pLysS for production of isomaltulose, a new generation of sugar

    SciTech Connect

    Moeis, Maelita R. Berlian, Liska Suhandono, Sony Prima, Alex Komalawati, Eli Kristianti, Tati

    2014-03-24

    Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, palI gene from Klebsiella oxytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length palI gene sequence of Klebsiella oxytoca was cloned in E. coli DH5α. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomerisation {sup 325}RLDRD{sup 329} and 97% identical to palI gene from Klebsiella sp. LX3 (GenBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNA sequencing result shows that palI gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of palI gene from Klebsiella oxytoca.

  19. Cloning and construction of recombinant palI gene from Klebsiella oxytoca on pET-32b into E. coli BL21 (DE3) pLysS for production of isomaltulose, a new generation of sugar

    NASA Astrophysics Data System (ADS)

    Moeis, Maelita R.; Berlian, Liska; Suhandono, Sony; Prima, Alex; Komalawati, Eli; Kristianti, Tati

    2014-03-01

    Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, palI gene from Klebsiella oxytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length palI gene sequence of Klebsiella oxytoca was cloned in E. coli DH5α. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomerisation 325RLDRD329 and 97% identical to palI gene from Klebsiella sp. LX3 (GenBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNA sequencing result shows that palI gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of palI gene from Klebsiella oxytoca.

  20. Rapid Isolation of Nuclear Transport-Competent Xenopus Nucleoplasmin Produced in Escherichia coli Strain BL21(DE3)

    DTIC Science & Technology

    1994-01-01

    method to produce nuclear transport-competent nucleo-plasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony.

  1. Production of 3-O-xylosyl quercetin in Escherichia coli.

    PubMed

    Pandey, Ramesh Prasad; Malla, Sailesh; Simkhada, Dinesh; Kim, Byung-Gee; Sohng, Jae Kyung

    2013-03-01

    Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/∆pgi, E. coli BL21(DE3)/∆zwf, E. coli BL21(DE3)/∆pgi∆zwf, and E. coli BL21(DE3)/∆pgi∆zwf∆ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/∆pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/∆pgi∆zwf∆ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.

  2. Efficacy evaluation of live Escherichia coli expression Brucella P39 protein combined with CpG oligodeoxynucleotides vaccine against Brucella melitensis 16M, in BALB/c mice.

    PubMed

    Al-Mariri, Ayman; Mahmoud, Nermeen Haj; Hammoud, Razan

    2012-03-01

    Brucella is gram-negative bacteria responsible for brucellosis in a wide variety of animals and humans. BALB/c mice were immunized with live Escherichia coli expression the p39 gene of Brucella melitensis, a gene coding for the periplasmic binding protein. Mice were injected with either E. coli BL21 (DE3) pEt15b or E. coli BL21 (DE3) pEt15b-p39 alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. E. coli BL21 (DE3) pEt15b-p39 with CpG ODN or with non-CpG ODN mice groups showed a significant IFN-γ production and T-cell proliferation as a reaction to P39 antigen. In addition, antibody responses (IgG, IgG1 and IgG2a), were only found in these two mice groups. A higher level of protection against B. melitensis 16M were observed in mice immunized with E. coli BL21 (DE3) pEt15b-p39 and CpG ODN comparing with those immunized with E. coli BL21 (DE3) pEt15b-p39 alone or with non-CpG ODN. No protection against B. melitensis 16M was observed in mice immunized with E. coli BL21 (DE3) pEt15b alone or with the adjuvant. Rev.1 protection at 4 and 8 weeks post-challenge was more effective than that observed with E. coli BL21 (DE3) pEt15b-p39 and CpG ODN.

  3. Development of glycerol-utilizing Escherichia coli strain for the production of bioethanol.

    PubMed

    Thapa, Laxmi Prasad; Lee, Sang Jun; Yoo, Hah Young; Choi, Han Suk; Park, Chulhwan; Kim, Seung Wook

    2013-08-15

    The production of bioethanol was studied using recombinant Escherichia coli with glycerol as a carbon source. Glycerol is an attractive feedstock for biofuels production since it is generated as a major byproduct in biodiesel industry; therefore, we investigated the conversion of glycerol to bioethanol using E. coli BL21 (DE3) which harbors several genes in ethanol production pathway of Enterobacter aerogenes KCTC 2190. Fermentation was carried out at 34°C for 42h, pH 7.6, using defined production medium. Under optimal conditions, bioethanol production by the recombinant E. coli BL21 (DE3), strain pEB, was two-fold (3.01g/L) greater than that (1.45g/L) by the wild-type counterpart. The results obtained in this study will provide valuable guidelines for engineering bioethanol producers.

  4. The regulatory elements of araBAD operon, contrary to lac-based expression systems, afford hypersynthesis of murine, and human interferons in Escherichia coli.

    PubMed

    Stefan, Alessandra; Alfarano, Pietro; Merulla, Davide; Mattana, Paolo; Rolli, Eleonora; Mangino, Pierluigi; Masotti, Lanfranco; Hochkoeppler, Alejandro

    2009-01-01

    The overexpression of four different interferons, i.e., murine interferon alpha1 and human interferons alpha1, alpha 8, and alpha 21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon alpha1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining beta-galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis.

  5. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    PubMed

    Nie, Yao; Yan, Wei; Xu, Yan; Chen, Wen Bo; Mu, Xiao Qing; Wang, Xinye; Xiao, Rong

    2013-01-01

    Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful

  6. Synergistic effects of chromosomal ispB deletion and dxs overexpression on coenzyme Q(10) production in recombinant Escherichia coli expressing Agrobacterium tumefaciens dps gene.

    PubMed

    Choi, Jin-Ho; Ryu, Yeon-Woo; Park, Yong-Cheol; Seo, Jin-Ho

    2009-10-12

    For biotechnological production of coenzyme Q(10) (CoQ(10)) in recombinant Escherichia coli, three genetic manipulations were performed: heterologous expression of decaprenyl diphosphate synthase (Dps) from Agrobacterium tumefaciens, deletion of endogenous octaprenyl diphosphate synthase (IspB), and overexpression of 1-deoxy-d-xylulose synthase (Dxs). Expression of the dps gene and deletion of the ispB gene in E. coli BL21(DE3)DeltaispB/pAP1 allowed production of CoQ(10) only. Furthermore, coexpression of the dxs gene increased the specific content of CoQ(10) from 0.55-0.89mgg(-1) to 1.40mgg(-1). For mass production of CoQ(10), fed-batch fermentation of E. coli BL21(DE3)DeltaispB/pAP1+pDXS was carried out in a defined medium with 20gl(-1) initial glucose and by the glucose-feeding strategy of pH-stat. Finally, 99.4mgl(-1) CoQ(10) concentration, 1.41mgg(-1) specific CoQ(10) content and 3.11mgl(-1)h(-1) productivity were obtained in 33h of the fermentation, which were 78, 1.9, and 19 times higher than those for E. coli BL21(DE3)/pAP1 without the ispB deletion and dxs overexpression.

  7. Polycistronic expression of human platelet factor 4 with heparin-neutralizing activity in Escherichia coli.

    PubMed

    Duan, Yitao; Wang, Zhe; Wu, Wei; Fang, Zhenjiang; Huang, He

    2012-01-01

    Human platelet factor 4 (hPF4) was evaluated as a clinical alternative to protamine for heparin neutralization, a protector against radiation injury and an anti-neoplastic. To achieve high-level expression of hPF4, expression vectors pET-28a(+)-nf PF4 (n=4, 5, 6) containing n tandem repeats of PF4 were constructed and transformed into the Escherichia coli BL21(DE3) strain. A higher expression level, about 45% of the total proteins (TP), was obtained for E. coli BL21(DE3)/pET28a(+)-nf PF4 (n=4, 5, 6). The purified His-PF4 protein was further identified by cleavage with enterokinase and MS, and its heparin-neutralizing activity was determined by colony formation assay. This study represents a novel approach to large-scale production of PF4 in E. coli, one that might be applied to large-scale production of PF4 protein for possible clinical application. It also provides theoretical points for the expression and purification of other small-molecule peptides.

  8. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  9. [Optimization of fermentation of recombinant human Endostatin (rh-Endostatin) expression in Escherichia coli].

    PubMed

    Chang, Guo-Dong; Li, Zhuang-Lin; Qin, Jia-Yang; Ma, Cui-Qing; Luo, Yong-Zhang; Xu, Ping

    2005-07-01

    The fermentation process of recombinant human Endostatin expression in Escherichia coli BL21 (DE3) was studied. The effects of factors such as concentration of IPTG, induction time, cultivation temperature and feeding strategies were investigated. Beside that, by changing the temperature to 40 degrees C after induction, the high-density cultivation finished in a much shorter period. After 9 hours cultivation, the optical density (OD) at 600 nm reached 140 and the yield of inclusion body was 3 g/L. While E. coli system was used, protein with better activity and stability was obtained. The cost was much lower and the producing process was much steadier. It will meet the demands of the industrial production.

  10. Overexpression of transport proteins improves the production of 5-aminovalerate from l-lysine in Escherichia coli

    PubMed Central

    Li, Zhong; Xu, Jing; Jiang, Tongtong; Ge, Yongsheng; Liu, Pan; Zhang, Manman; Su, Zhiguo; Gao, Chao; Ma, Cuiqing; Xu, Ping

    2016-01-01

    Bacterial transporters mediate the exchanges between intracellular and extracellular environments. Modification of transport route could be applied to speed up the metabolic reactions and promote the production of aimed compounds. Herein, lysine 2-monooxygenase (DavB) and δ-aminovaleramidase (DavA) were co-expressed in Escherichia coli BL21(DE3) to produce nylon-5 monomer 5-aminovalerate from l-lysine. Then, PP2911 (4-aminobutyrate transporter in Pseudomonas putida) and LysP (the lysine specific permease in E. coli) were overexpressed to promote 5-aminovalerate production using whole cells of recombinant E. coli. The constructed E. coli strain overexpressing transport proteins exhibited good 5-aminovalerate production performance and might serve as a promising biocatalyst for 5-aminovalerate production from l-lysine. This strategy not only shows an efficient process for the production of nylon monomers but also might be used in production of other chemicals. PMID:27510748

  11. Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus.

    PubMed

    Che, Xuchun; Hu, Jinwei; Wang, Lijuan; Zhu, Zhifeng; Xu, Qiong; Lv, Junqiang; Fu, Zheng; Sun, Yajun; Sun, Jia; Lin, Gang; Lu, Rong; Yao, Zhi

    2011-11-01

    Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.

  12. The Escherichia coli Peripheral Inner Membrane Proteome*

    PubMed Central

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

    2013-01-01

    Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with

  13. Permeability enhancement of Escherichia coli by single-walled carbon nanotube treatment.

    PubMed

    Mosleh, Abdollah; Heintz, Anna; Lim, Ki-Taek; Kim, Jin-Woo; Beitle, Robert

    2017-03-06

    This research investigated the use of single-walled carbon nanotubes (SWNTs) as an additive to increase the permeability of a bacterial cell wall. Recombinant Escherichia coli BL21 (DE3) that expressed β-lactamase were exposed to SWNTs under various levels of concentration and agitation. Activity of β-lactamase in the culture fluid and Transmission Electron Microscopy (TEM) were used to determine the amount of released protein, and visually examine the permeability enhancement of the cells. It was found that β-lactamase release in the culture fluid occurred in a dose dependent manner with treatment by SWNTs and was also dependent on agitation rate. Based on TEM, this treatment successfully caused an increase in permeability without significant damage to the cell wall. Consequently, SWNTs can be used as an enhancement agent to cause the release of intracellular proteins. This article is protected by copyright. All rights reserved.

  14. Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production

    PubMed Central

    2013-01-01

    Background In the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in biotechnology has led to the development of many different strains, and selecting an ideal host to produce a specific protein of interest is an important step in developing a production process. E. coli B and K–12 strains are frequently employed in large-scale production processes, and therefore are of particular interest. We previously evaluated the individual cultivation characteristics of E. coli BL21 and the K–12 hosts RV308 and HMS174. To our knowledge, there has not yet been a detailed comparison of the individual performances of these production strains in terms of recombinant protein production and system stability. The present study directly compared the T7-based expression hosts E. coli BL21(DE3), RV308(DE3), and HMS174(DE3), focusing on evaluating the specific attributes of these strains in relation to high-level protein production of the model protein recombinant human superoxide dismutase (SOD). The experimental setup was an exponential carbon-limited fed-batch cultivation with minimal media and single-pulse induction. Results The host strain BL21(DE3) produced the highest amounts of specific protein, followed by HMS174(DE3) and RV308(DE3). The expression system HMS174(DE3) exhibited system stability by retaining the expression vector over the entire process time; however, it entirely stopped growing shortly after induction. In contrast, BL21(DE3) and RV308(DE3) encountered plasmid loss but maintained growth. RV308(DE3) exhibited the lowest ppGpp concentration, which is correlated with the metabolic stress level and lowest degradation of soluble protein fraction compared to both other strains. Conclusions Overall, this study provides

  15. Expression of Recombinant Human Insulin-like Growth Factor Type 1 (rhIGF-1) in Escherichia coli

    PubMed Central

    Iranpoor, Hamidreza; Omidinia, Eskandar; Vatankhah, Venus; Gharanjik, Vahid; Shahbazi, Majid

    2015-01-01

    Background: Human insulin-like growth factor type 1 (hIGF-1) is a protein consisting of 70 amino acids (MW=7.6 kDa) and mainly synthesized by liver. Mecasermin (Trade name INCRELEX) is the synthetic form of the protein which is used as an effective treatment for particular disorders such as short stature, type 1 and 2 diabetes, and wound healing. Current study was aimed to investigate the expression of human insulin-like growth factor type1 in Escherichia coli (E. coli) BL21 (DE3) expression system in order to produce an active recombinant form of the protein. Methods: For the purpose of the study, firstly codon optimization was done for hIGF-1 gene, using bioinformatics databases. Then, the gene was synthesized and inserted in pET-24a vector by a cutting strategy included NdeI and BamHI-HF enzymes. In the next step, gene was run in agarose gel and purified. The constructed expression cassette was transformed into E. coli BL21 (DE3) cells through CaCl 2 heat shock method. Identification and confirmation of the transformed colonies were performed using screening PCR method. Synthesis of hIGF-1 was induced by IPTG. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. Confirmation of cloning and IGF-1 expression cassette was carried out through genetic engineering procedures. Results: Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells. Molecular weight of the expressed protein was estimated to be 7.6 kDa. Conclusion: hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified. In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized. PMID:26306149

  16. Simultaneous biocatalyst production and Baeyer-Villiger oxidation for bioconversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase.

    PubMed

    Lee, Won-Heong; Park, Yong-Cheol; Lee, Dae-Hee; Park, Kyungmoon; Seo, Jin-Ho

    2005-01-01

    Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce epsilon-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. A fed-batch process was designed to obtain high cell density for improving production of epsilon-caprolactone. The fed-batch SPO process gave the best results, 10.2 g/L of epsilon-caprolactone and 0.34 g/(L.h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.

  17. Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

    PubMed Central

    Juhas, Mario; Ajioka, James W.

    2015-01-01

    Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci. PMID:25816013

  18. Hyperexpression of rat spermatidal protein TP2 in Escherichia coli by codon optimization and engineering the vector-encoded 5' UTR.

    PubMed

    Meetei, A R; Rao, M R

    1998-07-01

    We have recently reported the cDNA cloning of rat spermatidal protein TP2 and its expression in Escherichia coli using pTrc 99A as the expression vector. However, the expression level was very low. We have now improved the expression of TP2 over fivefold by (1) optimizing the codons for lysine, arginine, proline, leucine, glycine, valine, threonine, alanine, and tyrosine and (2) by engineering the vector-encoded 5' UTR. The expressed protein was in the soluble phase and could be purified to homogeneity by successive chromatography on Zinc-NTA-agarose affinity matrix and heparin agarose. Serendipitously, we have also observed a concomitant hyperinduction of vector encoded beta-lactamase gene along with TP2 in the E. coli BL21 (DE3) cells.

  19. High-level expression of Falcipain-2 in Escherichia coli by codon optimization and auto-induction.

    PubMed

    Sarduy, Emir Salas; Muñoz, Aymara Cabrera; Trejo, Sebastián Alejandro; de los A Chavéz Planes, María

    2012-05-01

    Falcipain-2, the major cysteine hemoglobinase from the human malaria parasite Plasmodium falciparum, is critical for parasite development and is considered a promising chemotherapeutic target. In order to facilitate the high-throughput screening of Falcipain-2 inhibitors from natural sources, we developed an economic and highly-productive overexpression system in Escherichia coli using a codon-optimized proFalcipain-2 construct. Very high expression levels (35-55% of total host proteins) were observed when proFalcaipain-2 expression was induced with 1mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) in several E. coli strains, with the highest level observed for BL21(DE3). A lower expression (~40% of total host proteins) was observed when BL21(DE3) was grown in ZYM-5052 auto-induction medium, containing 0.2% lactose as inducer. However, the culture grew to notably higher cellular density, increasing ~1.5 times the overall yield of the system when compared with conventional IPTG-induction. Although several conditions were modified to achieve the expression of soluble and active Falcipain-2, the enzyme was mainly obtained in the form of insoluble aggregates. After purification and refolding, ~50 mg of active enzyme were obtained per liter of culture at low cost using a regular incubator shaker, and recombinant Falcipain-2 exhibited structural and functional characteristics very similar to the natural counterpart. Due to its versatility and simplicity, this strategy can be straightforwardly adapted to other proteins from Plasmodium species or any other organism with an AT-rich genome.

  20. Vibration and glycerol-mediated plasmid DNA transformation for Escherichia coli.

    PubMed

    Shanehbandi, Dariush; Saei, Amir A; Zarredar, Habib; Barzegari, Abolfazl

    2013-11-01

    Escherichia coli transformation is an essential step in many molecular biology experiments. Despite earlier advances in the field, many studies including shotgun cloning still require more efficient transformation protocols. Chemical transformation has been the most popular method, in which competent cells are transformed following a brief period of heat shock. Here, we report a novel protocol with higher efficiency, in which competent E. coli cells (treated with CaCl2 ) grown in media containing glycerol experience a gentle vibration. Three E. coli strains DH5α, Jm107 and BL21 (DE3) and three plasmids pGEM-T, pET-28a and pCAMBIA with different sizes (3000, 5369 and 8428 bp, respectively) were used to test the protocol. The results indicated a significant increase in number of transformed colonies compared with heat-shock method. Our findings also demonstrated the favourable impacts of glycerol on transformation of E. coli.

  1. Cloning and Optimization of Soluble Vascular Endothelial Growth Factor165 Expression in Escherichia coli

    PubMed Central

    Salimi, Ali; Babashamsi, Mohammad

    2016-01-01

    Background: Vascular Endothelial Growth Factor (VEGF) is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF165. VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF165 in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor165 expression in Escherichia coli (E. coli). Methods: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF165 gene was amplified by reverse transcription polymerase chain reaction (RTPCR), and then VEGF165 was subcloned into prokaryotic expression vectors pET-32a(+) and transformed into BL21 (DE3) E. coli strain. VEGF165 expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF165 was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using His•tag specific polyclonal antibody. Results: Our results demonstrated that VEGF165 was successfully cloned and expressed in pET-32a(+) vector. Optimization of the expression procedure showed that, induction by 1 mM IPTG at OD600=0.7 and overnight incubation at 22°C resulted in the highest expression levels of soluble VEGF165. Conclusion: In this study, the expression of VEGF165 in a high soluble level was successfully cloned and optimized. PMID:26855732

  2. Triphenyltin degradation and proteomic response by an engineered Escherichia coli expressing cytochrome P450 enzyme.

    PubMed

    Yi, Wenying; Yang, Kunliang; Ye, Jinshao; Long, Yan; Ke, Jing; Ou, Huase

    2017-03-01

    Although triphenyltin (TPT) degradation pathway has been determined, information about the enzyme and protein networks involved was severely limited. To this end, a cytochrome P450 hydroxylase (CYP450) gene from Bacillus thuringiensis was cloned and expressed in Escherichia coli BL21 (DE3), namely E. coli pET32a-CYP450, whose dosage at 1gL(-1) could degrade 54.6% TPT at 1mgL(-1) within 6 d through attacking the carbon-tin bonds of TPT by CYP450. Sequence analysis verified that the CYP450 gene had a 1214bp open reading frame, encoding a protein with 404 amino acids. Proteomic analysis determined that 60 proteins were significantly differentially regulated expression in E. coli pET32a-CYP450 after TPT degradation. The up-regulated proteins enriched in a network related to transport, cell division, biosynthesis of amino acids and secondary metabolites, and microbial metabolism in diverse environments. The current findings demonstrated for the first time that P450 received electrons transferring from NADH could effectively cleave carbon-metal bonds.

  3. A comparison and optimization of methods and factors affecting the transformation of Escherichia coli.

    PubMed

    Chan, Weng-Tat; Verma, Chandra S; Lane, David P; Gan, Samuel Ken-En

    2013-12-12

    DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl2-CaCl2, CaCl2 and Hanahan's methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan's method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl2 method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria-Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109's competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.

  4. Improved production of adipate with Escherichia coli by reversal of β-oxidation.

    PubMed

    Kallscheuer, Nicolai; Gätgens, Jochem; Lübcke, Marvin; Pietruszka, Jörg; Bott, Michael; Polen, Tino

    2017-03-01

    The linear C6 dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.

  5. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGES

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; ...

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  6. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxygenase with a bacterial type-I fatty acid synthase in E. coli.

    PubMed

    Coursolle, Dan; Lian, Jiazhang; Shanklin, John; Zhao, Huimin

    2015-09-01

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg L(-1) long chain alcohol/alkane products including a 57 mg L(-1) titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  7. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    SciTech Connect

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  8. Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli.

    PubMed

    Tan, Haidong; Wang, Jinxia; Zhao, Zongbao Kent

    2007-11-01

    The nucleotide sequence encoding bovine enterokinase light chain (EK) from Chinese northern yellow bovine was isolated. Two single-nucleotide mutations, namely, C245G and A528T were identified. The gene encoding the Pro82Arg/Glu176Asp variant of known bovine EK was fused with glutathione S-transferase and overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with IPTG and glucose. Effective fusion protein purification, refolding, auto-catalytic cleavage and mature EK recovery were described. The specific activity of the purified EK was determined as 110+/- 10 U/mg, which was comparable to a specific activity of > or =20 U/mg of the E. coli expressed EK sample provided by Sigma (Cat. No. E4906). This procedure produced approximately 53 mg of EK per 500 mL of cell culture, which was much higher than previous reports, thus providing a basis for large-scale production of EK and for further applications in biotechnology.

  9. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology

    PubMed Central

    Akbari, V.; Sadeghi, H. Mir Mohammad; Jafarian-Dehkordi, A.; Chou, C. Perry; Abedi, D.

    2015-01-01

    Human epidermal growth factor receptor (HER) family plays an important role in various types of cancers. As a result, antibodies against HER and the mechanism of antigen-antibody binding action are under active investigation. We previously constructed a single-chain variable fragment (ScFv) against HER2, i.e. anti-Her2 ScFv, for expressing in the Escherichia coli. In the present study, we report the optimization of anti-Her2 ScFv expression in an E. coli host of BL21 (DE3) pLysS using response surface methodology based on tuning of three cultivation variables, including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. A model for protein expression according to the Box-Behnken design predicted a maximal anti-Her2 ScFv expression at 37 °C, a post-induction time of 10.45 h and 0.75 mM IPTG. In addition, strategies based on inclusion body isolation and affinity chromatography were applied to purify anti-Her2 ScFv. The purity of the final product for inclusion bodies isolation and purification by Ni-NTA resin were 70 % and 95 %, respectively. The solubilization of the inclusion bodies was carried out using two denaturant agents, guanidine hydrochloride and urea. The present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies. PMID:26430460

  10. Expression of Caenorhabditis elegans antimicrobial peptide NLP-31 in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Lim, Mei-Perng; Nathan, Sheila

    2014-09-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a fulminant disease endemic in Southeast Asia and Northern Australia. The standardized form of therapy is antibiotics treatment; however, the bacterium has become increasingly resistant to these antibiotics. This has spurred the need to search for alternative therapeutic agents. Antimicrobial peptides (AMPs) are small proteins that possess broad-spectrum antimicrobial activity. In a previous study, the nematode Caenorhabditis elegans was infected by B. pseudomallei and a whole animal transcriptome analysis identified a number of AMP-encoded genes which were induced significantly in the infected worms. One of the AMPs identified is NLP-31 and to date, there are no reports of anti-B. pseudomallei activity demonstrated by NLP-31. To produce NLP-31 protein for future studies, the gene encoding for NLP-31 was cloned into the pET32b expression vector and transformed into Escherichia coli BL21(DE3). Protein expression was induced with 1 mM IPTG for 20 hours at 20°C and recombinant NLP-31 was detected in the soluble fraction. Taken together, a simple optimized heterologous production of AMPs in an E. coli expression system has been successfully developed.

  11. Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Wang, Da; Hurst, Tamiika K.; Thompson, Richard B.; Fierke, Carol A.

    2011-08-01

    Zinc is an essential element for numerous cellular processes, therefore zinc homeostasis is regulated in living organisms. Fluorescent sensors have been developed as important tools to monitor the concentrations of readily exchangeable zinc in live cells. One type of biosensor uses carbonic anhydrase (CA) as the recognition element based on its tunable affinity, superior metal selectivity, and fluorescence signal from aryl sulfonamide ligands coupled to zinc binding. Here, we fuse carbonic anhydrase with a red fluorescent protein to create a series of genetically-encoded Förster resonance energy transfer-based excitation ratiometric zinc sensors that exhibit large signal increases in response to alterations in physiological-free zinc concentrations. These sensors were applied to the prokaryotic model organism Escherichia coli to quantify the readily exchangeable zinc concentration. In minimal media, E. coli BL21(DE3) cells expressing the CA sensor, exhibit a median intracellular readily exchangeable zinc concentration of 20 pM, much less than the total cellular zinc concentration of ~0.2 mM. Furthermore, the intracellular readily exchangeable zinc concentration varies with the concentration of environmental zinc.

  12. Investigation of CNT-induced Escherichia coli Lysis and Protein Release

    NASA Astrophysics Data System (ADS)

    Mosleh, Abdollah

    This research investigated the use of carbon nanotubes (CNTs) as a treatment to increase the permeability of a bacterial cell wall. Recombinant Escherichia coli BL21 (DE3) containing a plasmid that expressed Green Fluorescent Protein (GFP) and ?-lactamase were exposed to CNTs under various levels of agitation for different times. Fluorescence assay for GFP, optical absorbance for beta-lactamase activity, and Transmission Electron Microscopy (TEM) were used to determine the amount of released protein, and visually examine the permeability enhancement of the cells, respectively. It was found that more beta-lactamase was present in the culture fluid after treatment with CNTs in a dose dependent manner. Indeed, CNTs can lyse the cells up to 90% of maximum when compared to lysozyme treatment. Based on TEM, it is believed that this treatment damaged the cell walls to make E. coli permeable, causing periplasm proteins and enzymes to leak out into the medium. Consequently, CNTs can be used as lysis agents when it is undesirable to add an additional enzyme (lysozyme) to cause the release of intracellular proteins.

  13. Expression and purification of truncated diphtheria toxin, DT386, in Escherichia coli: An attempt for production of a new vaccine against diphtheria

    PubMed Central

    Shafiee, Fatemeh; Rabbani, Mohammad; Behdani, Mahdi; Jahanian-Najafabadi, Ali

    2016-01-01

    The aim of this study was to produce a recombinant protein consisting of the catalytic and translocation domains of diphtheria toxin for its later application as a vaccine candidate against Corynebacterium diphtheria. To achieve this goal, at first, the amino acid sequence of DT386 was used for prediction of T and B cell epitopes using on-line servers. The DT386 coding sequence was synthesized and subcloned into the NcoI and XhoI sites of pET28a plasmid and recombinant pET28a plasmid was used to transform Escherichia coli BL21 (DE3) host cells. Afterwards, recombinant cells were selected and subjected to induction of expression by 1 mM isopropyl β-D-1-thiogalactopyranoside, (IPTG). Expression of the desired protein was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and finally, the recombinant protein was purified using nickel affinity chromatography. The results of epitope prediction using on-line servers established the ability of DT386 for stimulation of immune system against diphtheria toxin. Restriction digestion of the recombinant plasmids using NcoI and XhoI enzymes confirmed the fidelity of cloning by producing a band of about 1200 bp. SDS-PAGE analysis following induction of expression and also purification step confirmed the expression of the desired protein by showing a band of about 45 kDa. In addition, Western blot analysis using anti-6X-His antibody confirmed the identity of the expected protein. In conclusion, in the present study we amplified and cloned the coding sequence of DT386 fragment, followed by its expression by E. coli BL21 (DE3) cells. Then, the expressed protein was purified and will be used for later studies of evaluation of its immunogenic properties. PMID:27920826

  14. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  15. PATHOGENIC ESCHERICHIA COLI

    EPA Science Inventory

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  16. Pathogenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli, a member of the Enterobacteriaceae family, is a part of the normal flora of the intestinal tract of humans and a variety of animals. E. coli strains are classified on the basis of antigenic differences in two surface components (serotyping), the somatic antigen (O) of the lipopoly...

  17. Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli.

    PubMed

    Yuan, Liu-Di; Hua, Zi-Chun

    2002-07-01

    Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp)(4)-Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx-EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K(m) of 0.17 mM, and K(cat) is 20.8s(-1). From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.

  18. Recombinant expression and purification of heparin binding proteins: midkine and pleiotrophin from Escherichia coli.

    PubMed

    Singh, Priyo K; Srivastava, Vivek

    2012-10-01

    Midkine (MDK) and Pleiotrophin (PTN) belong to a class of heparin-binding growth factors and are highly expressed in a number of cancers. Bioactive and recombinant MDK and PTN are critical reagent for cancer drug discovery studies. MDK and PTN belong to a newly evolving family of secreted neurotrophic and developmentally regulated heparin-binding molecules. PTN is related to MDK with 45% sequence identity and both proteins have been shown to be involved in promoting neurite outgrowth. MDK is a cysteine-rich 13kDa protein containing five disulfide bonds and PTN is 19kDa protein containing ten disulphide bonds. In this study, we expressed recombinant human MDK (rhMDK), mouse MDK (rmMDK) and human pleiotrophin (rhPTN) in Escherichia coli BL21(DE3)pLysS strain. Soluble rhMDK, rmMDK and rhPTN were expressed at a high-level in this strain and the protein was purified (∼90%) by a one-step purification using heparin affinity chromatography. A total of 4mg purified MDK and 7mg of purified PTN were obtained with the overall yield from 1L of bacterial culture. Activity of purified rhMDK and rhPTN was confirmed by a cell proliferation assay using NIH3T3 cells.

  19. Effects of medium composition on production of 5-aminolevulinic acid by recombinant Escherichia coli.

    PubMed

    Qin, Gang; Lin, Jianping; Liu, Xiaoxia; Cen, Peilin

    2006-10-01

    The recombinant Escherichia coli BL21(DE3) harboring hemA from Agrobacterium radiobacter, which was engineered in our previous work, was used for the extracellular production of 5-aminolevulinic acid (ALA). The effects of various physiological factors, such as the concentrations of precursors (glycine, succinic acid and glucose) and the inhibitor 5-aminolevulinate dehydratase (levulinic acid), on the ALA accumulation in the fermentation broth were investigated in both shake flasks and a jar fermentor. Among these precursors, glycine exhibited the strongest ability to inhibit cell growth, while glucose mainly inhibited ALA formation. The optimum initial concentrations of glycine, succinic acid and glucose were found to be 2.0, 10.0 and 2.0 g/l, respectively. Levulinic acid (LA; 30 mM) was fed to the fermentation broth at the end of the exponential cell growth phase (about 8 h), and the intracellular activity of ALA dehydratase was efficaciously suppressed. Repeating the optimum composition of the medium in a stirred tank fermenter resulted in 1.49 g/l ALA. Furthermore, the fed batch of the precursors and inhibitor further increased ALA production up to 3.01 g/l.

  20. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

    PubMed Central

    Koolivand, Davoud; Bashir, Nemat Sokhandan; Behjatnia, Seyed Aliakbar; Joozani, Raziallah Jafari

    2016-01-01

    The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection. PMID:27721695

  1. Molecular cloning, expression, and characterization of a Sophora alopecuroides lectin from Escherichia coli.

    PubMed

    Li, Yang; Li, Tingting; Li, Jinyao; Liu, Dongliang; Yang, Jie; Yang, Jianhua; Zhang, Fuchun; Sun, Surong

    2014-09-01

    Sophora alopecuroides lectin (SAL) has been isolated from the seeds and confirmed to have antifungal and antitumor activities, and presently the preparation of the natural lectin was cumbersome, time-consuming, and the yield was relatively low for further analysis. In this study, the signal peptide of lectin, the modification sites, and the secondary structure were analyzed, and the three-dimensional structures of SAL were modeled. The gene of SAL was amplified by the reverse transcription polymerase chain reaction, and cloned into the pET-30a vector and expressed in Escherichia coli BL21(DE3) by the induction of isopropyl-beta-d-thiogalactopyranoside. Totally, 400 mg of recombinant SAL (rSAL) was purified from 1 l of bacterial culture through Ni-NTA agarose column and the purity reached 95%. The recombinant protein was further confirmed by western blot using rSAL-specific antibody. The biological activity analysis results showed that rSAL exclusively bound to d-galactose and had universal hemagglutinating activities to human A, B, O, and AB, and rabbit and mouse erythrocytes. rSAL also inhibited the growth of fungi, the proliferation of cancer cells, and the HIV-I reverse transcriptase activity. In conclusion, this study indicates that rSAL can be produced in large quantities in the prokaryotic expression system and the recombinant protein still retains the various biological activities, which will make the large-scale production of SAL recombinant protein at dramatically reduced cost possible.

  2. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    PubMed

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

  3. Escherichia coli biofilms

    PubMed Central

    Beloin, Christophe; Roux, Agnès; Ghigo, Jean-Marc

    2008-01-01

    Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli. PMID:18453280

  4. Production of extracellular fatty acid using engineered Escherichia coli

    PubMed Central

    2012-01-01

    Background As an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics. Results In this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3) improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL) produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired product. The strain p

  5. Characterization of the sensor domain of QseE histidine kinase from Escherichia coli.

    PubMed

    Yeo, Kwon Joo; Park, Jin-Wan; Kim, Eun-Hee; Jeon, Young Ho; Hwang, Kwang Yeon; Cheong, Hae-Kap

    2016-10-01

    In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4-5.0, the folded protein existed in a monomeric form with a predominantly helical structure. (1)H-(15)N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE.

  6. Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

    PubMed

    Eggers, Jessica; Steinbüchel, Alexander

    2014-12-01

    The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay

  7. Diarrheagenic Escherichia coli.

    PubMed

    Gomes, Tânia A T; Elias, Waldir P; Scaletsky, Isabel C A; Guth, Beatriz E C; Rodrigues, Juliana F; Piazza, Roxane M F; Ferreira, Luís C S; Martinez, Marina B

    2016-12-01

    Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

  8. Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1,7-bisphosphatase in Escherichia coli.

    PubMed

    Vira, Chaitali; Prakash, Gunjan; Rathod, Jayant Pralhad; Lali, Arvind M

    2016-11-16

    Sedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)24VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl β-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel-nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.

  9. Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli.

    PubMed

    Kim, Jae-Eung; Huang, Rui; Chen, Hui; You, Chun; Zhang, Y-H Percival

    2016-09-01

    A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error-prone PCR or molecular shuffling, and a linear vector backbone was prepared by high-fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap-extension PCR with a pair of 5'-phosphorylated primers. Third, full-length linear plasmids with phosphorylated 5'-ends were self-ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high-efficiency transformation. Self-made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10(5) cfu/µg of the self-ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6-phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5-fold improved catalytic efficiency (kcat /Km ) on NAD(+) as compared to the wild-type. This protocol is DNA-sequence independent, and does not require restriction enzymes, special E. coli host, or labor-intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.

  10. Cloning of a fibrinolytic enzyme (subtilisin) gene from Bacillus subtilis in Escherichia coli.

    PubMed

    Ghasemi, Younes; Dabbagh, Fatemeh; Ghasemian, Abdollah

    2012-09-01

    Several investigations are being pursued to enhance the efficacy and specificity of fibrinolytic therapy. In this regard, microbial fibrinolytic enzymes attracted much more medical interests during these decades. Subtilisin, a member of subtilases (the superfamily of subtilisin-like serine proteases) and also a fibrinolytic enzyme is quite common in Gram-positive bacteria, and Bacillus species stand out in particular, as many extracellular and even intracellular variants have been identified. In the present work, the subtilisin gene from Bacillus subtilis PTCC 1023 was cloned into the vector pET-15b and expressed in Escherichia coli strain BL21 (DE3). Total genomic DNA were isolated and used for PCR amplification of the subtilisin gene by means of the specific primers. SDS-PAGE and enzyme assay were done for characterizing the expressed protein. A ~1,100 bp of the structural subtilisin gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of subtilisin showed high sequence identity with the other strains of B. subtilis, whereas significantly lower identity was observed with other bacterial subtilisins. The recombinant enzyme had the same molecular weight as other reported subtilisins and the E. coli transformants showed high subtilisin activity. This study provides evidence that subtilisin can be actively expressed in E. coli. The commercial availability of subtilisin is of great importance for industrial applications and also pharmaceutical purposes as thrombolytic agent. Thus, the characterization of new recombinant subtilisin and the development of rapid, simple, and effective production methods are not only of academic interest, but also of practical importance.

  11. Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli.

    PubMed

    Wang, Jian-Feng; Xiong, Zhi-Qiang; Li, Shi-Yuan; Wang, Yong

    2013-09-01

    Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

  12. Production of uroporphyrinogen III, which is the common precursor of all tetrapyrrole cofactors, from 5-aminolevulinic acid by Escherichia coli expressing thermostable enzymes.

    PubMed

    Hibino, Aiko; Petri, René; Büchs, Jochen; Ohtake, Hisao

    2013-08-01

    Uroporphyrinogen III (urogen III) was produced from 5-aminolevulinic acid (ALA), which is a common precursor of all metabolic tetrapyrroles, using thermostable ALA dehydratase (ALAD), porphobilinogen deaminase (PBGD), and urogen III synthase (UROS) of Thermus thermophilus HB8. The UROS-coding gene (hemD₂) of T. thermophilus HB8 was identified by examining the gene product for its ability to produce urogen III in a coupled reaction with ALAD and PBGD. The genes encoding ALAD, PBGD, and UROS were separately expressed in Escherichia coli BL21 (DE3). To inactivate indigenous mesophilic enzymes, the E. coli transformants were heated at 70 °C for 10 min. The bioconversion of ALA to urogen III was performed using a mixture of heat-treated E. coli transformants expressing ALAD, PBGD, and UROS at a cell ratio of 1:1:1. When the total cell concentration was 7.5 g/l, the mixture of heat-treated E. coli transformants could convert about 88 % 10 mM ALA to urogen III at 60 °C after 4 h. Since eight ALA molecules are required for the synthesis of one porphyrin molecule, approximately 1.1 mM (990 mg/l) urogen III was produced from 10 mM ALA. The present technology has great potential to supply urogen III for the biocatalytic production of vitamin B₁₂.

  13. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    SciTech Connect

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  14. Process development for production of recombinant human interferon-gamma expressed in Escherichia coli.

    PubMed

    Khalilzadeh, R; Shojaosadati, S A; Maghsoudi, N; Mohammadian-Mosaabadi, J; Mohammadi, M R; Bahrami, A; Maleksabet, N; Nassiri-Khalilli, M A; Ebrahimi, M; Naderimanesh, H

    2004-02-01

    A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma(hIFN-gamma). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20-30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l(-1) after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-gamma in the variable specific growth rate strategy were 0.35 g rHu-IFN-gamma g(-1) dry cell wt and 0.9 g rHu-IFN-gamma l(-1) h(-1), respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-gamma from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-gamma was ~30% (100 mg rHu-IFN-gamma g(-1) dry cell wt). The purity of the rHu-IFN-gamma was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN-gamma has a specific antiviral activity of ~2 x 10(7) IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.

  15. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    PubMed

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

  16. Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media.

    PubMed

    Willrodt, Christian; David, Christian; Cornelissen, Sjef; Bühler, Bruno; Julsing, Mattijs K; Schmid, Andreas

    2014-08-01

    The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)-limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)-limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth-dependent production of (S)-limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two-liquid phase fed-batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L(org) (-1) (S)-limonene. Specific activities of 75 mU g(cdw) (-1) were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g(cdw) (-1) ) leading to a final (S)-limonene concentration of 2,700 mg L(org) (-1) . Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)-limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)-limonene production. The two-liquid phase fed-batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date.

  17. Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli.

    PubMed

    Tsai, Wen-Che; Wu, Tzee-Chung; Chiang, Bor-Luen; Wen, Hsiao-Wei

    2017-02-01

    In recent years, the number of people around the world who suffer from fruit allergies has increased. Mango can induce anaphylaxis, and two major mango allergens have been identified - Man i 1 and Man i 2. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3' and 5' RACE assays were used to identify the cDNA fragment of Man i 1. Subsequently, the full length of Man i 1 cDNA was inserted into a pET-21a(+) vector, and the inserted plasmid was transformed to Escherichia coli BL21 (DE3) to express rMan i 1. The conditions for the expression of rMan i 1, including IPTG concentration, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16 °C for 20 h. The His-tagged rMan i 1 was purified using Ni-NTA agarose and its identity was verified using an anti-histidine antibody and the serum of a mango-allergic person. Additionally, rMan i 1 was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shared 86.2% identity in amino acid sequence of GAPDH from wheat. Finally, an E. coli expression system of rMan i 1 was established, with the potential to be used in immunotherapy against mango allergy or the development of assays for detecting the residues of mango allergens.

  18. Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli

    PubMed Central

    Morowvat, Mohammad Hossein; Babaeipour, Valiollah; Rajabi-Memari, Hamid; Vahidi, Hossein; Maghsoudi, Nader

    2014-01-01

    Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins. PMID:24711841

  19. Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli.

    PubMed

    Guggenbichler, Florian; Büttner, Carolin; Rudolph, Wolfram; Zimmermann, Kurt; Gunzer, Florian; Pöhlmann, Christoph

    2016-12-01

    Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid loss of function due to dimer dissociation, we designed a synthetic hIL-10 analog by bridging both monomers via a 15 amino acid-long peptide spacer in a C-terminal to N-terminal fashion. For secretory expression in Escherichia coli, a 1156 bp fragment was generated from template vector pAZ1 by fusion PCR encoding a T7 promoter region and the signal sequence of the E. coli outer membrane protein F fused in frame to two tandem E. coli codon-optimized mature hIL-10 genes connected via a 45 nucleotide linker sequence. The construct was cloned into pUC19 for high-level expression in E. coli BL21 (DE3). The mean concentrations of hIL-10 fusion protein in the periplasm and supernatant of E. coli at 37 °C growth temperature were 130 ± 40 and 2 ± 1 ng/ml, respectively. The molecular mass of the recombinant protein was assessed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, indicating correct processing of the signaling sequence in E. coli. In vitro biological activity was shown by phosphorylation of signal transducer and activator of transcription protein 3 and suppression of tumor necrosis factor α secretion in lipopolysaccharide-stimulated macrophages.

  20. Expression of a hemA gene from Agrobacterium radiobacter in a rare codon optimizing Escherichia coli for improving 5-aminolevulinate production.

    PubMed

    Fu, Weiqi; Lin, Jianping; Cen, Peilin

    2010-01-01

    The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.

  1. Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli

    SciTech Connect

    Chaurasia, Neha; Mishra, Yogesh; Rai, Lal Chand

    2008-11-07

    Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 deg. C), NaCl (6% w/v), carbofuron (0.025 mg ml{sup -1}), CdCl{sub 2} (4 mM), CuCl{sub 2} (1 mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.

  2. Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology

    PubMed Central

    Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

    2017-01-01

    Background: The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Materials and Methods: Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli (E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected. Results: Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. Conclusion: In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies. PMID:28349025

  3. Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli.

    PubMed

    Gasparian, Marine E; Ostapchenko, Valeriy G; Schulga, Alexey A; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2003-09-01

    Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.

  4. Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.

    PubMed

    Zafar, Asma; Aftab, Muhammad Nauman; ud Din, Zia; Aftab, Saima; Iqbal, Irfana; ul Haq, Ikram

    2016-02-01

    A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.

  5. Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli.

    PubMed

    Yang, Yu; Zhang, Dongxu; Liu, Song; Jia, Dongxu; Du, Guocheng; Chen, Jian

    2012-01-01

    Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.

  6. Protein expression and isotopic enrichment based on induction of the Entner-Doudoroff pathway in Escherichia coli

    SciTech Connect

    Refaeli, Bosmat; Goldbourt, Amir

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer The Entner-Doudoroff pathway is induced during protein expression in E. coli. Black-Right-Pointing-Pointer 1-{sup 13}C-gluconate and {sup 15}NH{sub 4}Cl provide a carbonyl-amide protein backbone labeling scheme. Black-Right-Pointing-Pointer The enrichment pattern is determined by nuclear magnetic resonance. -- Abstract: The Entner-Doudoroff pathway is known to exist in many organisms including bacteria, archea and eukarya. Although the common route for carbon catabolism in Escherichia coli is the Embden-Meyerhof-Parnas pathway, it was shown that gluconate catabolism in E. coli occurs via the Entner-Doudoroff pathway. We demonstrate here that by supplying BL21(DE3) competent E.coli cells with gluconate in a minimal growth medium, protein expression can be induced. Nuclear magnetic resonance data of over-expressed ubiquitin show that by using [1-{sup 13}C]-gluconate as the only carbon source, and {sup 15}N-enriched ammonium chloride, sparse isotopic enrichment in the form of a spin-pair carbonyl-amide backbone enrichment is obtained. The specific amino acid labeling pattern is analyzed and is shown to be compatible with Entner-Doudoroff metabolism. Isotopic enrichment serves as a key factor in the biophysical characterization of proteins by various methods including nuclear magnetic resonance, mass spectrometry, infrared spectroscopy and more. Therefore, the method presented here can be applied to study proteins by obtaining sparse enrichment schemes that are not based on the regular glycolytic pathway, or to study the Entner-Doudoroff metabolism during protein expression.

  7. Enhanced production of epsilon-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Jin-Byung; Park, Kyungmoon; Kim, Myoung-Dong; Seo, Jin-Ho

    2007-08-01

    Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

  8. Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

    PubMed

    Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

    2013-09-01

    Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins.

  9. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    PubMed

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production.

  10. Effect of culture conditions and signal peptide on production of human recombinant N-acetylgalactosamine-6-sulfate sulfatase in Escherichia coli BL21.

    PubMed

    Hernández, Alejandra; Velásquez, Olga; Leonardi, Felice; Soto, Carlos; Rodríguez, Alexander; Lizaraso, Lina; Mosquera, Ángela; Bohórquez, Jorge; Coronado, Alejandra; Espejo, Ángela; Sierra, Rocio; Sánchez, Oscar F; Alméciga-Díaz, Carlos J; Barrera, Luis A

    2013-05-01

    The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.

  11. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    PubMed Central

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  12. Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology

    PubMed Central

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Background Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes. Objectives This investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene in Escherichia coli BL21 (DE3). Materials and Methods The hES gene was amplified, cloned, and expressed in the E. coli expression system. Three factors, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, post-induction time, and cell density before induction, were selected as important factors. The mRNA expression level was determined using real-time PCR. The expression levels of hES mRNA under the different growth conditions were analyzed. SDS-PAGE and western blot analyses were carried out for further confirmation of interest-gene expression. Results A maximum rhES mRNA level of 376.16% was obtained under the following conditions: 0.6 mM IPTG, 7 hours post-induction time, and 0.9 cell density before induction. The level of rhES mRNA was significantly correlated with post-induction time, IPTG concentration, and cell density before induction (P < 0.05). The expression of the hES gene was confirmed by western blot. Conclusions The obtained results indicate that RSM is an effective method for the optimization of culture conditions for hES gene expression in E. coli. PMID:27800134

  13. Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948.

    PubMed

    Peng, Guan-Jhih; Kuan, Yi-Chia; Chou, Hsiao-Yi; Fu, Tze-Kai; Lin, Jia-Shin; Hsu, Wen-Hwei; Yang, Ming-Te

    2014-01-20

    (R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist and is widely used as a nasal decongestant to treat the common cold without the side effects of other ephedrine adrenergic drugs. We identified a short-chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC 10948 that was able to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-PE. The SM_SDR used NADPH and NADH as cofactors with specific activities of 17.35±0.71 and 5.57±0.07mU/mg protein, respectively, at 30°C and pH 7.0, thereby indicating that this enzyme could be categorized as an NADPH-preferring short-chain dehydrogenase/reductase. Escherichia coli strain BL21 (DE3) expressing SM_SDR could convert HPMAE into (R)-PE with more than 99% enantiomeric excess. The productivity and conversion yield were 0.57mmolPE/lh and 51.06%, respectively, using 10mM HPMAE. Fructose was the most effective carbon source for the conversion of HPMAE to (R)-PE.

  14. Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli.

    PubMed

    Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q S; Qiao, Chuanling

    2007-08-01

    A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His(6)-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.

  15. Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.

    PubMed Central

    Wang, C. C.; Badylak, J. A.; Lux, S. E.; Moriyama, R.; Dixon, J. E.; Low, P. S.

    1992-01-01

    The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase. PMID:1304397

  16. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants.

    PubMed

    Lin, J; Milase, R N

    2015-12-01

    This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49% recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe(3+) or Fe(2+) enhanced the activity of Y64 C1,2O while other compounds such as Ca(2+), and EDTA had an inhibitory effect. 80% of C1,2O activity remained using 4-nitrocatechol as substrate while 2% remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

  17. Optimization of Fermentation Conditions for Recombinant Human Interferon Beta Production by Escherichia coli Using the Response Surface Methodology

    PubMed Central

    Morowvat, Mohammad Hossein; Babaeipour, Valiollah; Rajabi Memari, Hamid; Vahidi, Hossein

    2015-01-01

    Background: The periplasmic overexpression of recombinant human interferon beta (rhIFN-β)-1b using a synthetic gene in Escherichia coli BL21 (DE3) was optimized in shake flasks using Response Surface Methodology (RSM) based on the Box-Behnken Design (BBD). Objectives: This study aimed to predict and develop the optimal fermentation conditions for periplasmic expression of rhIFN-β-1b in shake flasks whilst keeping the acetate excretion as the lowest amount and exploit the best results condition for rhIFN-β in a bench top bioreactor. Materials and Methods: The process variables studied were the concentration of glucose as carbon source, cell density prior the induction (OD 600 nm) and induction temperature. Ultimately, a three-factor three-level BBD was employed during the optimization process. The rhIFN-β production and the acetate excretion served as the evaluated responses. Results: The proposed optimum fermentation condition consisted of 7.81 g L-1 glucose, OD 600 nm prior induction 1.66 and induction temperature of 30.27°C. The model prediction of 0.267 g L-1 of rhIFN-β and 0.961 g L-1 of acetate at the optimum conditions was verified experimentally as 0.255 g L-1 and 0.981 g L-1 of acetate. This agreement between the predicted and observed values confirmed the precision of the applied method to predict the optimum conditions. Conclusions: It can be concluded that the RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli. PMID:26034535

  18. Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement.

    PubMed

    Huang, Di; Yang, Kexin; Liu, Jia; Xu, Yingying; Wang, Yuanyuan; Wang, Ru; Liu, Bin; Feng, Lu

    2017-03-09

    Fucosyllactoses, including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), are important oligosaccharides in human milk that are commonly used as nutritional additives in infant formula due to their biological functions, such as the promotion of bifidobacteria growth, inhibition of pathogen infection, and improvement of immune response. In this study, we developed a synthetic biology approach to promote the efficient biosynthesis of 2'-FL and 3-FL in engineered Escherichia coli. To boost the production of 2'-FL and 3-FL, multiple modular optimization strategies were applied in a plug-and-play manner. First, comparisons of various exogenous α1,2-fucosyltransferase and α1,3-fucosyltransferase candidates, as well as a series of E. coli host strains, demonstrated that futC and futA from Helicobacter pylori using BL21(DE3) as the host strain yielded the highest titers of 2'-FL and 3-FL. Subsequently, both the availability of the lactose acceptor substrate and the intracellular pool of the GDP-L-fucose donor substrate were optimized by inactivating competitive (or repressive) pathways and strengthening acceptor (or donor) availability to achieve overproduction. Moreover, the intracellular redox regeneration pathways were engineered to further enhance the production of 2'-FL and 3-FL. Finally, various culture conditions were optimized to achieve the best performance of 2'-FL and 3-FL biosynthesizing strains. The final concentrations of 2'-FL and 3-FL were 9.12 and 12.43g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.

  19. [Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli].

    PubMed

    2013-10-01

    The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.

  20. Characterization of WbiQ: An α1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen.

    PubMed

    Pettit, Nicholas; Styslinger, Thomas; Mei, Zhen; Han, Weiqing; Zhao, Guohui; Wang, Peng George

    2010-11-12

    Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an α1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-β1,3-GalNAc-α-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe, on a milligram scale.

  1. Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies.

    PubMed

    Papaneophytou, Christos P; Kontopidis, George A

    2012-11-01

    Tumor necrosis factor-α (TNF-α) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-α is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-α inhibitors. In this work, induction conditions for the production of human TNF-α fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD(600nm)), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-α. An 11% increase of TNF-α production was achieved after determination of the optimum induction conditions: OD(600nm) prior induction 0.55, a post induction temperature of 25°C, an IPTG concentration of 1mM and a post-induction time of 4h. We have also studied TNF-α oligomerization, the major property of this protein, and a K(d) value of 0.26nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable K(d) value for protein trimerization probably due to the complexibility of our model.

  2. Highly effective renaturation of a streptokinase from Streptococcus pyogenes DT7 as inclusion bodies overexpressed in Escherichia coli.

    PubMed

    Nguyen, Sy Le Thanh; Quyen, Dinh Thi; Vu, Hong Diep

    2014-01-01

    The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.

  3. Highly Effective Renaturation of a Streptokinase from Streptococcus pyogenes DT7 as Inclusion Bodies Overexpressed in Escherichia coli

    PubMed Central

    Nguyen, Sy Le Thanh; Quyen, Dinh Thi; Vu, Hong Diep

    2014-01-01

    The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction. PMID:24883307

  4. Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression.

    PubMed

    Ushasree, Mrudula Vasudevan; Vidya, Jalaja; Pandey, Ashok

    2014-01-01

    A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH4)2SO4 precipitation. The enzyme had optimum activity at 50 °C and pH 6.5.

  5. Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro

    SciTech Connect

    Zhao Guohui; Liu Jun; Liu Xiang; Chen Min; Zhang Houcheng Wang, Peng George

    2007-11-23

    GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157:H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157:H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K{sub m} and K{sub cat}/K{sub m} for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10{sup 5} M{sup -1} S{sup -1}, and those for L-glutamate were 2 mM and 0.52 x 10{sup 5} M{sup -1}S{sup -1}, respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157:H7.

  6. Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli.

    PubMed

    Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Herein, we report an Escherichia coli-based expression and purification method of recombinant cholera toxin B subunit (CTB). The CTB gene (E. coli codon optimized) is cloned into commercial pET-22b(+) vector using standard molecular biology techniques and the resulting vector is transformed into BL21(DE3) electrocompetent cells. The bacterial cells are grown and induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) results in accumulation of CTB in the culture medium. CTB is purified from the culture medium using a simple two-step chromatography process: immobilized metal affinity chromatography (IMAC) followed by ceramic hydroxyapatite (CHT). CTB is purified to >95 % homogeneity with a yield of over 10 mg per liter of culture. Depending on the application, endotoxin is removed using a commercially available endotoxin removal resin to <1 EU/mg.

  7. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    PubMed

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  8. Simulated microgravity affects growth of Escherichia coli and recombinant beta-D-glucuronidase production.

    PubMed

    Xiang, Liang; Qi, Feng; Dai, DaZhang; Li, Chun; Jiang, YuanDa

    2010-10-01

    Effects of simulated microgravity (SMG) on bacteria have been studied in various aspects. However, few reports are available about production of recombinant protein expressed by bacteria in SMG. In this study growth of E. coli BL21 (DE3) cells transformed with pET-28a (+)-pgus in double-axis clinostat that could model low shear SMG environment and the recombinant beta-D-glucuronidase (PGUS) expression have been investigated. Results showed that the cell dry weights in SMG were 16.47%, 38.06%, and 28.79% more than normal gravity (NG) control, and the efficiency of the recombinant PGUS expression in SMG were 18.33%, 19.36%, and 33.42% higher than that in NG at 19 degrees C, 28 degrees C, and 37 degrees C, respectively (P < 0.05).

  9. Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins.

    PubMed

    Sun, Yong; Feng, Xue; Qu, Jiao; Han, Wenqi; Liu, Zi; Li, Xu; Zou, Ming; Zhen, Yuhong; Zhu, Jie

    2015-06-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family. In this study, the whole extracellular domain gene of HER2 was amplified by RT-PCR from human breast cancer cell line SK-BR-3. The genes of membrane-distal region (A) and membrane proximal region (B) of HER2 extracellular domain were amplified from the cloned template, and then inserted into the expression vector pET-28a and pET-30a, respectively. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) cells and induced by isopropyl-b-D-thiogalactopyranoside (IPTG) for expression of proteins His-A and His-B. The expressed proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The optimization of culture conditions led us to accomplish the recombinant protein induction with 1.0 mM IPTG at 37 °C for 8 h, and both proteins were expressed in the insoluble form. Both proteins were purified under the denaturing condition using Ni-NTA sepharose column. Balb/c mice were immunized with the purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by ELISA testing and had good specificity by western blot detection. The HER2 ECD proteins His-A and His-B could be expressed in E. coli and were suitable for production of high titer antibodies against HER2 ECD.

  10. High-level SUMO-mediated fusion expression of ABP-dHC-cecropin A from multiple joined genes in Escherichia coli.

    PubMed

    Zhang, Jiaxin; Movahedi, Ali; Wei, Zhiheng; Sang, Ming; Wu, Xiaolong; Wang, Mengyang; Wei, Hui; Pan, Huixin; Yin, Tongming; Zhuge, Qiang

    2016-09-15

    The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve the expression level of ABP-dHC-cecropin A in E. coli, tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors (n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins (n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A.

  11. Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

    PubMed

    Hoang, Vinh Van; Zou, Yanshuang; Kurata, Kentaro; Enomoto, Keiichi

    2015-05-01

    Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

  12. An efficient approach for recombinant expression and purification of the viral capsid protein from beak and feather disease virus (BFDV) in Escherichia coli.

    PubMed

    Sarker, Subir; Ghorashi, Seyed A; Swarbrick, Crystall M D; Khandokar, Yogesh B; Himiari, Zainab; Forwood, Jade K; Raidal, Shane R

    2015-04-01

    Structural insights into the biology of viruses such as beak and feather disease virus (BFDV) which do not replicate in cell cultures are increasingly reliant on recombinant methods for protein production and purification. Development of efficient methods for homogenous production of BFDV capsid protein is also essential for vaccine development and diagnostic purposes. In this study, two different plasmids (pMCSG21 and pMCSG24), three homologous BFDV capsid proteins, and two unique expression media (auto-induction and IPTG-induced expression) were trialled for over-expression of the BFDV in Escherichia coli. Over-expression was observed for all three recombinant targets of BFDV capsid protein using E. coli BL21 (DE3) Rosetta 2 cell lines under IPTG induction. These proteins could be purified using an optimized, two-step purification process using a buffer containing 20mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 500 mM NaCl and supplemented with 200 mM L-arginine at pH 10.5, to yield a soluble and stable protein of greater than 95% purity. The final concentration of purified protein was approximately fourteen-to-eighteen fold greater than that reported previously. Initial crystallization and X-ray diffraction confirm that the protein is structured in a manner consistent with icosahedral symmetry. Antigenicity of recombinant Cap was confirmed by immunoassay, verifying its validity for use in continued experimentation as a potential DNA vaccine, a reagent in diagnostic assays, and purified concentrated protein for structural and functional biology.

  13. Distribution and phylogeny of immunoglobulin-binding protein G in Shiga toxin-producing Escherichia coli and its association with adherence phenotypes.

    PubMed

    Merkel, Viktor; Ohder, Barbara; Bielaszewska, Martina; Zhang, Wenlan; Fruth, Angelika; Menge, Christian; Borrmann, Erika; Middendorf, Barbara; Müthing, Johannes; Karch, Helge; Mellmann, Alexander

    2010-08-01

    eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG, and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae-negative STEC strains but in none of 157 eae-positive STEC strains. The 36 eibG-positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H(-) and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG-positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes (eibG-alpha, eibG-beta, and eibG-gamma). Strains expressing EibG-alpha and EibG-beta displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-gamma adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG-alpha, eibG-beta, and eibG-gamma subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.

  14. Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences.

    PubMed

    Carvalho, Rimenys Junior; Cabrera-Crespo, Joaquin; Tanizaki, Martha Massako; Gonçalves, Viviane Maimoni

    2012-05-01

    Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

  15. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli

    PubMed Central

    Novinrooz, Aytak; Zahraei Salehi, Taghi; Firouzi, Roya; Arabshahi, Sina; Derakhshandeh, Abdollah

    2017-01-01

    E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2–4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression

  16. Characterization of WbiQ: An {alpha}1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen

    SciTech Connect

    Pettit, Nicholas; Styslinger, Thomas; Mei, Zhen; Han, Weiqing; Zhao, Guohui; Wang, Peng George

    2010-11-12

    Research highlights: {yields} WbiQ is an {alpha}1,2-fucosyltransferase from Escherichia coli O127. {yields} WbiQ demonstrates strict substrate specificity for the Gal-{beta}1,3-GalNAc acceptor. {yields} WbiQ was used to synthesize milligram scale of the H-type 3 blood group antigen. -- Abstract: Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an {alpha}1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-{beta}1,3-GalNAc-{alpha}-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-{alpha}1,2-Gal-{beta}1,3-GalNAc-{alpha}-OMe, on a milligram scale.

  17. Exonuclease IX of Escherichia coli.

    PubMed Central

    Shafritz, K M; Sandigursky, M; Franklin, W A

    1998-01-01

    The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways. PMID:9592142

  18. Construction, expression, and characterization of Arabidopsis thaliana 4CL and Arachis hypogaea RS fusion gene 4CL::RS in Escherichia coli.

    PubMed

    Zhang, Erhao; Guo, Xuefeng; Meng, Zhifen; Wang, Jin; Sun, Jia; Yao, Xi; Xun, Hang

    2015-09-01

    Resveratrol is an important antioxidant that confers several beneficial effects on human health. 4-coumarate coenzyme A ligase (4CL) and resveratrol synthase (RS) are key rate-limiting enzymes in the biosynthetic pathway of resveratrol. Using gene fusion technology, the fusion gene, 4CL::RS, was constructed by the 4CL gene from Arabidopsis thaliana and RS gene from Arachis hypogaea. DNAMAN analysis showed that the fusion gene encoded a 964-amino acid protein with an approximate weight of 104.7 kDa and a pI of 5.63. A prokaryotic expression vector containing Nco-I and EcoR-I restriction sites, pET-30a/4CL::RS, was identified by liquid culture bacterial PCR, enzyme digestion, and sequencing, and then used in the induction of expression. Subsequently, a biosynthetic pathway of resveratrol was constructed in Escherichia coli BL21(DE3) that harbored pET-30a/4CL::RS. The recombinant strains were induced to express the fusion protein at 28 °C for 8 h. After bacterial cells were disrupted by hypothermic ultrasonication, the 4CL::RS fusion protein was thoroughly separated from tags using Ni-NTA affinity chromatography, and then detected by SDS-PAGE analysis. When the recombinant strains expressed the fusion protein, the precursor, p-coumaric acid, was converted to resveratrol. In the present study, the final concentration of resveratrol derived from 1 mM p-coumaric acid was 80.524 mg/L, with a 35.28 % (mol/mol) conversion yield.

  19. P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

    PubMed Central

    Lee, Pyung-Gang; Kim, Joonwon; Kim, Eun-Jung; Jung, EunOk; Pandey, Bishnu Prasad

    2016-01-01

    (S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%. PMID:26801575

  20. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    PubMed

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

  1. Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct

    PubMed Central

    2013-01-01

    Background Interleukin-10 homologues encoded by Herpes viruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) hold interesting structural and biological characteristics compared to human interleukin-10 (hIL-10) that render these proteins promising candidates for therapeutic application in inflammatory bowel disease (IBD). Intestinal delivery of cytokines using bacterial carriers as chassis represents a novel approach for treatment of IBD patients. For proof of concept, a Sec-dependent transporter construct was designed for secretory expression of recombinant viral IL-10 proteins in the periplasm of Escherichia coli laboratory strain BL21 (DE3), which might serve as part of a prospective lysis based delivery and containment system. Results The signal peptide of E. coli outer membrane protein F fused to the mature form of the viral IL-10 proteins enabled successful transport into the periplasm, a compartment which seems crucial for proper assembly of the dimeric configuration of the cytokines. Cytokine concentrations in different bacterial compartments were determined by ELISA and achieved yields of 67.8 ng/ml ± 24.9 ng/ml for HCMV IL-10 and 1.5 μg/ml ± 841.4 ng/ml for EBV IL-10 in the periplasm. Immunoblot analysis was used to confirm the correct size of the E. coli-derived recombinant cytokines. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the signal transduction cascade after IL-10 receptor interaction, as well as suppression of tumor necrosis factor α (TNF-α) release of lipopolysaccharide-stimulated mouse macrophages were used as read-out assays for proving in vitro biological activity of the E. coli derived, recombinant viral IL-10 counterparts. Conclusions In this study, proof of principle is provided that E. coli cells are a suitable chassis for secretory expression of viral IL-10 cytokines encoded by codon-optimized synthetic genes fused to the E. coli ompF signal sequence. In vitro

  2. Structure of Escherichia Coli Tryptophanase

    SciTech Connect

    Ku,S.; Yip, P.; Howell, P.

    2006-01-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the {alpha}-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the {alpha}-proton of the substrate for {beta}-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  3. Structure of Escherichia coli tryptophanase.

    PubMed

    Ku, Shao Yang; Yip, Patrick; Howell, P Lynne

    2006-07-01

    Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

  4. Succinate production in Escherichia coli

    PubMed Central

    Thakker, Chandresh; Martínez, Irene; San, Ka-Yiu; Bennett, George N.

    2012-01-01

    Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for production of the four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon that is channeled to the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose as well as other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, as well as the requirement for efficient recovery of succinate and the reliability of the performance under scale-up are important in the overall process. The costs of the overall biorefinery compatible process will determine the economical commercialization of succinate and its impact in larger chemical markets. PMID:21932253

  5. Strategies for Protein Overproduction in Escherichia coli.

    ERIC Educational Resources Information Center

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  6. Escherichia coli survival in waters: Temperature dependence

    EPA Science Inventory

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  7. One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

  8. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  9. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  10. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  11. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  12. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from...

  13. Clinical Implications of Enteroadherent Escherichia coli

    PubMed Central

    Arenas-Hernández, Margarita M.P.; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

    2012-01-01

    Pathogenic Escherichia coli that colonize the small intestine primarily cause gastrointestinal illness in infants and travelers. The main categories of pathogenic E. coli that colonize the epithelial lining of the small intestine are enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. These organisms accomplish their pathogenic process by a complex, coordinated multistage strategy, including non-intimate adherence mediated by various adhesins. These so called “enteroadherent E. coli ” categories subsequently produced toxins or effector proteins that are either secreted to the milieu or injected to the host cell. Finally, destruction of the intestinal microvilli results from the intimate adherence or the toxic effect exerted over the epithelia, resulting in water secretion and diarrhea. In this review, we summarize the current state of knowledge regarding these enteroadherent E. coli strains and the present clinical understanding of how these organisms colonize the human intestine and cause disease. PMID:22798032

  14. An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

    PubMed Central

    Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A. A.; Baumann, Marcus E. M.; Selmer, Thorsten

    2013-01-01

    Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the

  15. In-stream Escherichia coli Modeling

    NASA Astrophysics Data System (ADS)

    Pandey, P.; Soupir, M.

    2013-12-01

    Elevated levels of pathogenic bacteria indicators such as Escherichia coli (E. coli) in streams are a serious concern. Controlling E. coli levels in streams requires improving our existing understanding of fate and transport of E. coli at watershed scale. In-stream E. coli concentrations are potentially linked to non-point pollution sources (i.e., agricultural land). Water of a natural stream can receive E. coli by either through overland flow (via runoff from cropland) or resuspension from the streambed to the water column. Calculating in-stream total E. coli loads requires estimation of particle attached bacteria as well free floating E. coli transport. Currently water quality models commonly used for predicting E. coli levels in stream water have limited capability for predicting E. coli levels in the water column as well as in the streambed sediment. The challenges in calculating in-stream E. coli levels include difficulties in modeling the complex interactions between sediment particles and E. coli. Here we have developed a watershed scale model (integrated with Soil and Water Assessment Tool (SWAT)), which involves calculation of particle attached E. coli, to predict in-stream E. coli concentrations. The proposed model predicts E. coli levels in streambed bed sediment as well as in the water column. An extensive in-stream E. coli monitoring was carried out to verify the model predictions, and results indicate that the model performed well. The study proposed here will improve understanding on in-stream bacterial contamination, and help improving existing water quality models for predicting pathogenic bacteria levels in ambient water bodies.

  16. Native valve Escherichia coli endocarditis following urosepsis.

    PubMed

    Rangarajan, D; Ramakrishnan, S; Patro, K C; Devaraj, S; Krishnamurthy, V; Kothari, Y; Satyaki, N

    2013-05-01

    Gram-negative organisms are a rare cause of infective endocarditis. Escherichia coli, the most common cause of urinary tract infection and gram-negative septicemia involves endocardium rarely. In this case report, we describe infection of native mitral valve by E. coli following septicemia of urinary tract origin in a diabetic male; subsequently, he required prosthetic tissue valve replacement indicated by persistent sepsis and congestive cardiac failure.

  17. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45..., non-intact product, that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26,...

  18. Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli.

    PubMed

    Huang, Lei; Ruan, Hong; Gu, Weiyan; Xu, Zhinan; Cen, Peilin; Fan, Limei

    2007-01-01

    Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.

  19. [Cloning of CTB-PROIN fusion gene and its expression in Escherichia coli].

    PubMed

    Chen, Li; Ouyang, Feng-Xiu; Qian, Bing-Jun; Ren, Hong; Wang, Qiang; Jiang, Qing-Wu; Wang, Yu-Jiong; Liu, Jing-Bo; Liang, Wan-Qi; Zhang, Da-Bing

    2005-03-01

    A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.

  20. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

  1. Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif.

    PubMed

    Li, Mei; Liu, Xian-Wei; Shao, Jun; Shen, Jie; Jia, Qiang; Yi, Wen; Song, Jing K; Woodward, Robert; Chow, Christine S; Wang, Peng George

    2008-01-08

    The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.

  2. High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

    PubMed

    Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

    2014-01-01

    Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

  3. Escherichia Coli--Key to Modern Genetics.

    ERIC Educational Resources Information Center

    Bregegere, Francois

    1982-01-01

    Mid-nineteenth century work by Mendel on plant hybrids and by Pasteur on fermentation gave birth by way of bacterial genetics to modern-day molecular biology. The bacterium Escherichia Coli has occupied a key position in genetic studies leading from early gene identification with DNA to current genetic engineering using recombinant DNA technology.…

  4. Escherichia coli and Sudden Infant Death Syndrome

    PubMed Central

    Bettelheim, Karl A.; Goldwater, Paul N.

    2015-01-01

    This review examines the association of strains of Escherichia coli with sudden infant death syndrome (SIDS) and the possible role these bacteria play in this enigmatic condition. The review addresses evidence for E. coli in SIDS infants, potential sources of E. coli in the environment, colonization by commensal and pathogenic strains, the variety of currently accepted pathotypes, and how these pathotypes could compromise intestinal integrity and induce inflammation. Both intestinal and extraintestinal pathotypes are compared in relation to the apparent liability in which virulence traits can be gained or lost by strains of E. coli. The way in which E. coli infections fit with current views on infant sleeping position and other SIDS risk factors is highlighted. PMID:26191064

  5. Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

    PubMed Central

    Lytle, Darren A.; Rice, Eugene W.; Johnson, Clifford H.; Fox, Kim R.

    1999-01-01

    The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased. PMID:10388724

  6. Hydrogen production by recombinant Escherichia coli strains

    PubMed Central

    Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

    2012-01-01

    Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

  7. Uropathogenic Escherichia coli-Associated Exotoxins.

    PubMed

    Welch, Rodney A

    2016-06-01

    Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and bloodstream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many, but not all, of these strains are likely to aid the colonization and immune-evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin; cytotoxic-necrotizing factor-1; and the autotransporters, Sat, Pic, and Vat, to extraintestinal human disease.

  8. The evolution of the Escherichia coli phylogeny.

    PubMed

    Chaudhuri, Roy R; Henderson, Ian R

    2012-03-01

    Escherichia coli is familiar to biologists as a classical model system, ubiquitous in molecular biology laboratories around the world. Outside of the laboratory, E. coli strains exist as an almost universal component of the lower-gut flora of humans and animals. Although usually a commensal, E. coli has an alter ego as a pathogen, and is associated with diarrhoeal disease and extra-intestinal infections. The study of E. coli diversity predates the availability of molecular data, with strains initially distinguished by serotyping and metabolic profiling, and genomic diversity illustrated by DNA hybridisation. The quantitative study of E. coli diversity began with the application of multi-locus enzyme electrophoresis (MLEE), and has progressed with the accumulation of nucleotide sequence data, from single genes through multi-locus sequence typing (MLST) to whole genome sequencing. Phylogenetic methods have shed light on the processes of genomic evolution in this extraordinarily diverse species, and revealed the origins of pathogenic E. coli strains, including members of the phylogenetically indistinguishable "genus"Shigella. In May and June 2011, an outbreak of haemorrhagic uraemic syndrome in Germany was linked to a strain of enterohaemorrhagic E. coli (EHEC) O104:H4. Application of high-throughput sequencing technologies allowed the genome and origins of the outbreak strain to be characterised in real time as the outbreak was in progress.

  9. Automatic tracking of Escherichia coli bacteria.

    PubMed

    Xie, Jun; Khan, Shahid; Shah, Mubarak

    2008-01-01

    In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy videos. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in sequence of frames. Then a novel matching gain measure is introduced to cope with the challenges such as dramatic changes of cells' appearance and serious overlapping and occlusion. For multiple cell tracking, an optimal matching strategy is proposed to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually-determined trajectories, as well as those obtained from existing tracking methods. The stability of the algorithm with different parameter values is also analyzed and discussed.

  10. Systems Metabolic Engineering of Escherichia coli.

    PubMed

    Choi, Kyeong Rok; Shin, Jae Ho; Cho, Jae Sung; Yang, Dongsoo; Lee, Sang Yup

    2017-03-01

    Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

  11. Interaction between Escherichia coli and lunar fines

    NASA Technical Reports Server (NTRS)

    Johansson, K. R.

    1983-01-01

    A sample of mature lunar fines (10084.151) was solubilized to a high degree (about 17 percent) by the chelating agent salicylic acid (0.01. M). The neutralized (pH adjusted to 7.0) leachate was found to inhibit the growth of Escherichia coli (ATCC 259922) in a minimial mineral salts glucose medium; however, the inhibition was somewhat less than that caused by neutralized salicylic acid alone. The presence of lunar fines in the minimal medium was highly stimulatory to growth of E. coli following an early inhibitory response. The bacterium survived less well in the lunar leachate than in distilled water, no doubt because of the salicylate. It was concluded that the sample of lunar soil tested has nutritional value to E. coli and that certain products of fermentation helped to solubilize the lunar soil.

  12. Production of curcuminoids in engineered Escherichia coli.

    PubMed

    Kim, Eun Ji; Cha, Mi Na; Kim, Bog-Gyu; Ahn, Joong-Hoon

    2017-03-09

    Curcumin, a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa, possesses diverse pharmacological properties including anti-inflammatory, antioxidant, antiproliferative and antiangiogenic activity. Two curcuminoids (dicinnamoylmethane and bisdemethoxycurcumin) were synthesized from glucose in Escherichia coli. PAL (phenylalanine ammonia lyase) or TAL (tyrosine ammonia lyase), along with Os4CL (p-coumaroyl-CoA ligase) and CUS (curcumin synthase), were introduced in to E. coli, and each strain produced dicinnamoylmethane or bisdemethoxycurcumin, respectively. In order to increase the production of curcuminoids in E. coli, the shikimic acid biosynthesis pathway which increases the substrates for curcuminoid biosynthesis, was engineered. Using engineered strains, the production of bisdemethoxycurcumin increased from 0.32 to 4.63 mg/L, and that of dicinnamoylmethane from 1.24 mg/L and 6.95 mg/L.

  13. Frequency-Dependent Escherichia coli Chemotaxis Behavior

    NASA Astrophysics Data System (ADS)

    Zhu, Xuejun; Si, Guangwei; Deng, Nianpei; Ouyang, Qi; Wu, Tailin; He, Zhuoran; Jiang, Lili; Luo, Chunxiong; Tu, Yuhai

    2012-03-01

    We study Escherichia coli chemotaxis behavior in environments with spatially and temporally varying attractant sources by developing a unique microfluidic system. Our measurements reveal a frequency-dependent chemotaxis behavior. At low frequency, the E. coli population oscillates in synchrony with the attractant. In contrast, in fast-changing environments, the population response becomes smaller and out of phase with the attractant waveform. These observations are inconsistent with the well-known Keller-Segel chemotaxis equation. A new continuum model is proposed to describe the population level behavior of E. coli chemotaxis based on the underlying pathway dynamics. With the inclusion of a finite adaptation time and an attractant consumption rate, our model successfully explains the microfluidic experiments at different stimulus frequencies.

  14. Thymineless death in Escherichia coli: strain specificity.

    PubMed

    Cummings, D J; Mondale, L

    1967-06-01

    Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, B(s-12), K-12 rec-21, and possibly K-12 Lon(-), all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation.

  15. Thymineless Death in Escherichia coli: Strain Specificity

    PubMed Central

    Cummings, Donald J.; Mondale, Lee

    1967-01-01

    Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, Bs−12, K-12 rec-21, and possibly K-12 Lon−, all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation. Images PMID:5337772

  16. Diversity of CRISPR loci in Escherichia coli.

    PubMed

    Díez-Villaseñor, C; Almendros, C; García-Martínez, J; Mojica, F J M

    2010-05-01

    CRISPR (clustered regularly interspaced short palindromic repeats) and CAS (CRISPR-associated sequence) proteins are constituents of a novel genetic barrier that limits horizontal gene transfer in prokaryotes by means of an uncharacterized mechanism. The fundamental discovery of small RNAs as the guides of the defence apparatus arose as a result of Escherichia coli studies. However, a survey of the system diversity in this species in order to further contribute to the understanding of the CRISPR mode of action has not yet been performed. Here we describe two CRISPR/CAS systems found in E. coli, following the analysis of 100 strains representative of the species' diversity. Our results substantiate different levels of activity between loci of both CRISPR types, as well as different target preferences and CRISPR relevances for particular groups of strains. Interestingly, the data suggest that the degeneration of one CRISPR/CAS system in E. coli ancestors could have been brought about by self-interference.

  17. Prodigiosin - A Multifaceted Escherichia coli Antimicrobial Agent

    PubMed Central

    Zorec, Maša; Stopar, David

    2016-01-01

    Despite a considerable interest in prodigiosin, the mechanism of its antibacterial activity is still poorly understood. In this work, Escherichia coli cells were treated with prodigiosin to determine its antimicrobial effect on bacterial physiology. The effect of prodigiosin was concentration dependent. In prodigiosin treated cells above MIC value no significant DNA damage or cytoplasmic membrane disintegration was observed. The outer membrane, however, becomes leaky. Cells had severely decreased respiration activity. In prodigiosin treated cells protein and RNA synthesis were inhibited, cells were elongated but could not divide. Pre-treatment with prodigiosin improved E. coli survival rate in media containing ampicillin, kanamycin and erythromycin but not phleomycin. The results suggest that prodigiosin acts as a bacteriostatic agent in E. coli cells. If prodigiosin was diluted, cells resumed growth. The results indicate that prodigiosin has distinct mode of antibacterial action in different bacteria. PMID:27612193

  18. Biodegradation of Aromatic Compounds by Escherichia coli

    PubMed Central

    Díaz, Eduardo; Ferrández, Abel; Prieto, María A.; García, José L.

    2001-01-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications. PMID:11729263

  19. Biodegradation of aromatic compounds by Escherichia coli.

    PubMed

    Díaz, E; Ferrández, A; Prieto, M A; García, J L

    2001-12-01

    Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications.

  20. The 503nm pigment of Escherichia coli

    PubMed Central

    Kamitakahara, Joyce R.; Polglase, W. J.

    1970-01-01

    The yield of cell protein was one-third less for streptomycin-dependent Escherichia coli B than for the wild-type parent strain when both were grown aerobically on a medium with limiting glucose, but anaerobically the yield of protein was similar for both strains. The transient pigment absorbing at 503nm that is known to be present in E. coli and other organisms was not detectable in streptomycin-dependent mutants nor in a non-dependent (energy-deficient) revertant. When wild-type E. coli B was grown on limiting glucose–salts medium containing 2,4 dinitrophenol, the yield of cell protein was decreased and formation of the 503nm pigment was inhibited. Fumarase, aconitase and glucose 6-phosphate dehydrogenase were de-repressed in E. coli B cells grown with excess of glucose in a medium containing 2,4-dinitrophenol. In air-oxidized, wild-type E. coli B cells, the 503nm pigment appeared before reduced cytochromes when gluconate was the substrate but failed to appear when succinate was the substrate. The results provide evidence for a role of the 503nm pigment in aerobic energy metabolism, possibly as an electron acceptor from NADPH. PMID:4395501

  1. 77 FR 6826 - Notice of Permit Applications Received Under the Antarctic Conservation Act of 1978 (Pub. L. 95-541)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-09

    ... certain animals and certain geographic areas a requiring special protection. The regulations establish... Palmer Station for experimentation. The applicant will use Escherichia coli strain BL21DE3 for...

  2. ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

    EPA Science Inventory

    The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

  3. Designed phosphoprotein recognition in Escherichia coli.

    PubMed

    Sawyer, Nicholas; Gassaway, Brandon M; Haimovich, Adrian D; Isaacs, Farren J; Rinehart, Jesse; Regan, Lynne

    2014-11-21

    Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.

  4. Escherichia coli growth under modeled reduced gravity

    NASA Technical Reports Server (NTRS)

    Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

    2004-01-01

    Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

  5. Detection of Escherichia coli enterotoxins in stools.

    PubMed Central

    Merson, M H; Yolken, R H; Sack, R B; Froehlich, J L; Greenberg, H B; Huq, I; Black, R W

    1980-01-01

    We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method. PMID:6995331

  6. Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation.

    PubMed

    Chen, Gen-Hung; Chen, Wei-Ming; Huang, Ya-Chi; Jiang, Shann-Tzong

    2012-03-21

    A cDNA encoding mature human tyrosinase was cloned into pET-23a(+) and transformed into E. coli BL21(DE3). Three major recombinant proteins, mature human tyrosinase (RHT₂₀₋₅₃₁), N-terminal truncated human tyrosinase (RHT₁₆₈₋₅₃₁), and β-lactamase, were overexpressed as inclusion bodies in E. coli after 12 h of induction with 1.0 mM isopropyl-β-D-thiogalactopyranoside at 37 °C. After sonication and centrifugation, the inclusion body was harvested, solubilized, dialyzed, and refolded into the active form with monophenolase and diphenolase activities. It was purified to homogeneity by DEAE-Sepharose FF and Sephadex G-75. The molecular mass and N-terminal sequence were 57.0 kDa and GHFPRAC, respectively, and corresponded to those of mature human tyrosinase. The RHT was active in a broad range of temperature and pH, and with optimum activity at 70 °C and pH 8.5.

  7. Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides.

    PubMed

    Zhang, Yanan; Li, Dan; Li, Yan

    2017-01-20

    L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from Echerichia coli and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene ansB encoding L-asparaginase was amplified from the genome of E. coli BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB. The expression of recombinant protein was purified by affinity chromatography using a nickel resin followed by anion exchange chromatography. The purity and quality of the recombinant L-asparaginase were optimised. The results indicated that km for the recombinant L-asparaginase was 3.02 × 10(-4) mol/L. Both cyclo-(Pro-Tyr) and cyclo-(Pro-Phe) could inhibit the activity of recombinant L-asparaginase at the level of 10(-5) mol/L.

  8. Escherichia coli O157:H7.

    PubMed

    Mead, P S; Griffin, P M

    1998-10-10

    Escherichia coli O157 was first identified as a human pathogen in 1982. One of several Shiga toxin-producing serotypes known to cause human illness, the organism probably evolved through horizontal acquisition of genes for Shiga toxins and other virulence factors. E. coli O157 is found regularly in the faeces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. Human infection is associated with a wide range of clinical illness, including asymptomatic shedding, non-bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome, and death. Since laboratory practices vary, physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens. Treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and other studies suggest no effect or even a protective effect. Physicians can help to prevent E. coli O157 infections by counselling patients about the hazards of consuming undercooked ground meat or unpasteurised milk products and juices, and about the importance of handwashing to prevent the spread of diarrhoeal illness, and by informing public-health authorities when they see unusual numbers of cases of bloody diarrhoea or haemolytic uraemic syndrome.

  9. Transport proteins promoting Escherichia coli pathogenesis.

    PubMed

    Tang, Fengyi; Saier, Milton H

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

  10. Transport proteins promoting Escherichia coli pathogenesis

    PubMed Central

    Tang, Fengyi; Saier, Milton H.

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

  11. Extracellular recombinant protein production from Escherichia coli.

    PubMed

    Ni, Ye; Chen, Rachel

    2009-11-01

    Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

  12. Engineering Escherichia coli for methanol conversion.

    PubMed

    Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Potthoff, Eva; Heux, Stéphanie; Quax, Wim J; Wendisch, Volker F; Brautaset, Trygve; Portais, Jean-Charles; Vorholt, Julia A

    2015-03-01

    Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

  13. Engineering Escherichia coli to bind to cyanobacteria.

    PubMed

    Zhang, Zijian; Meng, Liuyi; Ni, Congjian; Yao, Lanqiu; Zhang, Fengyu; Jin, Yuji; Mu, Xuelang; Zhu, Shiyu; Lu, Xiaoyu; Liu, Shiyu; Yu, Congyu; Wang, Chenggong; Zheng, Pu; Wu, Jie; Kang, Li; Zhang, Haoqian M; Ouyang, Qi

    2017-03-01

    We engineered Escherichia coli cells to bind to cyanobacteria by heterologously producing and displaying lectins of the target cyanobacteria on their surface. To prove the efficacy of our approach, we tested this design on Microcystis aeruginosa with microvirin (Mvn), the lectin endogenously produced by this cyanobacterium. The coding sequence of Mvn was C-terminally fused to the ice nucleation protein NC (INPNC) gene and expressed in E. coli. Results showed that E. coli cells expressing the INPNC::Mvn fusion protein were able to bind to M. aeruginosa and the average number of E. coli cells bound to each cyanobacterial cell was enhanced 8-fold. Finally, a computational model was developed to simulate the binding reaction and help reconstruct the binding parameters. To our best knowledge, this is the first report on the binding of two organisms in liquid culture mediated by the surface display of lectins and it may serve as a novel approach to mediate microbial adhesion.

  14. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  15. Role of Escherichia coli in Biofuel Production

    PubMed Central

    Koppolu, Veerendra; Vasigala, Veneela KR

    2016-01-01

    Increased energy consumption coupled with depleting petroleum reserves and increased greenhouse gas emissions have renewed our interest in generating fuels from renewable energy sources via microbial fermentation. Central to this problem is the choice of microorganism that catalyzes the production of fuels at high volumetric productivity and yield from cheap and abundantly available renewable energy sources. Microorganisms that are metabolically engineered to redirect renewable carbon sources into desired fuel products are contemplated as best choices to obtain high volumetric productivity and yield. Considering the availability of vast knowledge in genomic and metabolic fronts, Escherichia coli is regarded as a primary choice for the production of biofuels. Here, we reviewed the microbial production of liquid biofuels that have the potential to be used either alone or in combination with the present-day fuels. We specifically highlighted the metabolic engineering and synthetic biology approaches used to improve the production of biofuels from E. coli over the past few years. We also discussed the challenges that still exist for the biofuel production from E. coli and their possible solutions. PMID:27441002

  16. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1989-01-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. We are also investigating the control of other genes required for fermentation and anaerobic growth. We have isolated both structural and regulatory mutations affecting the expression of alcohol dehydrogenase, the enzyme responsible for the final step in alcohol synthesis. Some of these regulatory mutations also affect other anaerobically induced genes. The adh gene has been cloned and sequenced. The ADH protein is one of the largest highly expressed proteins in E. coli and requires approximately 2700bp of DNA for its cloning sequence. We have also isolated mutations affecting the fermentative lactate dehydrogenase. In consequence it is now possible to construct E. coli strains defective in the production of any one or more of their normal fermentation products (i.e. formate, acetate, lactate, ethanol and succinate). The factors affecting the ratio of fermentation products are being investigated by in vivo NMR spectroscopy.

  17. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1990-01-01

    The purpose of this project is to elucidate the way in which the synthesis of ethanol and related fermentation products are regulated in the facultative anaerobe Escherichia coli. We are also investigating the control of other genes required for anaerobic growth. We have isolated both structural and regulatory mutations affecting the expression of alcohol dehydrogenase, the enzyme responsible for the final step in alcohol synthesis. Some of these regulatory mutations also affect other anaerobically induced genes. The adh gene has been cloned and sequenced. The ADH protein is one of the largest highly expressed proteins in E. coli and requires approximately 2700bp of DNA for its coding sequence. We have also isolated mutations affecting the fermentative lactate dehydrogenase and have recently cloned the ldh gene. In consequence it is now possible to construct E. coli strains defective in the production of any one or more of their normal fermentation products (i.e. formate, acetate, lactate, ethanol and succinate). The factors affecting ratio of fermentation products are being investigated by in vivo NMR spectroscopy.

  18. Long term effects of Escherichia coli mastitis.

    PubMed

    Blum, Shlomo E; Heller, Elimelech D; Leitner, Gabriel

    2014-07-01

    Escherichia coli is one of the most frequently diagnosed causes of bovine mastitis, and is typically associated with acute, clinical mastitis. The objective of the present study was to evaluate the long term effects of intramammary infections by E. coli on milk yield and quality, especially milk coagulation. Twenty-four Israeli Holstein cows diagnosed with clinical mastitis due to intramammary infection by E. coli were used in this study. Mean lactation number, days in milk (DIM) and daily milk yield (DMY) at the time of infection was 3.3 ± 1.3, 131.7 days ± 78.6 and 45.7 L ± 8.4, respectively. DMY, milk constituents, somatic cells count (SCC), differential leukocytes count and coagulation parameters were subsequently assessed. Two patterns of inflammation were identified: 'short inflammation', characterized by <15% decrease in DMY and <30 days until return to normal (n = 5), and 'long inflammation', characterized by >15% decrease in DMY and >30 days to reach a new maximum DMY (n = 19). The estimated mean loss of marketable milk during the study was 200 L/cow for 'short inflammation' cases, and 1,500 L/cow for 'long inflammation' ones. Significant differences between 'short' and 'long inflammation' effects were found in almost all parameters studied. Long-term detrimental effects on milk quality were found regardless of clinical or bacteriological cure of affected glands.

  19. Assessment of the Fusion Tags on Increasing Soluble Production of the Active TEV Protease Variant and Other Target Proteins in E. coli.

    PubMed

    Yu, Xuelian; Sun, Jiaqi; Wang, Weiyu; Jiang, Li; Cheng, Beijiu; Fan, Jun

    2016-12-17

    In this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (TEV) protease (TEVp) variant in Escherichia coli strains BL21 (DE3) and Rosetta™ (DE3) are investigated. Combination of the augmenting rare transfer RNAs (tRNAs) and the fused expressivity tag (N-terminal seven amino acid residues of E. coli translation initiation factor II) promotes the soluble TEVp partner expressed at relatively high level. Attachment of the maltose-binding protein (MBP) tag increases soluble expression of the protease released from the fusion protein in E. coli cells, but the incorporated TEVp recognition sequence slightly decreases expressivity of the fusion construct. Except for the green fluorescent protein, the attached expressivity tag shows less efficiency than the MBP tag in enhancing expression levels of the selected five target proteins in the Rosetta™ (DE3) cells under different induction conditions. Our results identified that high-level production of the functional target protein as the fusion partner in E. coli is combined with the intrinsic property of fusion tag, fusion protein stability, inherent folding of target protein, rare tRNA abundance, and the incorporated linker. Purified TEVp fusion constructs with the N-terminal expressivity tag, as well as the MBP partner, are the ideal alternatives for removing fusion tag.

  20. WGS accurately predicts antimicrobial resistance in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: To determine the effectiveness of whole-genome sequencing (WGS) in identifying resistance genotypes of multidrug-resistant Escherichia coli (E. coli) and whether these correlate with observed phenotypes. Methods: Seventy-six E. coli strains were isolated from farm cattle and measured f...

  1. Evaluation of Recombinant Human Growth Hormone Secretion in E. coli using the L-asparaginase II Signal Peptide

    PubMed Central

    Zamani, Mozhdeh; Nezafat, Navid; Ghasemi, Younes

    2016-01-01

    Background: In the recent years, there has been an increasing interest in secretory production of recombinant proteins, due to its various advantages compared with intracellular expression. Signal peptides play a critical role in prosperous secretion of recombinant proteins. Accordingly, different signal peptides have been assessed for their ability to produce secretory proteins by trial-and-error experiments. The aim of this study was to evaluate the effect of L-asparaginase II signal peptide on the recombinant human Growth Hormone (hGH) protein secretion in the Escherichia coli (E. coli) host. Methods: Cloning and expression of a synthetic hGH gene, containing L-asparaginase II signal sequence was performed in E. coli BL21 (DE3) using 0.1mM IPTG as an inducer at 23°C overnight. Periplasmic protein extraction was performed using three methods, including osmotic shock, osmotic shock in the presence of glycine and combined Lysozyme/EDTA osmotic shock. Afterwards, the hGH expression was determined by SDS-PAGE. Results: Based on experimentally obtained results, hGH protein is expressed as inclusion body even in the presence of L-asparaginase II signal peptide. Conclusion: Therefore, this signal peptide is not effective for secretory production of the recombinant hGH. PMID:27920886

  2. Mechanism of Escherichia coli Resistance to Pyrrhocoricin

    PubMed Central

    Narayanan, Shalini; Modak, Joyanta K.; Ryan, Catherine S.; Garcia-Bustos, Jose; Davies, John K.

    2014-01-01

    Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10−7. Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials. PMID:24590485

  3. Animal models of enteroaggregative Escherichia coli infection

    PubMed Central

    Philipson, Casandra W.; Bassaganya-Riera, Josep; Hontecillas, Raquel

    2013-01-01

    Enteroaggregative Escherichia coli (EAEC) has been acknowledged as an emerging cause of gastroenteritis worldwide for over two decades. Epidemiologists are revealing the role of EAEC in diarrheal outbreaks as a more common occurrence than ever suggested before. EAEC induced diarrhea is most commonly associated with travelers, children and immunocompromised individuals however its afflictions are not limited to any particular demographic. Many attributes have been discovered and characterized surrounding the capability of EAEC to provoke a potent pro-inflammatory immune response, however cellular and molecular mechanisms underlying initiation, progression and outcomes are largely unknown. This limited understanding can be attributed to heterogeneity in strains and the lack of adequate animal models. This review aims to summarize current knowledge about EAEC etiology, pathogenesis and clinical manifestation. Additionally, current animal models and their limitations will be discussed along with the value of applying systems-wide approaches such as computational modeling to study host-EAEC interactions. PMID:23680797

  4. An overview of atypical enteropathogenic Escherichia coli.

    PubMed

    Hernandes, Rodrigo T; Elias, Waldir P; Vieira, Mônica A M; Gomes, Tânia A T

    2009-08-01

    The enteropathogenic Escherichia coli (EPEC) pathotype is currently divided into two groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The property that distinguishes these two groups is the presence of the EPEC adherence factor plasmid, which is only found in tEPEC. aEPEC strains are emerging enteropathogens that have been detected worldwide. Herein, we review the serotypes, virulence properties, genetic relationships, epidemiology, reservoir and diagnosis of aEPEC, including those strains not belonging to the classical EPEC serogroups (nonclassical EPEC serogroups). The large variety of serotypes and genetic virulence properties of aEPEC strains from nonclassical EPEC serogroups makes it difficult to determine which strains are truly pathogenic.

  5. Escherichia coli fliAZY operon.

    PubMed Central

    Mytelka, D S; Chamberlin, M J

    1996-01-01

    We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY. Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro. We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters. FliZ and FliY are not required for motility but may regulate sigma F activity, perhaps in response to a putative cell density signal that may be detected by FliY, a member of the bacterial extracellular solute-binding protein family 3. PMID:8550423

  6. Expanding ester biosynthesis in Escherichia coli.

    PubMed

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2014-04-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.

  7. [Enteroinvasive Escherichia coli. Pathogenesis and epidemiology].

    PubMed

    Prats, G; Llovet, T

    1995-03-01

    Enteroinvasive Escherichia coli (EIEC) is an intestinal pathogen causing enteritis, with a similar pathogenic mechanism to that of Shigella, which causes an epithelial invasion of the large bowel leading to inflammation and ulceration of the mucosa. The patients often develop the symptoms of bacillary dysentery. The EIEC strains are atypical in their biochemical reactions and may ferment lactose late or not at all, are lysine decarboxilase negative, and non motile. In addition, most EIEC strains express somatic antigens which are either strongly related or identical to Shigella antigens. EIEC invasion is mediated by a large plasmid (140 MDa) coding for the production of several outer membrane proteins involved in invasiveness. These strains have been isolated with some regularity in South America, the Extreme Orient, and Eastern Europe. In Spain the incidence of enteroinvasive E. coli is extraordinarily low (0.2%), the serogroup O124 being the most frequently isolated. EIEC enteritis has been associated to sporadic cases occurring in travellers. Occasional outbreaks related to ingestion of contaminated water or food and person to person have been reported.

  8. Independence of replisomes in Escherichia coli chromosomalreplication

    SciTech Connect

    Breier, Adam M.; Weier, Heinz-Ulrich G.; Cozzarelli, Nicholas R.

    2005-03-13

    In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.

  9. Nucleotide excision repair in Escherichia coli.

    PubMed Central

    Van Houten, B

    1990-01-01

    One of the best-studied DNA repair pathways is nucleotide excision repair, a process consisting of DNA damage recognition, incision, excision, repair resynthesis, and DNA ligation. Escherichia coli has served as a model organism for the study of this process. Recently, many of the proteins that mediate E. coli nucleotide excision have been purified to homogeneity; this had led to a molecular description of this repair pathway. One of the key repair enzymes of this pathway is the UvrABC nuclease complex. The individual subunits of this enzyme cooperate in a complex series of partial reactions to bind to and incise the DNA near a damaged nucleotide. The UvrABC complex displays a remarkable substrate diversity. Defining the structural features of DNA lesions that provide the specificity for damage recognition by the UvrABC complex is of great importance, since it represents a unique form of protein-DNA interaction. Using a number of in vitro assays, researchers have been able to elucidate the action mechanism of the UvrABC nuclease complex. Current research is devoted to understanding how these complex events are mediated within the living cell. PMID:2181258

  10. Chemotaxis Toward Sugars in Escherichia coli

    PubMed Central

    Adler, Julius; Hazelbauer, Gerald L.; Dahl, M. M.

    1973-01-01

    Using a quantitative assay for measuring chemotaxis, we tested a variety of sugars and sugar derivatives for their ability to attract Escherichia coli bacteria. The most effective attractants, i.e., those that have thresholds near 10−5 M or below, are N-acetyl-d-glucosamine, 6-deoxy-d-glucose, d-fructose, d-fucose, 1-d-glycerol-β-d-galactoside, galactitol, d-galactose, d-glucosamine, d-glucose, α-d-glucose-1-phosphate, lactose, maltose, d-mannitol, d-mannose, methyl-β-d-galactoside, methyl-β-d-glucoside, d-ribose, d-sorbitol, and trehalose. Lactose, and probably d-glucose-1-phosphate, are attractive only after conversion to the free monosaccharide, while the other attractants do not require breakdown for taxis. Nine different chemoreceptors are involved in detecting these various attractants. They are called the N-acetyl-glucosamine, fructose, galactose, glucose, maltose, mannitol, ribose, sorbitol, and trehalose chemoreceptors; the specificity of each was studied. The chemoreceptors, with the exception of the one for d-glucose, are inducible. The galactose-binding protein serves as the recognition component of the galactose chemoreceptor. E. coli also has osmotically shockable binding activities for maltose and d-ribose, and these appear to serve as the recognition components for the corresponding chemoreceptors. PMID:4580570

  11. Expanding ester biosynthesis in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M; Tashiro, Yohei; Atsumi, Shota

    2015-01-01

    To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l−1). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters. PMID:24609358

  12. The thermal impulse response of Escherichia coli

    PubMed Central

    Paster, Eli; Ryu, William S.

    2008-01-01

    Swimming Escherichia coli responds to changes in temperature by modifying its motor behavior. Previous studies using populations of cells have shown that E. coli accumulate in spatial thermal gradients, but these experiments did not cleanly separate thermal responses from chemotactic responses. Here we have isolated the thermal response by studying the behavior of single, tethered cells. The motor output of cells grown at 33°C was measured at constant temperature, from 10° to 40°C, and in response to small, impulsive increases in temperature, from 23° to 43°C. The thermal impulse response at temperatures < 31°C is similar to the chemotactic impulse response: Both follow a similar time course, share the same directionality, and show biphasic characteristics. At temperatures > 31°C, some cells show an inverted response, switching from warm- to cold-seeking behavior. The fraction of inverted responses increases nonlinearly with temperature, switching steeply at the preferred temperature of 37°C. PMID:18385380

  13. Cyclomodulins in urosepsis strains of Escherichia coli.

    PubMed

    Dubois, Damien; Delmas, Julien; Cady, Anne; Robin, Frédéric; Sivignon, Adeline; Oswald, Eric; Bonnet, Richard

    2010-06-01

    Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin.

  14. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  15. Escherichia coli survival in waters: temperature dependence.

    PubMed

    Blaustein, R A; Pachepsky, Y; Hill, R L; Shelton, D R; Whelan, G

    2013-02-01

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q₁₀ model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q₁₀ equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies "log concentration vs. time." There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q₁₀ equation was more accurate in rivers and coastal waters than in lakes making the value of

  16. Polymorphisms in the umuDC region of Escherichia species. [Escherichia coli; Escherichia alkalescens; Escherichia dispar; Escherichia aurescens

    SciTech Connect

    Sedgwick, S.G.; Robson, M.; Malik, F.

    1988-04-01

    The umuDC operon of Escherichia coli encodes mutagenic DNA repair. The umuDC regions of multiple isolates of E. coli, E. alkalescens, and E. dispar and a single stock of E. aurescens were mapped by nucleotide hybridization. umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long. Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract. Restriction site polymorphisms were not found around the DNA repair gene recA or polA. The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons. Possible mechanisms for the generation of these variants are discussed.

  17. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    Sewage samples from seven locations in the United States were analyzed for Escherichia coli isolates which were resistant to trimethoprim-sulfamethoxazole (SXT). The prevalence rate of SXT resistant organisms varied between the different geographical locales. The majority of th...

  18. Genome Sequence of Escherichia coli Tailed Phage Utah

    PubMed Central

    Leavitt, Justin C.; Heitkamp, Alexandra J.; Bhattacharjee, Ananda S.; Gilcrease, Eddie B.

    2017-01-01

    ABSTRACT Escherichia coli bacteriophage Utah is a member of the chi-like tailed phage cluster in the Siphoviridae family. We report here the complete 59,024-bp sequence of the genome of phage Utah. PMID:28360173

  19. Shigella strains are not clones of Escherichia coli but sister species in the genus Escherichia.

    PubMed

    Zuo, Guanghong; Xu, Zhao; Hao, Bailin

    2013-02-01

    Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.

  20. Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli

    PubMed Central

    2013-01-01

    Background Chromobacterium violaceum is a free-living β-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the β-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. Results The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-β-D-N,N’-diacetylchitobiose and p-nitrophenyl-β-D-N,N’,N”-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30

  1. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI.

    PubMed

    FREIFELDER, D; MAALOE, O

    1964-10-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987-990. 1964.-Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process.

  2. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI

    PubMed Central

    Freifelder, David; Maaløe, Ole

    1964-01-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987–990. 1964.—Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process. PMID:14219063

  3. Escherichia coli Unsaturated Fatty Acid Synthesis

    PubMed Central

    Feng, Youjun; Cronan, John E.

    2009-01-01

    Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA. PMID:19679654

  4. Mono and diterpene production in Escherichia coli.

    PubMed

    Reiling, K Kinkead; Yoshikuni, Yasuo; Martin, Vincent J J; Newman, Jack; Bohlmann, Jörg; Keasling, Jay D

    2004-07-20

    Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

  5. Microdiesel: Escherichia coli engineered for fuel production.

    PubMed

    Kalscheuer, Rainer; Stölting, Torsten; Steinbüchel, Alexander

    2006-09-01

    Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l(-1) and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

  6. Efficient Process Development of Recombinant Human Granulocyte Colony-Stimulating Factor (rh-GCSF) Production in Escherichia coli

    PubMed Central

    Babaeipour, Valiollah; Khanchezar, Sirwan; Mofid, Mohammad Reza; Pesaran Hagi Abbas, Mahdi

    2015-01-01

    Background: The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. Methods: An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h-1 at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. Results: Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l-1 and 32.1 g.l-1. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g-1 DCW and 1.83 g.l-1.h-1, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen® and PD-Grastim showed that all proteins have a similar secondary structure. Conclusion: Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity. PMID:25864815

  7. Regulation of Glutamine Transport in Escherichia coli.

    PubMed Central

    Willis, R C; Iwata, K K; Furlong, C E

    1975-01-01

    The formation of the high-affinity (Km equal to 0.2 muM) L-glutamine transport system of Escherichia coli strain 7 (Lin) appears to be subject to the same major control as the glutamine synthetase (EC 6.3.1.2) of this gram-negative organism. Culture of cells under nitrogen-limited conditions provides maximum derepression of both the glutamine synthetase and the glutamine transport system. Nutritional conditions providing a rich supply of ammonium salts or available sources of nitrogen, i.e., conditions which repress the formation of glutamine synthetase, provide three- and 20-fold repression, respectively, of the glutamine transport system. Culture of cells with glutamine supplements of 2 mM does not increase the repression of high-affinity glutamine transport system beyond the level observed in the absence of glutamine. A second kinetically distinct low-affinity component of glutamine. A second kinetically distinct low-affinity component of glutamine uptake is observed in cells cultured with a glutamine-depleted nutrient broth. This second component is associated with the appearance of glutaminase A (EC 3.5.1.2) and asparaginase I (EC 3.5.1.1), a periplasmic enzyme. Parallel changes were observed in the levels of the high-affinity glutamine transport system and the glutamine synthetase when cells were cultured with the carbon sources: glucose, glycerol, or succinate. PMID:238938

  8. ESCHERICHIA COLI Gene Induction by Alkylation Treatment

    PubMed Central

    Volkert, Michael R.; Nguyen, Dinh C.; Beard, K. Christopher

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased β-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes. PMID:3080354

  9. Escherichia coli gene induction by alkylation treatment.

    PubMed

    Volkert, M R; Nguyen, D C; Beard, K C

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.

  10. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1986-03-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. Focus is on the two final steps in alcohol synthesis, which are catalyzed by alcohol dehydrogenase and acetaldehyde CoA dehydrogenase. We have isolated a series of mutations affecting the expression of these enzymes. Some of these mutations are in the structural genes for these enzymes; others affect the regulation of the adh operon. We have recently cloned the genes coding for these enzymes and are now studying the effect of multiple copies of the adh gene on fermentative growth and its regulation. A recently invented technique, proton suicide has allowed the selection of a variety of novel mutants affecting fermentation which are presently being characterized. We have isolated a comprehensive collection of operon fusions in which the lacZ structural gene is fused to promoters that are inactive aerobically but active anaerobically. Although these genes (like adh) are only expressed under anaerobic conditions, the level of induction varies from two-fold to nearly 100-fold. The nitrogen source, medium pH, nature of the buffer, presence of alternative electron acceptors (e.g., nitrate), and other factors exert a great effect on the expression of many of these genes. In the near future we will investigate control mechanisms common to the adh operon and other anaerobically regulated genes.

  11. Antimicrobial-resistant Invasive Escherichia coli, Spain

    PubMed Central

    Oteo, Jesús; Lázaro, Edurne; de Abajo, Francisco J.; Baquero, Fernando; Campos, José

    2005-01-01

    To address the public health problem of antimicrobial resistance, the European Union founded the European Antimicrobial Resistance Surveillance System. A network of 32 Spanish hospitals, serving ≈9.6 million persons, submitted antimicrobial-susceptibility data on 7,098 invasive Escherichia coli species (2001–2003). Resistance to ampicillin, cotrimoxazole, ciprofloxacin, gentamicin, and tobramycin was found at rates of 59.9%, 32.6%, 19.3%, 6.8%, and 5.3%, respectively. Resistance to multiple drugs increased from 13.8% in 2001 to 20.6% in 2003 (p <0.0001). Antimicrobial consumption data were obtained from the Spanish National Health System. In spite of decreased cephalosporin and β-lactam use, overall extended-spectrum β-lactamase production increased from 1.6% (2001) to 4.1% (2003) (p <0.0001), mainly due to the rising prevalence of cefotaximases. Resistance to ciprofloxacin significantly increased, mostly in community-onset infections, which coincided with a rise in community quinolone use. Cotrimoxazole resistance remained stable at ≈30%, even though its use was dramatically reduced. PMID:15829192

  12. Genotoxicity of Graphene in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Sharma, Ananya

    Rapid advances in nanotechnology necessitate assessment of the safety of nanomaterials in the resulting products and applications. One key nanomaterial attracting much interest in many areas of science and technology is graphene. Graphene is a one atom thick carbon allotrope arranged in a two-dimensional honeycomb lattice. In addition to being extremely thin, graphene has several extraordinary physical properties such as its exceptional mechanical strength, thermal stability, and high electrical conductivity. Graphene itself is relatively chemically inert and therefore pristine graphene must undergo a process called functionalization, which is combination of chemical and physical treatments that change the properties of graphene, to make it chemically active. Functionalization of graphene is of crucial importance as the end application of graphene depends on proper functionalization. In the field of medicine, graphene is currently a nanomaterial of high interest for building biosensors, DNA transistors, and probes for cancer detection. Despite the promising applications of graphene in several areas of biomedicine, there have been only few studies in recent years that focus on evaluating cytotoxicity of graphene on cells, and almost no studies that investigate how graphene exposure affects cellular genetic material. Therefore, in this study we used a novel approach to evaluate the genotoxicity, i.e., the effects of graphene on DNA, using Escherichia coli as a prokaryotic model organism.

  13. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  14. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  15. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  16. Ribonuclease Sensitivity of Escherichia coli Ribosomes

    PubMed Central

    Santer, Melvin; Smith, Josephine R.

    1966-01-01

    Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099–1110. 1966.—The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10−4m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities. Images PMID:5332866

  17. Enterotoxigenic Escherichia coli Multilocus Sequence Types in Guatemala and Mexico

    PubMed Central

    Klena, John; Rodas, Claudia; Bourgeois, August Louis; Torres, Olga; Svennerholm, Ann-Mari; Sjöling, Åsa

    2010-01-01

    The genetic backgrounds of 24 enterotoxigenic Escherichia coli (ETEC) strains from Mexico and Guatemala expressing heat-stable toxin (ST) and coli surface antigen 6 (CS6) were analyzed. US travelers to these countries and resident children in Guatemala were infected by ETEC strains of sequence type 398, expressing STp and carrying genetically identical CS6 sequences. PMID:20031063

  18. Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterohemorrhagic E. coli (EHEC) are Shiga toxin–producing Escherichia coli (STEC) associated with the most severe forms of foodborne illnesses. The United States Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) has identified a higher percentage of non-O157 EHEC compared to E....

  19. Draft Genome Sequence of Uropathogenic Escherichia coli Strain NB8

    PubMed Central

    Mi, Zu-huang; Wang, Chun-xin; Zhu, Jian-ming

    2016-01-01

    Escherichia coli NB8 is a clinical pyelonephritis isolate. Here, we report the draft genome sequence of uropathogenic E. coli NB8, which contains drug resistance genes encoding resistance to beta-lactams, aminoglycosides, quinolones, macrolides, colistin, sulfonamide-trimethoprim, and tetracycline. NB8 infects the kidney and bladder, making it an important tool for studying E. coli pathogenesis. PMID:27609920

  20. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

    PubMed Central

    Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel; Löw, Mirjam; Eriksson, Jonas; Bonde, Ida; Herrgård, Markus J.; Heipieper, Hermann J.; Nørholm, Morten H. H.; Slotboom, Dirk Jan; de Gier, Jan-Willem

    2017-01-01

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production. PMID:28338018

  1. Soil solarization reduces Escherichia coli O157:H7 and total Escherichia coli on cattle feedlot pen surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feedlot pen soils are a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM)....

  2. Free RNA polymerase in Escherichia coli.

    PubMed

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

  3. The Melibiose Transporter of Escherichia coli

    PubMed Central

    Fuerst, Oliver; Lin, Yibin; Granell, Meritxell; Leblanc, Gérard; Padrós, Esteve; Lórenz-Fonfría, Víctor A.; Cladera, Josep

    2015-01-01

    We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na+-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na+ and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200–330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na+ ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na+ ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na+ binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na+ binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions. PMID:25971963

  4. Novel Mechanism of Escherichia coli Porin Regulation

    PubMed Central

    Castillo-Keller, Maria; Vuong, Phu; Misra, Rajeev

    2006-01-01

    A novel mechanism of Escherichia coli porin regulation was discovered from multicopy suppressors that permitted growth of cells expressing a mutant OmpC protein in the absence of DegP. Analyses of two suppressors showed that both substantially lowered OmpC expression. Suppression activities were confined to a short DNA sequence, which we designated ipeX for inhibition of porin expression, and to DNA containing a 3′-truncated ompR gene. The major effect of ipeX on ompC expression was exerted posttranscriptionally, whereas the truncated OmpR protein reduced ompC transcription. ipeX was localized within an untranslated region of 247 base pairs between the stop codon of nmpC—a remnant porin gene from the cryptic phage qsr′ (DLP12) genome—and its predicted Rho-independent transcriptional terminator. Interestingly, another prophage, PA-2, which encodes a porin similar to NmpC, known as Lc, has sequences downstream from lc identical to that of ipeX. PA-2 lysogenization leads to Lc expression and OmpC inhibition. Our data show that the synthesis of the lc transcript, whose 3′ end contains the corresponding ipeX sequence, inhibits OmpC expression. Overexpression of ipeX RNA inhibited both OmpC and OmpF expression but not that of OmpA. ompC-phoA chimeric gene constructs revealed a 248-bp untranslated region of ompC required for ipeX-mediated inhibition. However, no sequence complementarity was found between ipeX and this region of ompC, indicating that inhibition may not involve simple base pairing between the two RNA molecules. The effect of ipeX on ompC, but not on ompF, was independent of the RNA chaperone Hfq. PMID:16385048

  5. Serogroups of Escherichia coli from drinking water.

    PubMed

    Ramteke, P W; Tewari, Suman

    2007-07-01

    Fifty seven isolates of thermotolerant E. coli were recovered from 188 drinking water sources, 45 (78.9%) were typable of which 15 (26.3%) were pathogenic serotypes. Pathogenic serogroup obtained were 04 (Uropathogenic E. coli, UPEC), 025 (Enterotoxigenic E. coli, ETEC), 086 (Enteropathogenic E. coli, EPEC), 0103 (Shiga-toxin producing E. coli, STEC), 0157 (Shiga-toxin producing E. coli, STEC), 08 (Enterotoxigenic E. coli, ETEC) and 0113 (Shiga-toxin producing E. coli, STEC). All the pathogenic serotypes showed resistance to bacitracin and multiple heavy metal ions. Resistance to streptomycin and cotrimazole was detected in two strains whereas resistance to cephaloridine, polymixin-B and ampicillin was detected in one strain each. Transfer of resistances to drugs and metallic ions was observed in 9 out of 12 strains studied. Resistances to bacitracin were transferred in all nine strains. Among heavy metals resistance to As(3+) followed by Cr(6+) were transferred more frequently.

  6. Environmental Escherichia coli: Ecology and public health implications - A review

    USGS Publications Warehouse

    Jang, Jeonghwan; Hur, Hor-Gil; Sadowsky, Michael J.; Byappanahalli, Muruleedhara; Yan, Tao; Ishii, Satoshi

    2017-01-01

    Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through feces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent fecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extra-intestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a fecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics provide the diversity and complexity of E. coli strains in various environments, affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments in regards to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

  7. Investigation of ’Escherichia coli’ Enterotoxins

    DTIC Science & Technology

    1978-05-01

    E . coli diarrheal disease in man and domestic animals. Fundamentally, the design of the vaccine is based on the well- documented ability of cholera antitoxin to neutralize both cholera and heat- labile E . coli enterotoxins and on the ability of certain E . coli antigens to enhance the immune response to cholera toxoid and possibly whole-cell Cholera Vaccine, as

  8. [Cloning and expression of F18 fimbrial operon gene clusters from enterotoxigenic Escherichia coli and their bioactivity].

    PubMed

    Zhang, Jian-Jun; Zhu, Guo-Qiang

    2007-10-01

    The fed operon gene clusters with each size of 5.6kb, encoding the F18ab or F18ac fimbriae, was amplified respectively by high fidelity PCR using the genomic DNA templates from F18 fimbriae E. coli strains 107/86 or 2134P. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the vector pET-22b (+), the recombinant plamids with the inserts of both type of fed gene clusters were constructed and screened, further confirmed by the means of combination with restriction endonuclease analysis and sequencing. The both types of fimbriae F18ab and F18ac were expressed efficiently in the E. coli BL21 (DE3) after proper concentration of IPTG induction. Expressed fimbriae were revealed and confirmed by transmissible electromicroscope observation. The both fimbriae F18ab and F18ac were isolated and purified from the recombinant E. coli, and only a single major band of protein with size of approximately 15kDa was visualized in Coomassie blue-stained gels after SDS-PAGE. The rabbits sera with high titer of anti-F18 fimbriae were detected after being immunized with the purified F18ab or F18ac fimbriae. The results of combination of agglutination assay with Western blotting showed that the sera directed against both fimbriae F18ab and F18ac reacted positively with the F18 fimbriae from both wild E. coli 107/86 and 2134P. Small intestine epithelial cells with F18 fimbriae receptors, which were from post-weaning piglets with the genotypes of FUT1 gene both M307(GG) and M307(AG), were prepared and tested for the adherence of E. coli expressing F18 fimbriae under the microscopic examination. Adhesion and adhesion inhibition test showed both of the recombinant E. coli expressing F18ab or F18ac fimbriae respectively could adhere to the jejunal epithelial cells in vitro as E. coli 107/86 and 2134p did. The both of anti-sera directed against fimbriae F18ab or F18ac respectively can efficiently inhibit the fimbriae-mediated post-weaning piglet

  9. Rapid Sterilization of Escherichia coli by Solution Plasma Process

    NASA Astrophysics Data System (ADS)

    Andreeva, Nina; Ishizaki, Takahiro; Baroch, Pavel; Saito, Nagahiro

    2012-12-01

    Solution plasma (SP), which is a discharge in the liquid phase, has the potential for rapid sterilization of water without chemical agents. The discharge showed a strong sterilization performance against Escherichia coli bacteria. The decimal value (D value) of the reduction time for E. coli by this system with an electrode distance of 1.0 mm was estimated to be approximately 1.0 min. Our discharge system in the liquid phase caused no physical damage to the E. coli and only a small increase in the temperature of the aqueous solution. The UV light generated by the discharge was an important factor in the sterilization of E. coli.

  10. A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks.

    PubMed

    Cai, Mengli; Huang, Ying; Yang, Renbin; Craigie, Robert; Clore, G M

    2016-10-01

    We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl β-D-1-thiogalactopyranoside at an OD600 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.

  11. Infection by verocytotoxin-producing Escherichia coli.

    PubMed Central

    Karmali, M A

    1989-01-01

    Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a newly recognized group of enteric pathogens which are increasingly being recognized as common causes of diarrhea in some geographic settings. Outbreak studies indicate that most patients with VTEC infection develop mild uncomplicated diarrhea. However, a significant risk of two serious and potentially life-threatening complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes VTEC infection a public health problem of serious concern. The main reservoirs of VTEC appear to be the intestinal tracts of animals, and foods of animal (especially bovine) origin are probably the principal sources for human infection. The term VT refers to a family of subunit exotoxins with high biological activity. Individual VTEC strains elaborate one or both of at least two serologically distinct, bacteriophage-mediated VTs (VT1 and VT2) which are closely related to Shiga toxin and are thus also referred to as Shiga-like toxins. The holotoxins bind to cells, via their B subunits, to a specific receptor which is probably the glycolipid, globotriosyl ceramide (Gb3). Binding is followed by internalization of the A subunit, which, after it is proteolytically nicked and reduced to the A1 fragment, inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28S ribosomal ribonucleic acid. The mechanism of VTEC diarrhea is still controversial, and the relative roles of locally acting VT and "attaching and effacing adherence" of VTEC to the mucosa have yet to be resolved. There is increasing evidence that hemolytic uremic syndrome and possibly hemorrhagic colitis result from the systemic action of VT on vascular endothelial cells. The role of antitoxic immunity in preventing the systemic complications of VTEC infection is being explored. Antibiotics appear to be contraindicated in the treatment of VTEC infection. The most common VTEC serotype associated

  12. Intestinal Colonization by Enterotoxigenic Escherichia coli.

    DTIC Science & Technology

    1980-09-01

    E . coli is mediated by specific types of pili. These pili are antigenic and can be used in diagnosing enterotoxigenic E . coli infections. They are also good protective antigens. When pregnant dams are vaccinated parenterally or orally with pili on live piliated bacteria, they secrete antibodies against the pili in their milk. Neonates suckling dams so vaccinated are passively protected against fatal challenge by enterotoxigenic E . coli . Pili are also good candidate protective antigens for the development of vaccines to protect by

  13. Transcription of foreign DNA in Escherichia coli.

    PubMed

    Warren, René L; Freeman, John D; Levesque, Roger C; Smailus, Duane E; Flibotte, Stephane; Holt, Robert A

    2008-11-01

    Propagation of heterologous DNA in E. coli host cells is central to molecular biology. DNA constructs are often engineered for expression of recombinant protein in E. coli, but the extent of incidental transcription arising from natural regulatory sequences in cloned DNA remains underexplored. Here, we have used programmable microarrays and RT-PCR to measure, comprehensively, the transcription of H. influenzae, P. aeruginosa, and human DNA propagating in E. coli as bacterial artificial chromosomes. We find evidence that at least half of all H. influenzae genes are transcribed in E. coli. Highly transcribed genes are principally involved in energy metabolism, and their proximal promoter regions are significantly enriched with E. coli sigma(70) (also known as RpoD) binding sites. H. influenzae genes acquired from an ancient bacteriophage Mu insertion are also highly transcribed. Compared with H. influenzae, a smaller proportion of P. aeruginosa genes are transcribed in E. coli, and in E. coli there is punctuated transcription of human DNA. The presence of foreign DNA in E. coli disturbs the host transcriptional profile, with expression of the E. coli phage shock protein operon and the flagellar gene cluster being particularly strongly up-regulated. While cross-species transcriptional activation is expected to be enabling for horizontal gene transfer in bacteria, incidental expression of toxic genes can be problematic for DNA cloning. Ongoing characterization of cross-expression will help inform the design of biosynthetic gene clusters and synthetic microbial genomes.

  14. Recurrent Hemolytic and Uremic Syndrome Induced by Escherichia Coli

    PubMed Central

    Commereuc, Morgane; Weill, Francois-Xavier; Loukiadis, Estelle; Gouali, Malika; Gleizal, Audrey; Kormann, Raphaël; Ridel, Christophe; Frémeaux-Bacchi, Véronique; Rondeau, Eric; Hertig, Alexandre

    2016-01-01

    Abstract A widespread belief is that typical hemolytic and uremic syndrome (HUS) does not recur. We report the case of a patient infected twice with raw milk taken from his own cow and containing a Shiga toxin–producing Escherichia coli O174:H21 that induced recurrent HUS causing severe renal and cerebral disorders. A genomic comparison of the human and bovine Shiga toxin–producing Escherichia coli O174:H21 isolates revealed that they were identical. Typical HUS may recur. Since milk from this animal was occasionally distributed locally, thereby posing a serious threat for the whole village, this particular cow was destroyed. PMID:26735524

  15. [Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli].

    PubMed

    Ptitsyn, L R; Fatova, M A; Stepanov, A I

    1990-02-01

    Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.

  16. Diarrheagenic Escherichia coli in Children from Costa Rica

    PubMed Central

    Pérez, Cristian; Gómez-Duarte, Oscar G.; Arias, María L.

    2010-01-01

    More than 5,000 diarrheal cases per year receive medical care at the National Children's Hospital of Costa Rica, and nearly 5% of them require hospitalization. A total of 173 Escherichia coli strains isolated from children with diarrhea were characterized at the molecular, serologic, and phenotypic level. Multiplex and duplex polymerase chain reactions were used to detect the six categories of diarrheagenic E. coli. Thirty percent (n = 52) of the strains were positive, indicating a high prevalence among the pediatric population. Enteropathogenic E. coli and enteroinvasive E. coli pathotypes were the most prevalent (21% and 19%, respectively). Pathogenic strains were distributed among the four E. coli phylogenetic groups A, B1, B2, and D, with groups A and B1 the most commonly found. This study used molecular typing to evaluate the prevalence of diarrheagenic E. coli reported in Costa Rica and demonstrated the importance of these pathotypes in the pediatric population. PMID:20682870

  17. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

    PubMed Central

    2013-01-01

    Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures. PMID:23557146

  18. Large Surface Blebs on Escherichia coli Heated to Inactivating Temperatures

    PubMed Central

    Scheie, Paul; Ehrenspeck, Susan

    1973-01-01

    Large surface blebs were observed with phase-contrast optics on Escherichia coli B/r and Bs-1 heated to temperatures at which colony-forming ability was lost. Characterization of such blebs was consistent with the view that they were formed by a physical process and were bounded by the outer membrane of the cell. A hypothesis for thermal inactivation of E. coli is presented that places membrane damage near the primary lethal event. Images PMID:4196258

  19. Expression of staphylococcal enterotoxin C1 in Escherichia coli.

    PubMed Central

    Bohach, G A; Schlievert, P M

    1987-01-01

    The structural gene encoding staphylococcal enterotoxin C1 was cloned into Escherichia coli and localized on a 1.5-kilobase HindIII-ClaI DNA fragment by subcloning. The toxin was partially purified from E. coli clones and shown to be immunologically identical to enterotoxin C1 from Staphylococcus aureus. The cloned toxin also had the same molecular weight (26,000) and charge heterogeneity as staphylococcus-derived enterotoxin. Toxins from both sources were equally biologically active. Images PMID:3542834

  20. 76 FR 72331 - Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-23

    ... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... methods for controlling non-O157 Shiga toxin-producing Escherichia coli in raw, intact and non-intact beef... Escherichia coli in raw, intact and non-intact beef products and product components on or before December...

  1. Multidrug-resistant Escherichia coli in Asia: epidemiology and management.

    PubMed

    Sidjabat, Hanna E; Paterson, David L

    2015-05-01

    Escherichia coli has become multiresistant by way of production of a variety of β-lactamases. The prevalence of CTX-M-producing E. coli has reached 60-79% in certain parts of Asia. The acquisition of CTX-M plasmids by E. coli sequence type 131, a successful clone of E. coli, has caused further dissemination of CTX-M-producing E. coli. The prevalence of carbapenemase-producing E. coli, especially Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase (NDM)-producing E. coli has been increasing in Asia. K. pneumoniae carbapenemase and NDM have now been found in E. coli sequence type 131. The occurrence of NDM-producing E. coli is a major concern particularly in the Indian subcontinent, but now elsewhere in Asia as well. There are multiple reasons why antibiotic resistance in E. coli in Asia has reached such extreme levels. Approaches beyond antibiotic therapy, such as prevention of antibiotic resistance by antibiotic stewardship and protecting natural microbiome, are strategies to avoid further spread of antibiotic resistance.

  2. The quantitative and condition-dependent Escherichia coli proteome

    PubMed Central

    Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

    2016-01-01

    Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

  3. An integrated database to support research on Escherichia coli

    SciTech Connect

    Baehr, A.; Dunham, G.; Matsuda, Hideo; Michaels, G.; Taylor, R.; Overbeek, R.; Rudd, K.E.; Ginsburg, A.; Joerg, D.; Kazic, T.; Hagstrom, R.; Zawada, D.; Smith, C.; Yoshida, Kaoru

    1992-01-01

    We have used logic programming to design and implement a prototype database of genomic information for the model bacterial organism Escherichia coli. This report presents the fundamental database primitives that can be used to access and manipulate data relating to the E. coli genome. The present system, combined with a tutorial manual, provides immediate access to the integrated knowledge base for E. coli chromosome data. It also serves as the foundation for development of more user-friendly interfaces that have the same retrieval power and high-level tools to analyze complex chromosome organization.

  4. YeeO from Escherichia coli exports flavins.

    PubMed

    McAnulty, Michael J; Wood, Thomas K

    2014-01-01

    Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters.

  5. Heat-stable Escherichia coli enterotoxin production in vivo.

    PubMed Central

    Whipp, S C; Moon, H W; Lyon, N C

    1975-01-01

    Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life. Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity. Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E. coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro. It is concluded that heat-stable enterotoxigenic E. coli induce diarrhea by production of heat-stable enterotoxin in vivo. PMID:1097335

  6. Reassessing Escherichia coli as a cell factory for biofuel production.

    PubMed

    Wang, Chonglong; Pfleger, Brian F; Kim, Seon-Won

    2017-03-11

    Via metabolic engineering, industrial microorganisms have the potential to convert renewable substrates into a wide range of biofuels that can address energy security and environmental challenges associated with current fossil fuels. The user-friendly bacterium, Escherichia coli, remains one of the most frequently used hosts for demonstrating production of biofuel candidates including alcohol-, fatty acid- and terpenoid-based biofuels. In this review, we summarize the metabolic pathways for synthesis of these biofuels and assess enabling technologies that assist in regulating biofuel synthesis pathways and rapidly assembling novel E. coli strains. These advances maintain E. coli's position as a prominent host for developing cell factories for biofuel production.

  7. Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

    PubMed Central

    Toledo, M. Regina F.; Alvariza, M. do Carmo B.; Murahovschi, Jayme; Ramos, Sonia R. T. S.; Trabulsi, Luiz R.

    1983-01-01

    Enteropathogenic Escherichia coli serotypes were searched for in feces of 550 children with endemic diarrhea and in 129 controls, in São Paulo, in 1978 and 1979; serotypes O111ab:H−, O111ab:H2, and O119:H6 were significantly associated with diarrhea in children 0 to 5 months old and were the most frequent agents of diarrhea in this age group as compared with enterotoxigenic and enteroinvasive E. coli, Salmonella sp., Shigella sp., and Yersinia enterocolitica. It is concluded that various enteropathogenic E. coli serotypes may be agents of endemic infantile diarrhea. PMID:6339384

  8. Sources of Escherichia coli in a Coastal Subtropical Environment

    PubMed Central

    Solo-Gabriele, Helena M.; Wolfert, Melinda A.; Desmarais, Timothy R.; Palmer, Carol J.

    2000-01-01

    Sources of Escherichia coli in a coastal waterway located in Ft. Lauderdale, Fla., were evaluated. The study consisted of an extensive program of field measurements designed to capture spatial and temporal variations in E. coli concentrations as well as experiments conducted under laboratory-controlled conditions. E. coli from environmental samples was enumerated by using a defined substrate technology (Colilert-18). Field sampling tasks included sampling the length of the North Fork to identify the river reach contributing high E. coli levels, autosampler experiments at two locations, and spatially intense sampling efforts at hot spots. Laboratory experiments were designed to simulate tidal conditions within the riverbank soils. The results showed that E. coli entered the river in a large pulse during storm conditions. After the storm, E. coli levels returned to baseline levels and varied in a cyclical pattern which correlated with tidal cycles. The highest concentrations were observed during high tide, whereas the lowest were observed at low tide. This peculiar pattern of E. coli concentrations between storm events was caused by the growth of E. coli within riverbank soils which were subsequently washed in during high tide. Laboratory analysis of soil collected from the riverbanks showed increases of several orders of magnitude in soil E. coli concentrations. The ability of E. coli to multiply in the soil was found to be a function of soil moisture content, presumably due to the ability of E. coli to outcompete predators in relatively dry soil. The importance of soil moisture in regulating the multiplication of E. coli was found to be critical in tidally influenced areas due to periodic wetting and drying of soils in contact with water bodies. Given the potential for growth in such systems, E. coli concentrations can be artificially elevated above that expected from fecal impacts alone. Such results challenge the use of E. coli as a suitable indicator of water

  9. Structure of Water in Escherichia Coli B

    DTIC Science & Technology

    structure broadening of the NMR water spectrum. Using bacteria grown in the special chemically defined medium, we showed that the water in E. coli B was highly ordered and was very different from ’free’ water and from polywater .

  10. [Fusion expression of SLT-IIeB gene and FedF gene of Ee in Escherichia coli and its immunogenicity].

    PubMed

    Liu, Guo-Ping; Wu, Bin; Lin, Yi-Yuan; Liu, Meng-Yuan; Chen, Huan-Chun

    2007-12-01

    The DNA fragment encoding the truncated SLT-IIeB and FedF of Ee strain were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKSF. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-SF fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with mouse anti-SLT-IIeB antiserum, mouse anti-FedF antiserum and moue anti-GST monoclonal antibody respectively. The fusion protein was further purified and used as an antigen for preparation of immune serum. The anti-sera of GST-SF were able to restrain the toxicity of SLT-IIe to Vero-E6 cells and inhibit the adhesin of F18 fimbriae to brush borders of swine in vitro. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-SF, GST-B, GST-F and challenged intraperitoneally with volume of 5 LD50 Ee strain. The results show the fusion protein GST-SF had more shrong immunogenicity and better protection against Ee strain.

  11. Slugs: Potential Novel Vectors of Escherichia coli O157

    PubMed Central

    Sproston, Emma L.; Macrae, M.; Ogden, Iain D.; Wilson, Michael J.; Strachan, Norval J. C.

    2006-01-01

    Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157. PMID:16391036

  12. Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004.

    PubMed

    Qadri, Firdausi; Khan, Ashraful I; Faruque, Abu Syed G; Begum, Yasmin Ara; Chowdhury, Fahima; Nair, Gopinath B; Salam, Mohammed A; Sack, David A; Svennerholm, Ann-Mari

    2005-07-01

    Flooding in Dhaka in July 2004 caused epidemics of diarrhea. Enterotoxigenic Escherichia coli (ETEC) was almost as prevalent as Vibrio cholerae O1 in diarrheal stools. ETEC that produced heat-stable enterotoxin alone was most prevalent, and 78% of strains had colonization factors. Like V. cholerae O1, ETEC can cause epidemic diarrhea.

  13. armA and aminoglycoside resistance in Escherichia coli.

    PubMed

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C; Moreno, Miguel A; Courvalin, Patrice; Domínguez, Lucas

    2005-06-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

  14. armA and Aminoglycoside Resistance in Escherichia coli

    PubMed Central

    González-Zorn, Bruno; Teshager, Tirushet; Casas, María; Porrero, María C.; Courvalin, Patrice; Domínguez, Lucas

    2005-01-01

    We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant. PMID:15963296

  15. Escherichia coli as other Enterobacteriaceae: food poisoning and health effects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many Escherichia coli strains are harmless, and they are an important commensal in the intestinal microflora; however, pathogenic strains also exist. The pathogenic strains can be divided into diarrhea-inducing strains and strains that reside in the intestines but only cause disease in bodily sites...

  16. Escherichia coli growth studied by dual-parameter flow cytophotometry.

    PubMed Central

    Steen, H B; Boye, E

    1981-01-01

    The growth of Escherichia coli cells has been analyzed for the first time by dual-parameter flow cytophotometry, in which the deoxyribonucleic acid and protein contents of single bacteria have been measured simultaneously with an accuracy of a few percent and at a rate of 3,000 cells/s. PMID:7007339

  17. More than a locomotive organelle: flagella in Escherichia coli.

    PubMed

    Zhou, Mingxu; Yang, Yang; Chen, Panlin; Hu, Huijie; Hardwidge, Philip R; Zhu, Guoqiang

    2015-11-01

    The flagellum is a locomotive organelle that allows bacteria to respond to chemical gradients. This review summarizes the current knowledge regarding Escherichia coli flagellin variants and the role of flagella in bacterial functions other than motility, including the relationship between flagella and bacterial virulence.

  18. Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332.

    PubMed

    Saldaña-Ahuactzi, Zeus; Cruz-Córdova, Ariadnna; Rodea, Gerardo E; Porta, Helena; Navarro-Ocaña, Armando; Eslava-Campos, Carlos; Cevallos, Miguel A; Xicohtencatl-Cortes, Juan

    2017-02-23

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México.

  19. Stringent control of FLP recombinase in Escherichia coli.

    PubMed

    Bowden, Steven D; Palani, Nagendra P; Libourel, Igor G L

    2017-02-01

    Site specific recombinases are invaluable tools in molecular biology, and are emerging as powerful recorders of cellular events in synthetic biology. We have developed a stringently controlled FLP recombinase system in Escherichia coli using an arabinose inducible promoter combined with a weak ribosome binding site.

  20. Enteroinvasive Escherichia coli severe dysentery complicated by rotavirus gastroenteritis.

    PubMed

    Pacheco-Gil, Leova; Ochoa, Theresa J; Flores-Romo, Leopoldo; DuPont, Herbert L; Estrada-Garcia, Teresa

    2006-11-01

    Enteroinvasive Escherichia coli (EIEC) is an important agent of pediatric diarrhea and dysentery in developing countries. We report a life-threatening severe dysentery case due to EIEC in a malnourished 4-month-old male, native Indian infant co-infected with rotavirus. The severe gastrointestinal bleeding anemia and hypovolemic shock was successfully treated with IV blood transfusions, rehydration and antibiotic therapy.

  1. TRIMETHOPRIM-SULFAMETHOXAZOLE RESISTANCE IN SEWAGE ISOLATES OF ESCHERICHIA COLI

    EPA Science Inventory

    The increase in resistance rates to trimehtoprim-sulfamethoxazole (TMP/SMX) in isolates of Escherichia coli has become a matter of increasing concern. This has been particularly true in reference to community acquired urinary tract infections (UTI). This study utilized sewage i...

  2. Escherichia coli and other Enterobacteriaceae: Food poisoning and health effects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The family Enterobactericeae consists of rod-shaped, Gram-negative, facultatively anaerobic, non-spore forming bacteria and also includes the food-borne pathogens, Cronobacter spp., Escherichia coli, Salmonella enterica, Shigella spp., and Yersinia spp. Illness caused by these pathogens is acquired...

  3. Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332

    PubMed Central

    Saldaña-Ahuactzi, Zeus; Cruz-Córdova, Ariadnna; Rodea, Gerardo E.; Porta, Helena; Navarro-Ocaña, Armando; Eslava-Campos, Carlos

    2017-01-01

    ABSTRACT   Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México. PMID:28232434

  4. New types of Escherichia coli recombination-deficient mutants.

    PubMed

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  5. New types of Escherichia coli recombination-deficient mutants.

    PubMed Central

    Freifelder, D

    1976-01-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency. PMID:789362

  6. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

    EPA Science Inventory

    Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfe...

  7. rRNA transcription rate in Escherichia coli.

    PubMed Central

    Gotta, S L; Miller, O L; French, S L

    1991-01-01

    The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. Images FIG. 1 PMID:1717439

  8. Multidrug-Resistant Escherichia coli in Bovine Animals, Europe

    PubMed Central

    Brennan, Evan; Martins, Marta; McCusker, Matthew P.; Wang, Juan; Alves, Bruno Martins; Hurley, Daniel; El Garch, Farid; Woehrlé, Frédérique; Miossec, Christine; McGrath, Leisha; Srikumar, Shabarinath; Wall, Patrick

    2016-01-01

    Of 150 Escherichia coli strains we cultured from specimens taken from cattle in Europe, 3 had elevated MICs against colistin. We assessed all 3 strains for the presence of the plasmid-mediated mcr-1 gene and identified 1 isolate as mcr-1–positive and co-resistant to β-lactam, florfenicol, and fluoroquinolone antimicrobial compounds. PMID:27533105

  9. EcoCyc: Encyclopedia of Escherichia coli genes and metabolism.

    PubMed

    Karp, P D; Riley, M; Paley, S M; Pellegrini-Toole, A; Krummenacker, M

    1998-01-01

    The encyclopedia of Escherichia coli genes and metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of E.coli. The database describes 3030 genes of E.coli , 695 enzymes encoded by a subset of these genes, 595 metabolic reactions that occur in E.coli, and the organization of these reactions into 123 metabolic pathways. The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways. EcoCyc can be thought of as an electronic review article because of its copious references to the primary literature, and as a (qualitative) computational model of E.coli metabolism. EcoCyc is available at URL http://ecocyc.PangeaSystems.com/ecocyc/

  10. Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples

    PubMed Central

    Morcatti Coura, Fernanda; Diniz, Soraia de Araújo; Silva, Marcos Xavier; Mussi, Jamili Maria Suhet; Barbosa, Silvia Minharro; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2015-01-01

    This study analyzes the occurrence and distribution of phylogenetic groups of 391 strains of Escherichia coli isolated from poultry, cattle, and water buffalo. The frequency of the phylogroups was A = 19%, B1 = 57%, B2 = 2.3%, C = 4.6%, D = 2.8%, E = 11%, and F = 3.3%. Phylogroups A (P < 0.001) and F (P = 0.018) were associated with E. coli strains isolated from poultry, phylogroups B1 (P < 0.001) and E (P = 0.002) were associated with E. coli isolated from cattle, and phylogroups B2 (P = 0.003) and D (P = 0.017) were associated with E. coli isolated from water buffalo. This report demonstrated that some phylogroups are associated with the host analyzed and the results provide knowledge of the phylogenetic composition of E. coli from domestic animals. PMID:26421310

  11. Glycerol elicits energy taxis of Escherichia coli and Salmonella typhimurium.

    PubMed

    Zhulin, I B; Rowsell, E H; Johnson, M S; Taylor, B L

    1997-05-01

    Escherichia coli and Salmonella typhimurium show positive chemotaxis to glycerol, a chemical previously reported to be a repellent for E. coli. The threshold of the attractant response in both species was 10(-6) M glycerol. Glycerol chemotaxis was energy dependent and coincident with an increase in membrane potential. Metabolism of glycerol was required for chemotaxis, and when lactate was present to maintain energy production in the absence of glycerol, the increases in membrane potential and chemotactic response upon addition of glycerol were abolished. Methylation of a chemotaxis receptor was not required for positive glycerol chemotaxis in E. coli or S. typhimurium but is involved in the negative chemotaxis of E. coli to high concentrations of glycerol. We propose that positive chemotaxis to glycerol in E. coli and S. typhimurium is an example of energy taxis mediated via a signal transduction pathway that responds to changes in the cellular energy level.

  12. Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

    PubMed

    Chung, Mu-En; Yeh, I-Hsin; Sung, Li-Yu; Wu, Meng-Ying; Chao, Yun-Peng; Ng, I-Son; Hu, Yu-Chen

    2017-01-01

    Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.

  13. Polyerositis and Arthritis Due to Escherichia coli in Gnotobiotic Pigs

    PubMed Central

    Waxler, G. L.; Britt, A. L.

    1972-01-01

    Forty gnotobiotic pigs from six litters were exposed orally to Escherichia coli 083:K·:NM at 69 to 148 hours of age, while 17 pigs from the same litters served as unexposed controls. Clinical signs of infection included fever, anorexia, diarrhea, lameness, and reluctance to move. Eighty-four percent of the exposed pigs in four litters died, while only 13% in two litters died. Gross and microscopic lesions included serofibrinous to fibrinopurulent polyserositis in 96% of the exposed pigs in four litters and 33% of the exposed pigs in two litters. A few pigs had gross and/or microscopic lesions of arthritis. Escherichia coli was routinely isolated from the serous and synovial cavities of infected pigs. Anti-hog cholera serum administered orally as a colostrum substitute gave partial protection against E. coli infection. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8. PMID:4261837

  14. Cytotoxic Escherichia coli strains encoding colibactin colonize laboratory mice.

    PubMed

    García, Alexis; Mannion, Anthony; Feng, Yan; Madden, Carolyn M; Bakthavatchalu, Vasudevan; Shen, Zeli; Ge, Zhongming; Fox, James G

    2016-12-01

    Escherichia coli strains have not been fully characterized in laboratory mice and are not currently excluded from mouse colonies. Colibactin (Clb), a cytotoxin, has been associated with inflammation and cancer in humans and animals. We performed bacterial cultures utilizing rectal swab, fecal, and extra intestinal samples from clinically unaffected or affected laboratory mice. Fifty-one E. coli were isolated from 45 laboratory mice, identified biochemically, and selected isolates were serotyped. The 16S rRNA gene was amplified and sequenced for specific isolates, PCR used for clbA and clbQ gene amplification, and phylogenetic group identification was performed on all 51 E. coli strains. Clb genes were sequenced and selected E. coli isolates were characterized using a HeLa cell cytotoxicity assay. Forty-five of the 51 E. coli isolates (88%) encoded clbA and clbQ and belonged to phylogenetic group B2. Mouse E. coli serotypes included: O2:H6, O-:H-, OM:H+, and O22:H-. Clb-encoding O2: H6 mouse E. coli isolates were cytotoxic in vitro. A Clb-encoding E. coli was isolated from a clinically affected genetically modified mouse with cystic endometrial hyperplasia. Our findings suggest that Clb-encoding E. coli colonize laboratory mice and may induce clinical and subclinical diseases that may impact experimental mouse models.

  15. Using zebra mussels to monitor Escherichia coli in environmental waters.

    PubMed

    Selegean, J P; Kusserow, R; Patel, R; Heidtke, T M; Ram, J L

    2001-01-01

    Use of the zebra mussel (Dreissena polymorpha) as an indicator of previously elevated bacteria concentrations in a watershed was examined. The ability of the zebra mussel to accumulate and purge Escherichia coli over several days was investigated in both laboratory and field experiments. In laboratory experiments, periodic enumeration of E. coli in mussels that had been exposed to a dilute solution of raw sewage demonstrated that (i) maximum concentrations of E. coli are reached within a few hours of exposure to sewage, (ii) the tissue concentration attained is higher than the concentration in the ambient water, and (iii) the E. coli concentrations take several days to return to preexposure concentrations when mussels are subsequently placed in sterile water. In field experiments conducted in southeast Michigan in the Clinton River watershed, brief increases in E. coli concentrations in the water were accompanied by increases in mussel concentrations of E. coli that lasted 2 or 3 d. The ability of mussels to retain and to concentrate E. coli made it possible to detect E. coli in the environment under conditions that conventional monitoring may often miss. Sampling caged mussels in a river and its tributaries may enable watershed managers to reduce the sampling frequency normally required to identify critical E. coli sources, thereby providing a more cost-effective river monitoring strategy for bacterial contamination.

  16. Lytic bacteriophages reduce Escherichia coli O157

    PubMed Central

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

  17. Experimental Escherichia coli O157:H7 carriage in calves.

    PubMed Central

    Brown, C A; Harmon, B G; Zhao, T; Doyle, M P

    1997-01-01

    Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding. PMID:8979335

  18. Travelers' diarrhea and toxigenic Escherichia coli.

    PubMed

    Gorbach, S L; Kean, B H; Evans, D G; Evans, D J; Bessudo, D

    1975-05-01

    In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.

  19. Genomic Comparative Study of Bovine Mastitis Escherichia coli

    PubMed Central

    Kempf, Florent; Slugocki, Cindy; Blum, Shlomo E.; Leitner, Gabriel; Germon, Pierre

    2016-01-01

    Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes. PMID:26809117

  20. [Acute diarrheal disease caused by enteropathogenic Escherichia coli in Colombia].

    PubMed

    Gómez-Duarte, Oscar G

    2014-10-01

    Intestinal Escherichia coli pathogens are leading causes of acute diarrheal disease in children less than 5 years in Latin America, Africa and Asia and a leading cause of death in children living in poorest communities in Africa and South East Asia. Studies on the role of E. coli pathogens in childhood diarrhea in Colombia and other countries in Latin America are limited due to the lack of detection assays in clinical laboratories at the main urban medical centers. Recent studies report that enterotoxigenic E. coli is the most common E. coli pathogens associated with diarrhea in children less than 5 years of age. Other E. coli pathotypes have been detected in children with diarrhea including enteropathogenic, enteroaggregative, shiga-toxin producing and diffusely adherent E. coli. It was also found that meat and vegetables at retail stores are contaminated with Shiga-toxin producing E. coli and enteroaggregative E. coli, suggesting that food products are involved in transmission and infection of the susceptible host. More studies are necessary to evaluate the mechanisms of transmission, the impact on the epidemiology of diarrheal disease, and management strategies and prevention of these pathogens affecting the pediatric population in Colombia.

  1. Proton-linked D-xylose transport in Escherichia coli.

    PubMed Central

    Lam, V M; Daruwalla, K R; Henderson, P J; Jones-Mortimer, M C

    1980-01-01

    The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli. PMID:6995439

  2. Biosynthesis of Two Flavones, Apigenin and Genkwanin, in Escherichia coli.

    PubMed

    Lee, Hyejin; Kim, Bong Gyu; Kim, Mihyang; Ahn, Joong-Hoon

    2015-09-01

    The flavonoid apigenin and its O-methyl derivative, genkwanin, have various biological activities and can be sourced from some vegetables and fruits. Microorganisms are an alternative for the synthesis of flavonoids. Here, to synthesize genkwanin from tyrosine, we first synthesized apigenin from p-coumaric acid using four genes (4CL, CHS, CHI, and FNS) in Escherichia coli. After optimization of different combinations of constructs, the yield of apigenin was increased from 13 mg/l to 30 mg/l. By introducing two additional genes (TAL and POMT7) into an apigenin-producing E. coli strain, we were able to synthesize 7-O-methyl apigenin (genkwanin) from tyrosine. In addition, the tyrosine content in E. coli was modulated by overexpressing aroG and tyrA. The engineered E. coli strain synthesized approximately 41 mg/l genkwanin.

  3. [Escherichia coli R live vaccine Suicolplex "Dessau"].

    PubMed

    Michael-Meese, M; Klie, H; Schöll, W

    1980-01-01

    Immunisation of pregnant sows prior to parturition has long proved to be a good method to forestall coli dysentery in piglets before weaning. Inactivated vaccines of the pathogenetically important E. coli serogroups with and without adjuvant so far were primarily used at international level. A vaccine of that kind has become available in the GDR more than eight years ago. Its name is Coliporc "Dessau". A live vaccine has been developed from two R-mutants at the authors' institute. The effectiveness of that live vaccine on laboratory animals and in field experiments is reported in this paper together with possibilities of differential diagnosis to distinguish wild strains from the mutants. The live vaccine was commercially registered under the name of Suicolpex "Dessau", in spring 1976.

  4. Compilation of DNA sequences of Escherichia coli

    PubMed Central

    Kröger, Manfred

    1989-01-01

    We have compiled the DNA sequence data for E.coli K12 available from the GENBANK and EMBO databases and over a period of several years independently from the literature. We have introduced all available genetic map data and have arranged the sequences accordingly. As far as possible the overlaps are deleted and a total of 940,449 individual bp is found to be determined till the beginning of 1989. This corresponds to a total of 19.92% of the entire E.coli chromosome consisting of about 4,720 kbp. This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and the various insertion sequences. This compilation may be available in machine readable form from one of the international databanks in some future. PMID:2654890

  5. Genotypic Characterization of Egypt Enterotoxigenic Escherichia coli Isolates Expressing Coli Surface Antigen 6

    DTIC Science & Technology

    2013-02-01

    USA Abstract Introduction: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on...results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface...has received considerable attention as a target for vaccine development [11-14]. CS6 is immunogenic in humans both after natural infection and

  6. Metabolic engineering of Escherichia coli for 1-butanol production.

    PubMed

    Atsumi, Shota; Cann, Anthony F; Connor, Michael R; Shen, Claire R; Smith, Kevin M; Brynildsen, Mark P; Chou, Katherine J Y; Hanai, Taizo; Liao, James C

    2008-11-01

    Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.

  7. Functional role of bdm during flagella biogenesis in Escherichia coli.

    PubMed

    Kim, Ji-Sun; Kim, Yu Jin; Seo, Sojin; Seong, Maeng-Je; Lee, Kangseok

    2015-03-01

    The biofilm-dependent modulation gene (bdm) has recently been shown to play a role in osmotic-induced formation of biofilm in Escherichia coli. In this study, we demonstrated that deletion of bdm results in down-regulation of flagella biosynthesis genes and, consequently, a defect in E. coli motility. In addition, we employed atomic force microscopy to confirm the absence of flagella-like structures on the surface of bdm-null cells. These findings indicate that bdm plays a key role in regulatory pathway for the formation of flagella.

  8. PROPERTIES OF A BACTERIOPHAGE DERIVED FROM ESCHERICHIA COLI K235

    PubMed Central

    Jesaitis, Margeris A.; Hutton, John J.

    1963-01-01

    A temperate bacteriophage was isolated from the colicinogenic strain of Escherichia coli K235 and characterized. This phage, termed PK, is related to P2 virus morphologically, serologically, and, possibly, genetically and it bears no relationship to the T-even phages. It was also demonstrated that PK virus and colicine K differ both in their host range and in their immunological specificity, and that PK prophage does not induce the colicinogenesis in its host bacterium. It was concluded that the formation of colicine K. and PK phage in E. coli K235 are controlled by different genetic determinants. PMID:14029160

  9. Nitric oxide donor-mediated killing of bioluminescent Escherichia coli.

    PubMed Central

    Virta, M; Karp, M; Vuorinen, P

    1994-01-01

    The antimicrobial activities of two nitric oxide-releasing compounds against Escherichia coli were investigated by using recombinant E. coli cloned with a luciferase gene from Pyrophorus plagiophthalamus. Since luciferase uses intracellular ATP to generate visible light which can be measured from living cells in real time, we wanted to compare the extent to which cell viability parallels light emission. Results from luminescence measurements and CFU counts were in good agreement, and the decrease in light emission was shown to provide a rapid and more sensitive indication of cytotoxicity. PMID:7695261

  10. Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption.

    PubMed

    Richter, Katrin; Gescher, Johannes

    2014-06-01

    Escherichia coli can ferment glycerol anaerobically only under very defined restrictive conditions. Hence, it was the aim of this study to overcome this limitation via a co-cultivation approach. Anaerobic glycerol fermentation by a pure E. coli culture was compared to a co-culture that also contained the formate-oxidizing methanogen Methanobacterium formicicum. Co-cultivation of the two strains led to a more than 11-fold increased glycerol consumption. Furthermore, it supported a constantly neutral pH and a shift from ethanol to succinate production. Moreover, M. formicicum was analyzed for its ability to grow on different standard media and a surprising versatility could be demonstrated.

  11. Bacterial self-defence: how Escherichia coli evades serum killing.

    PubMed

    Miajlovic, Helen; Smith, Stephen G

    2014-05-01

    The ability to survive the bactericidal action of serum is advantageous to extraintestinal pathogenic Escherichia coli that gain access to the bloodstream. Evasion of the innate defences present in serum, including complement and antimicrobial peptides, involves multiple factors. Serum resistance mechanisms utilized by E. coli include the production of protective extracellular polysaccharide capsules and expression of factors that inhibit or interfere with the complement cascade. Recent studies have also highlighted the importance of structural integrity of the cell envelope in serum survival. These survival strategies are outlined in this review with particular attention to novel findings and recent insights into well-established resistance mechanisms.

  12. Escherichia coli as a model active colloid: A practical introduction.

    PubMed

    Schwarz-Linek, Jana; Arlt, Jochen; Jepson, Alys; Dawson, Angela; Vissers, Teun; Miroli, Dario; Pilizota, Teuta; Martinez, Vincent A; Poon, Wilson C K

    2016-01-01

    The flagellated bacterium Escherichia coli is increasingly used experimentally as a self-propelled swimmer. To obtain meaningful, quantitative results that are comparable between different laboratories, reproducible protocols are needed to control, 'tune' and monitor the swimming behaviour of these motile cells. We critically review the knowledge needed to do so, explain methods for characterising the colloidal and motile properties of E. coli cells, and propose a protocol for keeping them swimming at constant speed at finite bulk concentrations. In the process of establishing this protocol, we use motility as a high-throughput probe of aspects of cellular physiology via the coupling between swimming speed and the proton motive force.

  13. Inducible repair of oxidative DNA damage in Escherichia coli.

    PubMed

    Demple, B; Halbrook, J

    Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.

  14. Sedimentation and gravitational instability of Escherichia coli Suspension

    NASA Astrophysics Data System (ADS)

    Douarche, Carine; Salin, Dominique; Collaboration between Laboratory FAST; LPS Collaboration

    2016-11-01

    The successive run and tumble of Escherichia coli bacteria provides an active matter suspension of rod-like particles with a large swimming diffusion. As opposed to inactive elongated particles, this diffusion prevents clustering and instability in the gravity field. We measure the time dependent E . coli concentration profile during their sedimentation. After some hours, due to the dioxygen consumption, a motile / non-motile front forms leading to a Rayleigh-Taylor type gravitational instability. Analyzing both sedimentation and instability in the framework of active particle suspensions, we can measure the relevant bacteria hydrodynamic characteristics such as its single particle sedimentation velocity and its hindrance volume.

  15. The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

    PubMed

    Wurm, David Johannes; Veiter, Lukas; Ulonska, Sophia; Eggenreich, Britta; Herwig, Christoph; Spadiut, Oliver

    2016-10-01

    Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-D-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q s,lac) and q s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q s,lac and q s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.

  16. Advances in molecular serotyping and subtyping of Escherichia coli

    DOE PAGES

    Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong; ...

    2016-05-03

    Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtypingmore » and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsedfield gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. Furthermore, a variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.« less

  17. Gentamicin resistance among Escherichia coli strains isolated in neonatal sepsis.

    PubMed

    Hasvold, J; Bradford, L; Nelson, C; Harrison, C; Attar, M; Stillwell, T

    2013-01-01

    Neonatal sepsis is a significant cause of morbidity and mortality among term and preterm infants. Ampicillin and gentamicin are standard empiric therapy for early onset sepsis. Four cases of neonatal sepsis secondary to Escherichia coli (E. coli) found to be gentamicin resistant occurred within a five week period in one neonatal intensive care unit (NICU). To determine whether these cases could be tied to a single vector of transmission, and to more broadly evaluate the incidence of gentamicin resistant strains of E. coli in the neonatal population at our institution compared to other centers, we reviewed the charts of the four neonates (Infants A through D) and their mothers. The E. coli isolates were sent for Pulse Field Gel Electrophoresis (PFGE) to evaluate for genetic similarity between strains. We also reviewed all positive E. coli cultures from one NICU over a two year period. Infants A and B had genetically indistinguishable strains which matched that of urine and placental cultures of Infant B's mother. Infant C had a genetically distinct organism. Infant D, the identical twin of Infant C, did not have typing performed. Review of all cultures positive for E. coli at our institution showed a 12.9 percent incidence of gentamicin-resistance. A review of other studies showed that rates of resistance vary considerably by institution. We conclude that gentamicin-resistant E. coli is a relatively uncommon cause of neonatal sepsis, but should remain a consideration in patients who deteriorate despite initiation of empiric antibiotics.

  18. Inactivation of Escherichia coli using atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Kuwahata, Hiroshi; Yamaguchi, Takeshi; Ohyama, Ryu-ichiro; Ito, Atsushi

    2015-01-01

    An atmospheric-pressure argon (Ar) plasma jet was applied to the inactivation of Escherichia coli. The Ar plasma jet was generated at a frequency of 10 kHz, an applied voltage of 10 kV, and an Ar gas flow rate of 10 L/min at atmospheric pressure. E. coli cells seeded on an agar medium in a Petri dish were inactivated by Ar plasma jet irradiation for 1 s. Scanning electron microscopy (SEM) revealed that E. coli cells were killed because their cell wall and membrane were disrupted. To determine the causes of the disruption of the cell wall and membrane of E. coli, we performed the following experiments: the measurement of the surface temperature of an agar medium using a thermograph, the analysis of an emission spectrum of a plasma jet obtained using a multichannel spectrometer, and the determination of the distribution of the concentration of hydrogen peroxide (H2O2) generated on an agar medium by plasma jet irradiation using semiquantitative test strips. Moreover, H2O2 solutions of different concentrations were dropped onto an agar medium seeded with E. coli cells to examine the contribution of H2O2 to the death of E. coli. The results of these experiments showed that the cell wall and membrane of E. coli were disrupted by electrons in the plasma jet, as well as by electroneutral excited nitrogen molecules (N2) and hydroxyl (OH) radicals in the periphery of the plasma jet.

  19. Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli.

    PubMed

    Chang, Y Y; Kennedy, E P

    1967-09-01

    An enzyme (L-glycerol 3-phosphate: CMP phosphatidyltransferase) catalyzing the synthesis of phosphatidyl glycerophosphate from CDP-diglyceride and L-glycerol 3-phosphate has been rendered soluble by treatment of the particulate, membrane-containing fraction of E. coli with Triton X-100 and has been partially purified. The enzyme, devoid of phosphatidyl glycerophosphatase activity, is specific for L-glycerol 3-phosphate and is completely dependent upon added Mg(++) or Mn(++) for activity. It has high affinity for CDP-diglyceride and can be used for the assay of this nucleotide. Other properties of the enzyme are also described.

  20. Growth and Division of Filamentous Forms of Escherichia coli.

    PubMed

    Adler, H I; Hardigree, A A

    1965-07-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.

  1. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua.

    PubMed

    Tajkarimi, Mehrdad; Harrison, Scott H; Hung, Albert M; Graves, Joseph L

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.

  2. Enterotoxigenic Escherichia coli infection in captive black-footed ferrets.

    PubMed

    Bradley, G A; Orr, K; Reggiardo, C; Glock, R D

    2001-07-01

    Enterotoxigenic Escherichia coli with genes for heat stabile toxins Sta and STb was isolated from the gastrointestinal tract and multiple visceral organs of three adult and three juvenile black-footed ferrets (Mustela nigripes) that died in a captive breeding colony between 24 May 1998 and 2 July 1998. Similar isolates were obtained from rectal swabs of one adult and one juvenile that were clinically ill. All were fed a diet composed of mink chow, raw rabbit meat, beef liver powder, blood meal and lard. Escherichia coli of the same toxin genotype was isolated from the mixed ration. Clinical signs included sudden death, dehydration, anorexia and diarrhea. Necropsy lesions included acute enteritis with large numbers of rod shaped bacteria microscopically visible on intestinal villi.

  3. Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

    PubMed Central

    Tajkarimi, Mehrdad; Harrison, Scott H.; Hung, Albert M.; Graves, Joseph L.

    2016-01-01

    A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism. PMID:26914334

  4. Thiolases of Escherichia coli: purification and chain length specificities.

    PubMed Central

    Feigenbaum, J; Schulz, H

    1975-01-01

    The presence of only one thiolase (EC 2.3.1.9) in wild-type Escherichia coli induced for enzymes of beta oxidation was demonstrated. A different thiolase was shown to be present in a mutant constitutive for the enzymes of butyrate degradation. The two thiolases were purified to near homogeneity by a simple two-step procedure and were found to be associated with different proteins as shown by gel electrophoresis. The thiolase isolated from induced wild-type Escherichia coli cell was active on beta-ketoacyl-coenzyme A derivatives containing 4 to 16 carbons, but exhibited optimal activity with medium-chain substrates. In contrast, the thiolase isolated from the constitutive mutant was shown to be specific for acetoacetyl-coenzyme A. PMID:236278

  5. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI III.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. III. Requirements for enzyme synthesis. J. Bacteriol. 84:996–1006. 1962.—The requirements for the formation of tryptophanase and tryptophan synthetase in Escherichia coli during repression release were studied. The kinetics of the formation of tryptophan synthetase differed in the two strains examined; this was attributed to differences in the endogenous level of tryptophan in the bacterial cells. The formation of both enzymes was inhibited by chloramphenicol, and by the absence of arginine in an arginine-requiring mutant. These results are indicative of a requirement for protein synthesis for enzyme formation. Requirements for nucleic acid synthesis were examined by use of a uracil- and thymine-requiring mutant, and with purine and pyrimidine analogues. The results obtained suggest that some type of ribonucleic acid synthesis was necessary for the formation of tryptophanase and tryptophan synthetase. PMID:13959620

  6. Shear alters motility of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Molaei, Mehdi; Jalali, Maryam; Sheng, Jian

    2013-11-01

    Understanding of locomotion of microorganisms in shear flows drew a wide range of interests in microbial related topics such as biological process including pathogenic infection and biophysical interactions like biofilm formation on engineering surfaces. We employed microfluidics and digital holography microscopy to study motility of E. coli in shear flows. We controlled the shear flow in three different shear rates: 0.28 s-1, 2.8 s-1, and 28 s-1 in a straight channel with the depth of 200 μm. Magnified holograms, recorded at 15 fps with a CCD camera over more than 20 minutes, are analyzed to obtain 3D swimming trajectories and subsequently used to extract shear responses of E.coli. Thousands of 3-D bacterial trajectories are tracked. The change of bacteria swimming characteristics including swimming velocity, reorientation, and dispersion coefficient are computed directly for individual trajectory and ensemble averaged over thousands of realizations. The results show that shear suppresses the bacterial dispersions in bulk but promote dispersions near the surface contrary to those in quiescent flow condition. Ongoing analyses are focusing to quantify effect of shear rates on tumbling frequency and reorientation of cell body, and its implication in locating the hydrodynamic mechanisms for shear enhanced angular scattering. NIH, NSF, GoMRI.

  7. Some factors affecting cyclopropane acid formation in Escherichia coli

    PubMed Central

    Knivett, V. A.; Cullen, Julia

    1965-01-01

    1. The fatty acid composition of the extractable lipids of Escherichia coli varied with growth conditions. 2. The principal fatty acids were palmitic acid, hexadecenoic acid, octadecenoic acid and the cyclopropane acids, methylenehexadecanoic acid and methyleneoctadecanoic acid. 3. Cyclopropane acid formation from monoenoic acids was increased by acid media, poor oxygen supply, or high growth temperature. 4. Cyclopropane acid formation was decreased by alkaline media, well oxygenated conditions, the presence of citrate, or lack of Mg2+. PMID:5324304

  8. Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.

    PubMed

    Watanabe, Jun; Tanaka, Hisaki; Akagawa, Takumi; Mogi, Yoshinobu; Yamazaki, Tatsuo

    2007-10-01

    To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.

  9. Polymorphous crystallization and diffraction of threonine deaminase from Escherichia coli.

    PubMed

    Gallagher, D T; Eisenstein, E; Fisher, K E; Zondlo, J; Chinchilla, D; Yu, H D; Dill, J; Winborne, E; Ducote, K; Xiao, G; Gilliland, G L

    1998-05-01

    The biosynthetic threonine deaminase from Escherichia coli, an allosteric tetramer with key regulatory functions, has been crystallized in several crystal forms. Two distinct forms, both belonging to either space group P3121 or P3221, with different sized asymmetric units that both contain a tetramer, grow under identical conditions. Diffraction data sets to 2.8 A resolution (native) and 2. 9 A resolution (isomorphous uranyl derivative) have been collected from a third crystal form in space group I222.

  10. Positive regulation of the Escherichia coli glycine cleavage enzyme system.

    PubMed Central

    Wilson, R L; Steiert, P S; Stauffer, G V

    1993-01-01

    A new mutation in Escherichia coli, designated gcvA1, that results in noninducible expression of both gcv and a gcvT-lacZ gene fusion was isolated. A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression. These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv. PMID:8423160

  11. Division pattern of a round mutant of Escherichia coli.

    PubMed Central

    Cooper, S

    1997-01-01

    A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

  12. Antibacterial efficacy of silver nanoparticles against Escherichia coli

    NASA Astrophysics Data System (ADS)

    Pattabi, Rani M.; Thilipan, G. Arun Kumar; Bhat, Vinayachandra; Sridhar, K. R.; Pattabi, Manjunatha

    2013-02-01

    Silver nanoparticles (AgNPs) synthesized by subjecting an aqueous solution of AgNO3 and polyvinyl alcohol to irradiation from an UV lamp has been studied for its antibacterial potential against Gram-negative bacteria (Escherichia coli). The diameter of the zone of inhibition is found to depend on both the irradiation time and the nanoparticle concentration. As the synthesis method adopted uses no toxic reagents, these particles may serve as promising candidates in the search for better antibacterial agents.

  13. Electric field induced bacterial flocculation of Enteroaggregative Escherichia coli 042

    SciTech Connect

    Kumar, Aloke; Mortensen, Ninell P; Mukherjee, Partha P; Retterer, Scott T; Doktycz, Mitchel John

    2011-01-01

    A response of the aggregation dynamics of enteroaggregative Escherichia coli under low magnitude steady and oscillating electric fields is presented. The presence of uniform electric fields hampered microbial adhesion and biofilm formation on a transverse glass surface, but instead promoted the formation of flocs. Extremely heterogeneous distribution of live and dead cells was observed among the flocs. Moreover, floc formation was largely observed to be independent of the frequency of alternating electric fields.

  14. Role for the female in bacterial conjugation in Escherichia coli.

    PubMed

    Freifelder, D

    1967-08-01

    Hfr and F' Lac male strains of Escherichia coli were mated with purine-requiring females which had been starved for purine. These females formed mating pairs with the males. However, a mating in the absence of purine markedly reduced the yield of recombinants. Transfer of F' Lac or of lambda prophage also occurred infrequently. It was concluded that deoxyribonucleic acid transfer from male to female requires some, as yet unknown, function of the female.

  15. Role for the Female in Bacterial Conjugation in Escherichia coli

    PubMed Central

    Freifelder, David

    1967-01-01

    Hfr and F′ Lac male strains of Escherichia coli were mated with purine-requiring females which had been starved for purine. These females formed mating pairs with the males. However, a mating in the absence of purine markedly reduced the yield of recombinants. Transfer of F′ Lac or of λ prophage also occurred infrequently. It was concluded that deoxyribonucleic acid transfer from male to female requires some, as yet unknown, function of the female. PMID:5341864

  16. Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli.

    PubMed

    Kong, Haishen; Hong, Xiaoping; Li, Xuefen

    2015-08-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen that causes acute and persistent diarrhea in children and adults. While the pathogenic mechanisms of EAEC intestinal colonization have been uncovered (including bacterial adhesion, enterotoxin and cytotoxin secretion, and stimulation of mucosal inflammation), those of severe extraintestinal infections remain largely unknown. The recent emergence of multidrug resistant EAEC represents an alarming public health threat and clinical challenge, and research on the molecular mechanisms of resistance is urgently needed.

  17. Lipophilic chelator inhibition of electron transport in Escherichia coli.

    PubMed Central

    Crane, R T; Sun, I L; Crane, F L

    1975-01-01

    The lipophilic chelator bathophenanthroline inhibits electron transport in membranes from Escherichia coli. The less lipophilic 1,10-phenanthroline, bathophenanthroline sulfonate, and alpha,alpha-dipyridyl have little effect. Reduced nicotinamide adenine dinucleotide oxidase is more sensitive to bathophenanthroline inhibition than lactate oxidase activity. Evidence for two sites of inhibition comes from the fact that both reduced nicotinamide adenine dinucleotide menadione reductase and duroquinol oxidase activities are inhibited. Addition of uncouplers of phosphorylation before bathophenanthroline protects against inhibition. PMID:1092663

  18. Effects of Acridine Orange on the Growth of Escherichia coli

    PubMed Central

    Southwick, Frederick S.; Carr, Howard S.; Carden, George A.; D'Alisa, Rose M.; Rosenkranz, Herbert S.

    1972-01-01

    Exposure of Escherichia coli to critical acridine orange (AO) concentrations did not result in loss of viability. However, the deoxyribonucleic acid (DNA) of cells exposed to such agents was rapidly degraded and repolymerized. On the other hand, a bacterium deficient in DNA repair (pol A1−, lacking DNA polymerase) was sensitive to the action of AO. The DNA of such cells was also degraded but it was not repaired. PMID:4553001

  19. Two Forms of d-Glycerate Kinase in Escherichia coli

    PubMed Central

    Ornston, M. K.; Ornston, L. N.

    1969-01-01

    Escherichia coli K-12 synthesizes two chromatographically distinct forms of glycerate kinase which differ both in their thermolability and in the dependence of their activity upon pH. One enzymatic form, GK I, is found in cells grown with glycerate, glucarate, or glycolate. Of these compounds, glycolate is the only carbon source that elicits the synthesis of the second enzymatic form, GK II. PMID:4887503

  20. Preparation of Soluble Proteins from Escherichia coli

    PubMed Central

    Wingfield, Paul T.

    2014-01-01

    Purification of human IL-1β is used in this unit as an example of the preparation of soluble proteins from E. coli. Bacteria containing IL-1β are lysed, and IL-1 β in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation and cation-exchange chromatography, and then concentrated. Finally, the IL-1 β protein is applied to a gel-filtration column to separate it from remaining higher- and lower-molecular-weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. Also, the purification procedure serves as an example of how use classical protein purifications methods which may also be used in conjunction with the affinity-based methods now more commonly used. PMID:25367009

  1. Fluorogenic assays for immediate confirmation of Escherichia coli.

    PubMed Central

    Feng, P C; Hartman, P A

    1982-01-01

    Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains). Images PMID:7049088

  2. Prevalence of Escherichia coli in apple cider manufactured in Connecticut.

    PubMed

    Dingman, D W

    1999-06-01

    Cider samples obtained from 11 cider mills operating in Connecticut during the 1997 to 1998 production season were tested for the presence of Escherichia coli. Cider production began in mid August and continued through March, with peak production in September and October. Of 314 cider samples tested, 11 (4%) were found to contain E. coli. Of the 11 mills, 6 (55%) tested positive for E. coli in the cider at least once during the production year. E. coli was first observed in cider samples produced in mid to late October and was not detected in samples made after January. A trend was observed for cider to decrease in acidity and increase in Brix (soluble sugars) throughout the production season. No correlation between pH and soluble sugars of cider and the presence of E. coli was detected. Eight mills used both dropped apples and tree-picked apples, whereas three mills used tree-picked apples only. The use of dropped apples in cider production began 5 weeks before the first detection of E. coli in cider. E. coli was isolated from cider samples produced using dropped apples and from samples produced using only tree-picked apples. No direct correlation between the use of dropped apples or tree-picked apples and the presence of E. coli in the cider was observed. An association between the time of apple harvest and the appearance of E. coli in cider was noted. For mills providing adequate records, all contaminated cider was produced from apples harvested between mid October and mid November.

  3. Thymineless Death in Escherichia coli: Inactivation and Recovery

    PubMed Central

    Cummings, Donald J.; Kusy, Alvin R.

    1969-01-01

    The effects of chloramphenicol (CAP) on the progress of thymineless death (TLD), nalidixic acid (NA) inactivation, ultraviolet (UV) irradiation, and mitomycin C (MC) inactivation were studied in Escherichia coli B, Bs−1, Bs−3, Bs−12, and B/r. This was done before, during, and after inactivation. During the progress of inactivation, it was found that at 10 to 20 μg of CAP per ml, up to 50% of the UV-sensitive bacteria survived TLD and about 10% survived NA. In E. coli B/r, at these concentrations of CAP, about 10 to 15% of the cells survived TLD and about 20 to 25% survived NA. Concentrations of CAP greater than 25 μg/ml actually increased the sensitivity of E. coli B, Bs−1, Bs−3, and Bs−12 to inactivation by either TLD or NA; at 150 μg of CAP per ml, the sensitivity of E. coli B/r to inactivation also increased. When E. coli B cells were incubated in CAP prior to inactivation, the longer the preincubation the longer onset of TLD was delayed; NA inactivation was also affected in that the rate of inactivation after CAP incubation was greatly decreased. Preincubation of E. coli B/r with CAP had much less effect on the progress of inactivation. After thymineless death, incubation in CAP plus thymine led to a rapid and almost complete recovery of E. coli B and Bs−12. Lesser recoveries were observed after inactivation due to UV, NA, or MC inactivation. E. coli Bs−1 and B/r did not recover viability after any mode of inactivation, and E. coli Bs−3 and Bs−12 recovered from UV to about 20% of the initial titer. It was suggested that protein synthesis, in particular proteins involved in deoxyribonucleic synthesis, was a determining factor in these inactivating and recovery events. PMID:4897115

  4. Interaction of Escherichia coli and Soil Particles in Runoff

    PubMed Central

    Muirhead, Richard William; Collins, Robert Peter; Bremer, Philip James

    2006-01-01

    A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (>45 μm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (<2 μm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix. PMID:16672484

  5. Measuring Escherichia coli Gene Expression during Human Urinary Tract Infections

    PubMed Central

    Mobley, Harry L. T.

    2016-01-01

    Extraintestinal Escherichia coli (E. coli) evolved by acquisition of pathogenicity islands, phage, plasmids, and DNA segments by horizontal gene transfer. Strains are heterogeneous but virulent uropathogenic isolates more often have specific fimbriae, toxins, and iron receptors than commensal strains. One may ask whether it is the virulence factors alone that are required to establish infection. While these virulence factors clearly contribute strongly to pathogenesis, bacteria must survive by metabolizing nutrients available to them. By constructing mutants in all major metabolic pathways and co-challenging mice transurethrally with each mutant and the wild type strain, we identified which major metabolic pathways are required to infect the urinary tract. We must also ask what else is E. coli doing in vivo? To answer this question, we examined the transcriptome of E. coli CFT073 in the murine model of urinary tract infection (UTI) as well as for E. coli strains collected and analyzed directly from the urine of patients attending either a urology clinic or a university health clinic for symptoms of UTI. Using microarrays and RNA-seq, we measured in vivo gene expression for these uropathogenic E. coli strains, identifying genes upregulated during murine and human UTI. Our findings allow us to propose a new definition of bacterial virulence. PMID:26784237

  6. Regulation of arabinose and xylose metabolism in Escherichia coli.

    PubMed

    Desai, Tasha A; Rao, Christopher V

    2010-03-01

    Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.

  7. Escherichia coli sequence type 131: epidemiology and challenges in treatment.

    PubMed

    Qureshi, Zubair A; Doi, Yohei

    2014-05-01

    Escherichia coli ST131 has emerged as a global epidemic, multidrug-resistant clone of E. coli causing extra-intestinal infections. It is now highly prevalent among fluoroquinolone-resistant and CTX-M ESBL-producing E. coli isolates worldwide. Humans are likely the primary reservoir of ST131. Factors associated with its acquisition include residence in long-term care facilities and recent receipt of antimicrobial agents. E. coli ST131 causes a wide array of infections ranging from cystitis to life-threatening sepsis. Fluoroquinolones and trimethoprim-sulfamethoxazole are no longer adequate options for empiric therapy when E. coli ST131 is suspected from risk factors and local epidemiology. Expanded-spectrum cephalosporins, piperacillin-tazobactam and carbapenems are options to treat serious non-ESBL-producing E. coli ST131 infections, while carbapenems are indicated for ESBL-producing infections. There is a growing interest in reevaluating oral agents including fosfomycin and pivmecillinam for less serious infections such as uncomplicated cystitis.

  8. Characterization of Shiga toxigenic Escherichia coli isolated from foods.

    PubMed

    Martínez, Aida Juliana; Bossio, Carolina Paba; Durango, Adriana Coral; Vanegas, Maria Consuelo

    2007-12-01

    The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.

  9. [Escherichia coli, a pathogen under fire from the news].

    PubMed

    Cohen, R; Raymond, J; Gendrel, D; Bingen, E

    2012-11-01

    Escherichia coli is both a gastrointestinal tract commensal and a major pathogen. In recent years, E. coli is under fire from the news due to a better understanding of pathogenic factors, outbreaks of infections caused by enterohaemorrhagic strains, and last but not least, the worrying development of antibiotic resistance. Due to the absence of new compounds active against these strains, producing extended-spectrum ß-lactamases (ESBL) and frequently multiresistant to other antibiotics, their emergence will pose therapeutic problems for practitioners of all pediatric specialties. The gold standard treatment for severe infections due to ESBL-E. coli family is the penem class. The frequent use of penems promotes the emergence of strains resistant to carbapenems. Sparing carbapenems should be a clear objective for non life-threatening infections.

  10. Functions of the gene products of Escherichia coli.

    PubMed Central

    Riley, M

    1993-01-01

    A list of currently identified gene products of Escherichia coli is given, together with a bibliography that provides pointers to the literature on each gene product. A scheme to categorize cellular functions is used to classify the gene products of E. coli so far identified. A count shows that the numbers of genes concerned with small-molecule metabolism are on the same order as the numbers concerned with macromolecule biosynthesis and degradation. One large category is the category of tRNAs and their synthetases. Another is the category of transport elements. The categories of cell structure and cellular processes other than metabolism are smaller. Other subjects discussed are the occurrence in the E. coli genome of redundant pairs and groups of genes of identical or closely similar function, as well as variation in the degree of density of genetic information in different parts of the genome. PMID:7508076

  11. Incidence of Escherichia coli in Black Walnut Meats

    PubMed Central

    Meyer, Melvin T.; Vaughn, Reese H.

    1969-01-01

    Examination of commercially shelled black walnut meats showed inconsistent numbers of total aerobic bacteria, coliforms, and Escherichia coli; variation occurred among different meat sizes and within each meat size. The incidence of E. coli on meats of commercially hulled black walnuts depended on the physical condition of the nuts. Apparently tightly sealed ones contained only a few or none, whereas those with visibly separated sutures and spoiled meats yielded the most. This contamination was in part correlated to a hulling operation. Large numbers of E. coli on the husk of the walnuts contaminated the hulling water, subsequently also contaminating the meats by way of separated sutures. Chlorination of the hulling wash water was ineffective. Attempts were made to decontaminate the walnut meats without subsequent deleterious changes in flavor or texture. A treatment in coconut oil at 100 C followed by removal of excess surface oil by centrifugation was best. PMID:4905608

  12. Quantitative method for enumeration of enterotoxigenic Escherichia coli.

    PubMed Central

    Calderon, R L; Levin, M A

    1981-01-01

    A rapid method was developed to quantify toxigenic Escherichia coli, using a membrane filter procedure. After filtration of samples, the membrane filter was first incubated on a medium selective for E. coli (24 h, 44 degrees C) and then transferred to tryptic soy agar (3%; 6 h, 37 degrees C). To assay for labile toxin-producing colonies, the filter was then transferred to a monolayer of Y-1 cells, the E. coli colonies were marked on the bottom of the petri dish, and the filter was removed after 15 min. The monolayer was observed for a positive rounding effect after a 15- to 24-h incubation. The method has an upper limit of detecting 30 toxigenic colonies per plate and can detect as few as one toxigenic colony per plate. A preliminary screening for these enterotoxigenic strains in polluted waters and known positive fecal samples was performed, and positive results were obtained with fecal samples only. PMID:7007415

  13. Mechanisms of the radioprotective effect of cysteamine in Escherichia coli

    SciTech Connect

    Korystov, Yu.N.; Vexler, F.B.

    1988-06-01

    The values of the oxygen effect (m) and the maximal protective effect of cysteamine (DMF*) were estimated for four Escherichia coli strains: AB1157 (wild type), AB1886 (uvrA), AB2463 (recA), and p3478 (polA). A correlation made between DMF* and m as well as the kinetics of the increase of DMF with oxygen depletion showed that the protective effect of cysteamine is realized by three mechanisms: (i) anoxia achieved by oxygen reduction, with the DMF varying from 2.2 to 4.2 for different E. coli strains (this protection is the major contribution to the entire mechanism); (ii) lowering of the indirect radiation effect; i.e., for 50 mM cysteamine DMF does not exceed 1.1; and (iii) increase of the efficiency of enzymatic repair. The latter effect of cysteamine is registered only with the wild-type E. coli, the DMF being not less than 1.4.

  14. Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli

    PubMed Central

    Laganenka, Leanid; Colin, Remy; Sourjik, Victor

    2016-01-01

    Bacteria communicate by producing and sensing extracellular signal molecules called autoinducers. Such intercellular signalling, known as quorum sensing, allows bacteria to coordinate and synchronize behavioural responses at high cell densities. Autoinducer 2 (AI-2) is the only known quorum-sensing molecule produced by Escherichia coli but its physiological role remains elusive, although it is known to regulate biofilm formation and virulence in other bacterial species. Here we show that chemotaxis towards self-produced AI-2 can mediate collective behaviour—autoaggregation—of E. coli. Autoaggregation requires motility and is strongly enhanced by chemotaxis to AI-2 at physiological cell densities. These effects are observed regardless whether cell–cell interactions under particular growth conditions are mediated by the major E. coli adhesin (antigen 43) or by curli fibres. Furthermore, AI-2-dependent autoaggregation enhances bacterial stress resistance and promotes biofilm formation. PMID:27687245

  15. Engineering Escherichia coli K12 MG1655 to use starch

    PubMed Central

    2014-01-01

    Background To attain a sustainable bioeconomy, fuel, or valuable product, production must use biomass as substrate. Starch is one of the most abundant biomass resources and is present as waste or as a food and agroindustry by-product. Unfortunately, Escherichia coli, one of the most widely used microorganisms in biotechnological processes, cannot use starch as a carbon source. Results We engineered an E. coli strain capable of using starch as a substrate. The genetic design employed the native capability of the bacterium to use maltodextrins as a carbon source plus expression and secretion of its endogenous α-amylase, AmyA, in an adapted background. Biomass production improved using 35% dissolved oxygen and pH 7.2 in a controlled bioreactor. Conclusion The engineered E. coli strain can use starch from the milieu and open the possibility of optimize the process to use agroindustrial wastes to produce biofuels and other valuable chemicals. PMID:24886307

  16. Effect of tannins on the in viro growth of Escherichia coli O157:H7 and in vivo growth of generic Escherichia coli excreted from steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement o...

  17. Bioremediation of trace cobalt from simulated spent decontamination solutions of nuclear power reactors using E. coli expressing NiCoT genes.

    PubMed

    Raghu, G; Balaji, V; Venkateswaran, G; Rodrigue, A; Maruthi Mohan, P

    2008-12-01

    Removal of radioactive cobalt at trace levels (approximately nM) in the presence of large excess (10(6)-fold) of corrosion product ions of complexed Fe, Cr, and Ni in spent chemical decontamination formulations (simulated effluent) of nuclear reactors is currently done by using synthetic organic ion exchangers. A large volume of solid waste is generated due to the nonspecific nature of ion sorption. Our earlier work using various fungi and bacteria, with the aim of nuclear waste volume reduction, realized up to 30% of Co removal with specific capacities calculated up to 1 microg/g in 6-24 h. In the present study using engineered Escherichia coli expressing NiCoT genes from Rhodopseudomonas palustris CGA009 (RP) and Novosphingobium aromaticivorans F-199 (NA), we report a significant increase in the specific capacity for Co removal (12 microg/g) in 1-h exposure to simulated effluent. About 85% of Co removal was achieved in a two-cycle treatment with the cloned bacteria. Expression of NiCoT genes in the E. coli knockout mutant of NiCoT efflux gene (rcnA) was more efficient as compared to expression in wild-type E. coli MC4100, JM109 and BL21 (DE3) hosts. The viability of the E. coli strains in the formulation as well as at different doses of gamma rays exposure and the effect of gamma dose on their cobalt removal capacity are determined. The potential application scheme of the above process of bioremediation of cobalt from nuclear power reactor chemical decontamination effluents is discussed.

  18. Effective medicinal plants against enterohaemorrhagic Escherichia coli O157:H7.

    PubMed

    Voravuthikunchai, Supayang; Lortheeranuwat, Amornrat; Jeeju, Wanpen; Sririrak, Trechada; Phongpaichit, Souwalak; Supawita, Thanomjit

    2004-09-01

    The stimulating effect of subinhibitory concentrations of antibiotics on the production of verocytotoxin (VT) by enterohaemorrhagic Escherichia coli (EHEC) O157:H7 has been claimed. The purpose of this study was to find an alternative, but bioactive medicine for the treatment of this organism. Fifty-eight preparations of aqueous and ethanolic extracts of 38 medicinal plant species commonly used in Thailand to cure gastrointestinal infections were tested for their antibacterial activity against different strains of Escherichia coli, including 6 strains of Escherichia coli O157:H7, Escherichia coli O26:H11, Escherichia coli O111:NM, Escherichia coli O22; 5 strains of Escherichia coli isolated from bovine; and Escherichia coli ATCC 25922. Inhibition of growth was primarily tested by the paper disc agar diffusion method. Among the medicinal plants tested, only 8 species (21.05%) exhibited antimicrobial activity against Escherichia coli O157:H7. Acacia catechu, Holarrhena antidysenterica, Peltophorum pterocarpum, Psidium guajava, Punica granatum, Quercus infectoria, Uncaria gambir, and Walsura robusta demonstrated antibacterial activity with inhibition zones ranging from 7 to 17 mm. The greatest inhibition zone against Escherichia coli O157:H7 (RIMD 05091083) was produced from the ethanolic extract of Quercus infectoria. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the agar microdilution method and agar dilution method in petri dishes with millipore filter. Both aqueous and ethanolic extracts of Quercus infectoria and aqueous extract of Punica granatum were highly effective against Escherichia coli O157:H7 with the best MIC and MBC values of 0.09, 0.78, and 0.19, 0.39 mg/ml, respectively. These plant species may provide alternative but bioactive medicines for the treatment of Escherichia coli O157:H7 infection.

  19. Soil solarization reduces Escherichia coli O157:H7 and total Escherichia coli on cattle feedlot pen surfaces.

    PubMed

    Berry, Elaine D; Wells, James E

    2012-01-01

    Feedlot pen soil is a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM). A feedlot pen was identified in which naturally occurring E. coli O157:H7 was prevalent and evenly distributed in the FSM. Forty plots 3 by 3 m were randomly assigned such that five plots of each of the solarization times of 0, 1, 2, 3, 4, 6, 8, and 10 weeks were examined. Temperature loggers were placed 7.5 cm below the surface of each plot, and plots to be solarized were covered with clear 6-mil polyethylene. At each sampling time, five FSM samples were collected from each of five solarized and five unsolarized plots. E. coli concentrations and E. coli O157:H7 presence by immunomagnetic separation and plating were determined for each FSM sample. Initial percentages of E. coli O157:H7-positive samples in control and solarized FSM were 84 and 80%, respectively, and did not differ (P > 0.05). E. coli O157:H7 was no longer detectable by 8 weeks of solarization, but was still detected in unsolarized FSM at 10 weeks. The average initial concentration of E. coli in FSM was 5.56 log CFU/g and did not differ between treatments (P > 0.05). There was a 2.0-log decrease of E. coli after 1 week of solarization, and a >3.0-log reduction of E. coli by week 6 of solarization (P, 0.05). E. coli levels remained unchanged in unsolarized FSM (P > 0.05). Daily peak FSM temperatures were on average 8.7°C higher for solarized FSM compared with unsolarized FSM, and reached temperatures as high as 57°C. Because soil solarization reduces E. coli O157:H7, this technique may be useful for reduction of persistence and transmission of this pathogen in cattle production, in addition to remediation of E. coli O157:H7-contaminated soil used to grow food crops.

  20. 77 FR 26725 - Changes to FSIS Traceback, Recall Procedures for Escherichia coli O157:H7 Positive Raw Beef...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-07

    ... Food Safety and Inspection Service Changes to FSIS Traceback, Recall Procedures for Escherichia coli... find raw ground beef presumptive positive for Escherichia coli (E. coli) O157:H7. This methodology will... Escherichia coli O157:H7'' and requested comments on these documents. FSIS also held a public meeting...

  1. Antibiotic Resistance in Urinary Isolates of Escherichia coli

    PubMed Central

    Abduzaimovic, Amila; Aljicevic, Mufida; Rebic, Velma; Vranic, Sabina Mahmutovic; Abduzaimovic, Kadrija; Sestic, Sabina

    2016-01-01

    Objectives: The aim of this study was to examine the presence of antimicrobial resistance / susceptibility strains of Escherichia coli in inpatients and outpatients. Materials and methods: It is a retrospective study carried out at the Department of Microbiology, Parasitology and Virology Faculty of Medicine, University of Sarajevo. In cooperation with the Microbiological laboratory of the Cantonal Hospital Zenica and the Microbiological laboratory of the General Hospital Tesanj, 3863 urine samples were processed in the period from March 1st to March 31st 2016. Results: Our study showed that E. coli had the highest antimicrobial resistance to trimethoprim / sulfamethoxazole (38.61%), followed by amoxicillin / clavulanic acid (19.62%), ciprofloxacin (9.49%), gentamicin (8.86%), cephalexin (8.23%), nitrofurantoin (8.23%), cefuroxime (7.52%), ceftazidime (6.33%), cefuroxime (89.87%), amikacin (4.43%). Conclusions: The isolated strains of E. coli showed the highest resistance to trimethoprim / sulfamethoxazole and amoxicillin / clavulanic acid. The isolated strains of E. coli showed the greatest susceptibility to amikacin and ceftazidime. Gender distribution of positive E. coli isolates showed statistically significant differences in favor of females. PMID:28144190

  2. Antibiotic Resistance of Escherichia coli Serotypes from Cochin Estuary

    PubMed Central

    Sukumaran, Divya P.; Durairaj, Srinivasan; Abdulla, Mohamed Hatha

    2012-01-01

    This study aimed at detecting the prevalence of antibiotic-resistant serotypes of Escherichia coli in Cochin estuary, India. E. coli strains were isolated during the period January 2010–December 2011 from five different stations set at Cochin estuary. Water samples from five different stations in Cochin estuary were collected on a monthly basis for a period of two years. Isolates were serotyped, antibiogram-phenotyped for twelve antimicrobial agents, and genotyped by polymerase chain reaction for uid gene that codes for β-D-glucuronidase. These E. coli strains from Cochin estuary were tested against twelve antibiotics to determine the prevalence of multiple antibiotic resistance among them. The results revealed that more than 53.33% of the isolates were multiple antibiotic resistant. Thirteen isolates showed resistance to sulphonamides and two of them contained the sul 1 gene. Class 1 integrons were detected in two E. coli strains which were resistant to more than seven antibiotics. In the present study, O serotyping, antibiotic sensitivity, and polymerase chain reaction were employed with the purpose of establishing the present distribution of multiple antibiotic-resistant serotypes, associated with E. coli isolated from different parts of Cochin estuary. PMID:23008708

  3. Escherichia coli ST131, an Intriguing Clonal Group

    PubMed Central

    Bertrand, Xavier; Madec, Jean-Yves

    2014-01-01

    SUMMARY In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum β-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131. PMID:24982321

  4. Recent Advances in Understanding Enteric Pathogenic Escherichia coli

    PubMed Central

    Croxen, Matthew A.; Law, Robyn J.; Scholz, Roland; Keeney, Kristie M.; Wlodarska, Marta

    2013-01-01

    SUMMARY Although Escherichia coli can be an innocuous resident of the gastrointestinal tract, it also has the pathogenic capacity to cause significant diarrheal and extraintestinal diseases. Pathogenic variants of E. coli (pathovars or pathotypes) cause much morbidity and mortality worldwide. Consequently, pathogenic E. coli is widely studied in humans, animals, food, and the environment. While there are many common features that these pathotypes employ to colonize the intestinal mucosa and cause disease, the course, onset, and complications vary significantly. Outbreaks are common in developed and developing countries, and they sometimes have fatal consequences. Many of these pathotypes are a major public health concern as they have low infectious doses and are transmitted through ubiquitous mediums, including food and water. The seriousness of pathogenic E. coli is exemplified by dedicated national and international surveillance programs that monitor and track outbreaks; unfortunately, this surveillance is often lacking in developing countries. While not all pathotypes carry the same public health profile, they all carry an enormous potential to cause disease and continue to present challenges to human health. This comprehensive review highlights recent advances in our understanding of the intestinal pathotypes of E. coli. PMID:24092857

  5. The Escherichia coli Proteome: Past, Present, and Future Prospects†

    PubMed Central

    Han, Mee-Jung; Lee, Sang Yup

    2006-01-01

    Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

  6. Fumarate-Mediated Persistence of Escherichia coli against Antibiotics

    PubMed Central

    Kim, Jun-Seob; Cho, Da-Hyeong; Heo, Paul; Jung, Suk-Chae; Park, Myungseo; Oh, Eun-Joong; Sung, Jaeyun; Kim, Pan-Jun; Lee, Suk-Chan; Lee, Dae-Hee; Lee, Sarah; Lee, Choong Hwan; Shin, Dongwoo

    2016-01-01

    Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) in Escherichia coli led to a higher frequency of persister formation. The persister frequency of E. coli was increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-related hipA7 mutation indicated that surplus fumarate markedly elevated the E. coli persister frequency. An E. coli strain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears that SDH and FRD represent a paired system that gives rise to and maintains E. coli persisters by producing and utilizing fumarate, respectively. PMID:26810657

  7. Escherichia coli β-Lactamases: What Really Matters

    PubMed Central

    Bajaj, Priyanka; Singh, Nambram S.; Virdi, Jugsharan S.

    2016-01-01

    Escherichia coli strains belonging to diverse pathotypes have increasingly been recognized as a major public health concern. The β-lactam antibiotics have been used successfully to treat infections caused by pathogenic E. coli. However, currently, the utility of β-lactams is being challenged severely by a large number of hydrolytic enzymes – the β-lactamases expressed by bacteria. The menace is further compounded by the highly flexible genome of E. coli, and propensity of resistance dissemination through horizontal gene transfer and clonal spread. Successful management of infections caused by such resistant strains requires an understanding of the diversity of β-lactamases, their unambiguous detection, and molecular mechanisms underlying their expression and spread with regard to the most relevant information about individual bacterial species. Thus, this review comprises first such effort in this direction for E. coli, a bacterial species known to be associated with production of diverse classes of β-lactamases. The review also highlights the role of commensal E. coli as a potential but under-estimated reservoir of β-lactamases-encoding genes. PMID:27065978

  8. Effects of Escherichia coli hemolysin on endothelial cell function.

    PubMed Central

    Suttorp, N; Flöer, B; Schnittler, H; Seeger, W; Bhakdi, S

    1990-01-01

    Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin. Images PMID:2121650

  9. The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

    PubMed Central

    Miskinyte, Migla; Sousa, Ana; Ramiro, Ricardo S.; de Sousa, Jorge A. Moura; Kotlinowski, Jerzy; Caramalho, Iris; Magalhães, Sara; Soares, Miguel P.; Gordo, Isabel

    2013-01-01

    Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity. PMID:24348252

  10. Unusual "flesh-eating" strains of Escherichia coli.

    PubMed

    Shaked, Hila; Samra, Zmira; Paul, Michal; Madar-Shapiro, Liora; Cohen, Jonathan; Pitlik, Silvio; Bishara, Jihad

    2012-12-01

    Monomicrobial necrotizing fasciitis (type II) is typically caused by group A streptococcus alone or in combination with Staphylococcus aureus. Escherichia coli has been isolated from polymicrobial or Fournier's gangrene but has rarely been reported in monomicrobial necrotizing fasciitis. We describe the clinical characteristics and outcomes of seven cases of monomicrobial E. coli necrotizing fasciitis and/or severe soft tissue infection diagnosed at a single institution during an 18-month period. Four isolates from three patients and two isolates from two patients with type I polymicrobial severe soft tissue infection (controls) were assayed by the randomly amplified polymorphic DNA (RAPD) analysis for fingerprinting and PCR amplification of primers in order to detect cytotoxic necrotizing factor 1 and 2 (cnf1 and cnf2) genes. All patients had some type of immune suppression. The limb was the most commonly involved organ. In all cases, E. coli was isolated as a monomicrobial pathogen from blood, fascia, or both. All patients died during hospitalization, three within the first 48 h. The RAPD amplification assay showed a high degree of genetic diversity among the "flesh-eating" strains and controls. The cnf1 toxin gene was identified in two out of three cases, but not in the controls. cnf2 was not detected in any of the patients. E. coli may be responsible for life-threatening necrotizing fasciitis. Further research is needed to reveal relevant risk factors, reservoirs, and modes of transmission of cnf1 E. coli.

  11. Escherichia coli exports cyclic AMP via TolC.

    PubMed

    Hantke, Klaus; Winkler, Karin; Schultz, Joachim E

    2011-03-01

    In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

  12. Selective detection of Escherichia coli DNA using fluorescent carbon spindles.

    PubMed

    Roy, Anurag; Chatterjee, Sabyasachi; Pramanik, Srikrishna; Devi, Parukuttyamma Sujatha; Suresh Kumar, Gopinatha

    2016-04-28

    We investigate the interaction of hydrophilic blue emitting carbon spindles with various deoxyribonucleic acids (DNA) having different base pair compositions, such as Herring testes (HT), calf thymus (CT), Escherichia coli (EC) and Micrococcus lysodeikticus (ML) DNA, to understand the mode of interaction. Interestingly, the fluorescent carbon spindles selectively interacted with E. coli DNA resulting in enhanced fluorescence of the former. Interaction of the same carbon with other DNAs exhibited insignificant changes in fluorescence. In addition, in the presence of EC DNA, the D band in the Raman spectrum attributed to the defect state completely disappeared, resulting in enhanced crystallinity. Microscopy images confirmed the wrapping of DNA on the carbon spindles leading to the assembly of spindles in the form of flowers. Dissociation of double-stranded DNA occurred upon interaction with carbon spindles, resulting in selective E. coli DNA interaction. The carbon spindles also exhibited a similar fluorescence enhancement upon treating with E. coli bacteria. These results confirm the possibility of E. coli detection in water and other liquid foods using such fluorescent carbon.

  13. Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance

    PubMed Central

    Vega, Nicole M.; Allison, Kyle R.; Samuels, Amanda N.; Klempner, Mark S.; Collins, James J.

    2013-01-01

    Bacterial communication plays an important role in many population-based phenotypes and interspecies interactions, including those in host environments. These interspecies interactions may prove critical to some infectious diseases, and it follows that communication between pathogenic bacteria and commensal bacteria is a subject of growing interest. Recent studies have shown that Escherichia coli uses the signaling molecule indole to increase antibiotic tolerance throughout its population. Here, we show that the intestinal pathogen Salmonella typhimurium increases its antibiotic tolerance in response to indole, even though S. typhimurium does not natively produce indole. Increased antibiotic tolerance can be induced in S. typhimurium by both exogenous indole added to clonal S. typhimurium populations and indole produced by E. coli in mixed-microbial communities. Our data show that indole-induced tolerance in S. typhimurium is mediated primarily by the oxidative stress response and, to a lesser extent, by the phage shock response, which were previously shown to mediate indole-induced tolerance in E. coli. Further, we find that indole signaling by E. coli induces S. typhimurium antibiotic tolerance in a Caenorhabditis elegans model for gastrointestinal infection. These results suggest that the intestinal pathogen S. typhimurium can intercept indole signaling from the commensal bacterium E. coli to enhance its antibiotic tolerance in the host intestine. PMID:23946425

  14. Nonthermal atmospheric argon plasma jet effects on Escherichia coli biomacromolecules.

    PubMed

    Hosseinzadeh Colagar, Abasalt; Memariani, Hamed; Sohbatzadeh, Farshad; Valinataj Omran, Azadeh

    2013-12-01

    Nonthermal atmospheric plasma jet, a promising technology based on ionized gas at low temperatures, can be applied for disinfection of contaminated surfaces. In this study, Escherichia coli cells and their macromolecules were exposed to the nonthermal atmospheric argon plasma jet for different time durations. Total protein, genomic DNA, and malondialdehyde (MDA) levels of E. coli were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining; agarose gel electrophoresis; and measurement of absorbance at 534 nm, respectively. After exposure, the spectroscopic results of liquid samples indicated that the survival reduction of E. coli can reach to 100 % in an exposure time of 600 s. Moreover, inactivation zones of E. coli, DNA degradation, and MDA levels were significantly increased. Additionally, banding patterns of total protein were changed and amino acid concentrations increased following ninhydrin test. The experimental results suggest that the nonthermal plasma could serve as an effective instrument for both sterilizing E. coli and degrading macromolecules from the surface of the objects being sterilized.

  15. Paper-based ELISA to rapidly detect Escherichia coli.

    PubMed

    Shih, Cheng-Min; Chang, Chia-Ling; Hsu, Min-Yen; Lin, Jyun-Yu; Kuan, Chen-Meng; Wang, Hsi-Kai; Huang, Chun-Te; Chung, Mu-Chi; Huang, Kui-Chou; Hsu, Cheng-En; Wang, Chun-Yuan; Shen, Ying-Cheng; Cheng, Chao-Min

    2015-12-01

    Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 10(5)cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment.

  16. Pulsed-Plasma Disinfection of Water Containing Escherichia coli

    NASA Astrophysics Data System (ADS)

    Satoh, Kohki; MacGregor, Scott J.; Anderson, John G.; Woolsey, Gerry A.; Fouracre, R. Anthony

    2007-03-01

    The disinfection of water containing the microorganism, Escherichia coli (E. coli) by exposure to a pulsed-discharge plasma generated above the water using a multineedle electrode (plasma-exposure treatment), and by sparging the off-gas of the pulsed plasma into the water (off-gas-sparging treatment), is performed in the ambient gases of air, oxygen, and nitrogen. For the off-gas-sparging treatment, bactericidal action is observed only when oxygen is used as the ambient gas, and ozone is found to generate the bactericidal action. For the plasma-exposure treatment, the density of E. coli bacteria decreases exponentially with plasma-exposure time for all the ambient gases. It may be concluded that the main contributors to E. coli inactivation are particle species produced by the pulsed plasma. For the ambient gases of air and nitrogen, the influence of acidification of the water in the system, as a result of pulsed-plasma exposure, may also contribute to the decay of E. coli density.

  17. Susceptibility of Gnotobiotic Swine to Escherichia coli Isolated from Nonenteric Human Infections

    PubMed Central

    Meyer, R. C.; Rhoades, H. E.; Simon, J.

    1972-01-01

    Newborn, germfree piglets were susceptible to Escherichia coli associated with human, nonenteric infections and should provide a useful model in the study of generalized E. coli infections. PMID:4557565

  18. Diarrhea, bacteremia and multiorgan dysfunction due to an extraintestinal pathogenic Escherichia coli strain with enteropathogenic E. coli genes.

    PubMed

    Kessler, Robert; Nisa, Shahista; Hazen, Tracy H; Horneman, Amy; Amoroso, Anthony; Rasko, David A; Donnenberg, Michael S

    2015-11-01

    A 55-year-old man with well-controlled HIV had severe diarrhea for 3 weeks and developed multiorgan dysfunction and bacteremia due to Escherichia coli. The genome of the patient's isolate had features characteristic of extraintestinal pathogenic E. coli and genes distantly related to those defining enteropathogenic E. coli.

  19. Role of enteroaggregative Escherichia coli virulence factors in uropathogenesis.

    PubMed

    Boll, Erik J; Struve, Carsten; Boisen, Nadia; Olesen, Bente; Stahlhut, Steen G; Krogfelt, Karen A

    2013-04-01

    A multiresistant clonal Escherichia coli O78:H10 strain qualifying molecularly as enteroaggregative Escherichia coli (EAEC) was recently shown to be the cause of a community-acquired outbreak of urinary tract infection (UTI) in greater Copenhagen, Denmark, in 1991. This marks the first time EAEC has been associated with an extraintestinal disease outbreak. Importantly, the outbreak isolates were recovered from the urine of patients with symptomatic UTI, strongly implying urovirulence. Here, we sought to determine the uropathogenic properties of the Copenhagen outbreak strain and whether these properties are conferred by the EAEC-specific virulence factors. We demonstrated that through expression of aggregative adherence fimbriae, the principal adhesins of EAEC, the outbreak strain exhibited pronouncedly increased adherence to human bladder epithelial cells compared to prototype uropathogenic strains. Moreover, the strain was able to produce distinct biofilms on abiotic surfaces, including urethral catheters. These findings suggest that EAEC-specific virulence factors increase uropathogenicity and may have played a significant role in the ability of the strain to cause a community-acquired outbreak of UTI. Thus, inclusion of EAEC-specific virulence factors is warranted in future detection and characterization of uropathogenic E. coli.

  20. Genotyping and virulence factors assessment of bovine mastitis Escherichia coli.

    PubMed

    Blum, Shlomo E; Leitner, Gabriel

    2013-05-03

    Escherichia coli is a major agent of bovine mastitis worldwide. However, specific E. coli virulence factors associated to pathogenicity during intra-mammary infections are yet unknown and this pathotype remains uncharacterized. The objectives of the present work were to assess the presence of a wide range of known virulence factors in a large set of E. coli strains isolated from bovine mastitis (mastitis set) and to study the genotypic distribution of strains in the mastitis set in comparison to a set of strains isolated from cows' environment in dairy farms (environmental set). Virulence factors were assessed by DNA hybridization microarray. The three most prevalent virulence factors found in the mastitis set were lpfA (long polar fimbriae), iss (increased serum resistance) and astA (enteroaggregative E. coli heat-stable enterotoxin 1). None, however, characterized the majority of these strains. Genotyping was assessed by ECOR phylogenetic grouping, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Strains in the mastitis and environmental sets were differentially distributed into ECOR phylogenetic groups; groups A and B1 being the most prevalent ones. Multiple MLST strain types were found in the two sets of strains, but only a few were common to both, and diversity was higher in the environmental set. A variety of PFGE patterns were found in the mastitis and environmental sets. Two clusters comprising mostly highly similar mastitis strains were identified. The results confirm that mastitis E. coli strains mostly lack known E. coli virulence factors. In addition, it is shown that the genotypic diversity of mastitis strains does not reflect the diversity found in the environmental E. coli population.

  1. Distribution of core oligosaccharide types in lipopolysaccharides from Escherichia coli.

    PubMed

    Amor, K; Heinrichs, D E; Frirdich, E; Ziebell, K; Johnson, R P; Whitfield, C

    2000-03-01

    In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69. 4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coli serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.

  2. Reduction of verotoxigenic Escherichia coli in production of fermented sausages.

    PubMed

    Holck, Askild L; Axelsson, Lars; Rode, Tone Mari; Høy, Martin; Måge, Ingrid; Alvseike, Ole; L'abée-Lund, Trine M; Omer, Mohamed K; Granum, Per Einar; Heir, Even

    2011-11-01

    After a number of foodborne outbreaks of verotoxigenic Escherichia coli involving fermented sausages, some countries have imposed regulations on sausage production. For example, the US Food Safety and Inspection Service requires a 5 log(10) reduction of E. coli in fermented products. Such regulations have led to a number of studies on the inactivation of E. coli in fermented sausages by changing processing and post-processing conditions. Several factors influence the survival of E. coli such as pre-treatment of the meat, amount of NaCl, nitrite and lactic acid, water activity, pH, choice of starter cultures and addition of antimicrobial compounds. Also process variables like fermentation temperature and storage time play important roles. Though a large variety of different production processes of sausages exist, generally the reduction of E. coli caused by production is in the range 1-2 log(10). In many cases this may not be enough to ensure microbial food safety. By optimising ingredients and process parameters it is possible to increase E. coli reduction to some extent, but in some cases still other post process treatments may be required. Such treatments may be storage at ambient temperatures, specific heat treatments, high pressure processing or irradiation. HACCP analyses have identified the quality of the raw materials, low temperature in the batter when preparing the sausages and a rapid pH drop during fermentation as critical control points in sausage production. This review summarises the literature on the reduction verotoxigenic E. coli in production of fermented sausages.

  3. Persistence of Escherichia coli in batch and continuous vermicomposting systems.

    PubMed

    Hénault-Ethier, Louise; Martin, Vincent J J; Gélinas, Yves

    2016-10-01

    Vermicomposting is a biooxidation process in which epigeicearthworms act in synergy with microbial populations to degrade organic matter. Vermicomposting does not go through a thermophilic stage as required by North American legislations for pathogen eradication. We examined the survival of a Green Fluorescent Protein (GFP) labeled Escherichia coli MG1655 as a model for the survival of pathogenic bacteria in both small-scale batch and medium-scale continuously-operated systems to discern the influence of the earthworm Eisenia fetida, nutrient content and the indigenous vermicompost microbial community on pathogen abundance. In batch systems, the microbial community had the greatest influence on the rapid decline of E. coli populations, and the effect of earthworms was only visible in microbially-impoverishedvermicomposts. No significant earthworm density-dependent relationship was observed on E. coli survival under continuous operation. E. coli numbers decreased below the US EPA compost sanitation guidelines of 10(3)Colony Forming Units (CFU)/g (dry weight) within 18-21days for both the small-scale batch and medium-scale continuous systems, but it took up to 51days without earthworms and with an impoverished microbial community to reach the legal limit. Nutrient replenishment (i.e. organic carbon) provided by continuous feed input did not appear to extend E. coli survival. In fact, longer survival of E. coli was noticed in treatments where less total and labile sugars were available, suggesting that sugars may support potentially antagonist bacteria in the vermicompost. Total N, pH and humidity did not appear to affect E. coli survival. Several opportunistic human pathogens may be found in vermicompost, and their populations are likely kept in check by antagonists.

  4. Diarrhea, Urosepsis and Hemolytic Uremic Syndrome Caused by the Same Heteropathogenic Escherichia coli Strain.

    PubMed

    Ang, C Wim; Bouts, Antonia H M; Rossen, John W A; Van der Kuip, Martijn; Van Heerde, Marc; Bökenkamp, Arend

    2016-09-01

    We describe an 8-month-old girl with diarrhea, urosepsis and hemolytic uremic syndrome caused by Escherichia coli. Typing of cultured E. coli strains from urine and blood revealed the presence of virulence factors from multiple pathotypes of E. coli. This case exemplifies the genome plasticity of E. coli and the resulting heteropathogenic strains.

  5. Genome Sequence of the Enterohemorrhagic Escherichia coli Bacteriophage UFV-AREG1.

    PubMed

    Lopez, Maryoris E Soto; Batalha, Laís Silva; Vidigal, Pedro Marcus Pereira; Albino, Luiz Augusto A; Boggione, Delaine Meireles Gouveia; Gontijo, Marco Tulio Pardini; Bazzolli, Denise M Soares; Mendonca, Regina C Santos

    2016-10-13

    Here, we present the genome sequence of the Escherichia coli bacteriophage UFV-AREG1. This phage was isolated from cowshed wastewater and showed specificity for enterohemorrhagic E. coli O157:H7 (ATCC 43895), E. coli 0111 (CDC O11ab) and E. coli (ATCC 23229).

  6. Genome Sequence of the Enterohemorrhagic Escherichia coli Bacteriophage UFV-AREG1

    PubMed Central

    Batalha, Laís Silva; Albino, Luiz Augusto A.; Boggione, Delaine Meireles Gouveia; Gontijo, Marco Tulio Pardini; Bazzolli, Denise M. Soares; Mendonca, Regina C. Santos

    2016-01-01

    Here, we present the genome sequence of the Escherichia coli bacteriophage UFV-AREG1. This phage was isolated from cowshed wastewater and showed specificity for enterohemorrhagic E. coli O157:H7 (ATCC 43895), E. coli 0111 (CDC O11ab) and E. coli (ATCC 23229). PMID:27738021

  7. Multiplex PCR for Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

    PubMed Central

    Vidal, Roberto; Vidal, Maricel; Lagos, Rossana; Levine, Myron; Prado, Valeria

    2004-01-01

    A multiplex PCR for detection of three categories of diarrheagenic Escherichia coli was developed. With this method, enterohemorrhagic E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were identified in fecal samples from patients with hemorrhagic colitis, watery diarrhea, or hemolytic-uremic syndrome and from food-borne outbreaks. PMID:15071051

  8. Persistent colonization of sheep by Escherichia coli O157:H7 and other E. coli pathotypes.

    PubMed

    Cornick, N A; Booher, S L; Casey, T A; Moon, H W

    2000-11-01

    Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness in humans. Ruminants appear to be more frequently colonized by STEC than are other animals, but the reason(s) for this is unknown. We compared the frequency, magnitude, duration, and transmissibility of colonization of sheep by E. coli O157:H7 to that by other pathotypes of E. coli. Young adult sheep were simultaneously inoculated with a cocktail consisting of two strains of E. coli O157:H7, two strains of enterotoxigenic E. coli (ETEC), and one strain of enteropathogenic E. coli. Both STEC strains and ETEC 2041 were given at either 10(7) or 10(10) CFU/strain/animal. The other strains were given only at 10(10) CFU/strain. We found no consistent differences among pathotypes in the frequency, magnitude, and transmissibility of colonization. However, the STEC strains tended to persist to 2 weeks and 2 months postinoculation more frequently than did the other pathotypes. The tendency for persistence of the STEC strains was apparent following an inoculation dose of either 10(7) or 10(10) CFU. One of the ETEC strains also persisted when inoculated at 10(10) CFU. However, in contrast to the STEC strains, it did not persist when inoculated at 10(7) CFU. These results support the hypothesis that STEC is better adapted to persist in the alimentary tracts of sheep than are other pathotypes of E. coli.

  9. Effect of bile on growth, peritoneal absorption, and blood clearance of Escherichia coli in E coli peritonitis

    SciTech Connect

    Andersson, R.; Schalen, C.; Tranberg, K.G. )

    1991-06-01

    The effect of intraperitoneal bile on growth, peritoneal absorption, and clearance of Escherichia coli was determined in E coli peritonitis in the rat. In E coli peritonitis, intraperitoneal bacterial counts gradually decreased, whereas they increased (after 2 hours) with subsequent development of bacteremia in E coli plus bile peritonitis. After an intraperitoneal injection of labeled bacteria, blood radioactivity was only initially lower in E coli plus bile peritonitis compared with E coli peritonitis. Clearance from blood was lower in E coli plus bile peritonitis than in E coli peritonitis. Organ localization was similar in E coli peritonitis and E coli plus bile peritonitis with decreased splenic, increased pulmonary, and unchanged hepatic uptakes compared with controls. Impaired peritoneal absorption of bacteria, together with impaired local host defense, is likely to enhance the noxious effect of bile in E coli peritonitis.

  10. Structure of the Escherichia coli S10 ribosomal protein operon.

    PubMed Central

    Zurawski, G; Zurawski, S M

    1985-01-01

    The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data. PMID:3892488

  11. Genome-scale genetic engineering in Escherichia coli.

    PubMed

    Jeong, Jaehwan; Cho, Namjin; Jung, Daehee; Bang, Duhee

    2013-11-01

    Genome engineering has been developed to create useful strains for biological studies and industrial uses. However, a continuous challenge remained in the field: technical limitations in high-throughput screening and precise manipulation of strains. Today, technical improvements have made genome engineering more rapid and efficient. This review introduces recent advances in genome engineering technologies applied to Escherichia coli as well as multiplex automated genome engineering (MAGE), a recent technique proposed as a powerful toolkit due to its straightforward process, rapid experimental procedures, and highly efficient properties.

  12. Antitermination of transcription from an Escherichia coli ribosomal RNA promoter.

    PubMed

    Holben, W E; Morgan, E A

    1984-11-01

    The Escherichia coli lac and ara promoters and rrnC ribosomal RNA promoter-leader region were fused to lacZYA. Transcription termination signals were introduced into the lac genes of these fusions by Tn9 and IS1 insertions. Measurement of lac enzymes from upstream and downstream of the insertions showed that termination signals resulting from these insertions are very efficient when transcription begins at lac or ara promoters but are very inefficient when transcription begins at the rrnC promoter-leader region. The rrnC promoter-leader region must, therefore, modify RNA polymerase to enable it to read through transcription termination signals.

  13. Regulation of the L-arabinose operon of Escherichia coli.

    PubMed

    Schleif, R

    2000-12-01

    Over forty years of research on the L-arabinose operon of Escherichia coli have provided insights into the mechanism of positive regulation of gene activity. This research also discovered DNA looping and the mechanism by which the regulatory protein changes its DNA-binding properties in response to the presence of arabinose. As is frequently seen in focused research on biological subjects, the initial studies were primarily genetic. Subsequently, the genetic approaches were augmented by physiological and then biochemical studies. Now biophysical studies are being conducted at the atomic level, but genetics still has a crucial role in the study of this system.

  14. Studies on the Chick-lethal Toxin of Escherichia coli

    PubMed Central

    Truscott, R. B.

    1973-01-01

    A toxin which is lethal for two week old chicks has been recovered from strains of Escherichia coli O78:K80 of bovine and avian origin and from avian isolates of serogroups O2, O45 and O109. The toxin is heat-labile, antigenic, high in protein, inactivated by pronase, trypsin, amylase, and pancreatic lipase. The toxin may be precipitated by ammonium sulfate or TCA treatment from the supernatant obtained by repeated centrifugation of sonicated cells. Considerable purification has been obtained by column chromatography using Sepharose 6B. PMID:4270809

  15. Compilation and analysis of Escherichia coli promoter DNA sequences.

    PubMed Central

    Hawley, D K; McClure, W R

    1983-01-01

    The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence. PMID:6344016

  16. [Hemolytic uremic syndrome caused by enterohaemorrhagic Escherichia coli].

    PubMed

    Ibarra, Cristina; Goldstein, Jorge; Silberstein, Claudia; Zotta, Elsa; Belardo, Marcela; Repetto, Horacio A

    2008-10-01

    Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, plaquetopenia and kidney damage. It is the leading cause of acute renal failure in pediatric age and the second for chronic renal failure. Shiga toxin-producing Escherichia coli (STEC) is the first etiologic agent of HUS being its main reservoir cattle and transmitted via contaminated food. At present, there is no specific treatment to reduce the progression of HUS. The study of the mechanisms by which STEC infects and Shiga toxin induces HUS can help to find new strategies to prevent this disease.

  17. Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

    PubMed Central

    Salomón, R A; Farías, R N

    1992-01-01

    Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system. Images PMID:1429464

  18. Interaction of the exr and lon Genes in Escherichia coli

    PubMed Central

    Donch, John; Green, Michael H. L.; Greenberg, Joseph

    1968-01-01

    Strains of Escherichia coli carrying the gene lon typically produced excess capsular polysaccharide, and were sensitive to ultraviolet light (UV) irradiation, thymine starvation, and nalidixic acid, forming long filaments after these treatments. Sensitivity was reduced by a number of posttreatments. In the presence of a second UV sensitivity gene, exr, some of these properties were suppressed: long filaments were not formed, the effect of lon on UV and nalidixic acid sensitivity was greatly reduced, and irradiation posttreatments gave an enhancement of survival characteristic of exr rather than lon strains. Production of capsular polysaccharide was not affected by the exr gene. PMID:4882020

  19. CRISPR adaptation in Escherichia coli subtypeI-E system.

    PubMed

    Kiro, Ruth; Goren, Moran G; Yosef, Ido; Qimron, Udi

    2013-12-01

    The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR-Cas system of Escherichia coli.

  20. Synthesis of calf prochymosin (prorennin) in Escherichia coli.

    PubMed Central

    Emtage, J S; Angal, S; Doel, M T; Harris, T J; Jenkins, B; Lilley, G; Lowe, P A

    1983-01-01

    A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach. Images PMID:6304731

  1. DNA probes for identification of enteroinvasive Escherichia coli.

    PubMed Central

    Gomes, T A; Toledo, M R; Trabulsi, L R; Wood, P K; Morris, J G

    1987-01-01

    Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were tested with two distinct invasiveness probes (pMR17 and pSF55). All 54 Sereny test-positive strains and 5 strains that lost Sereny positivity during storage hybridized with both probes. Probe-positive strains carried a 120- to 140-megadalton plasmid, did not produce lysine decarboxylase, and, with the exception of certain serotypes, were nonmotile. Motile strains of serotype O144:H25 were for the first time characterized as invasive by hybridization with the probes. PMID:3312292

  2. Electric dipole moments of Escherichia coli HB 101.

    PubMed

    Stoylov, Stoyl P; Gyurova, Anna Y; Bunin, Viktor; Angersbach, Alexander; Georgieva, Ralitsa N; Danova, Svetla T

    2009-04-01

    The theoretical and experimental studies of the particles' electric dipole moments in the microscopic and submicroscopic size range show that in the case of polar and conductive media the interfacial components of the dipole moments are of greatest importance. While in the range of manometer's sizes there seems to be no important problems in the identification and in the estimation of the values of the dipole moments at present, in the micrometer range there are serious problems. In this communication these problems are considered and illustrated by electro-optic investigations of Escherichia coli HB 101.

  3. Infected abdominal aortic aneurysm due to Escherichia coli.

    PubMed

    Bouzas, Miguel; Tchana-Sato, Vincent; Lavigne, Jean Paul

    2016-10-19

    Early diagnosis of infected abdominal aortic aneurysm (IAAA) is still a medical challenge due to its diverse and non-specific symptoms and signs. The most common responsible pathogens are Salmonella, Staphylococcus, Campylobacter and Streptococcus species. The authors report the case of a 67-year-old man, admitted for high fever and finally diagnosed with Escherichia coli (E.coli)-related IAAA. The IAAA ruptured during the general anaesthesia induction, leading to an emergency surgery. The authors successfully proceeded to an open aneurysmectomy with extensive debridement and in situ graft replacement. This case emphasizes the potential for rapid IAAA expansion, its high-rupture risk and the importance of computed tomography as a diagnostic tool.

  4. Impact of cranberry on Escherichia coli cellular surface characteristics

    SciTech Connect

    Johnson, Brandy J.; Malanoski, Anthony P.; Ligler, Frances S.

    2008-12-19

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  5. Avian pathogenic Escherichia coli bind fibronectin and laminin.

    PubMed

    Ramírez, Rosa María; Almanza, Yolanda; González, Rafael; García, Santos; Heredia, Norma

    2009-04-01

    Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

  6. Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase.

    PubMed

    Kantrowitz, Evan R

    2012-03-15

    The allosteric enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli has been the subject of investigations for approximately 50 years. This enzyme controls the rate of pyrimidine nucleotide biosynthesis by feedback inhibition, and helps to balance the pyrimidine and purine pools by competitive allosteric activation by ATP. The catalytic and regulatory components of the dodecameric enzyme can be separated and studied independently. Many of the properties of the enzyme follow the Monod, Wyman Changeux model of allosteric control thus E. coli ATCase has become the textbook example. This review will highlight kinetic, biophysical, and structural studies which have provided a molecular level understanding of how the allosteric nature of this enzyme regulates pyrimidine nucleotide biosynthesis.

  7. Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli.

    PubMed

    Upadhyay, Vaibhav; Singh, Anupam; Panda, Amulya K

    2016-01-01

    Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications.

  8. Inversions between ribosomal RNA genes of Escherichia coli.

    PubMed Central

    Hill, C W; Harnish, B W

    1981-01-01

    It might be anticipated that the presence of redundant but oppositely oriented sequences in a chromosome could allow inversion of the intervening material through homologous recombination. For example, the ribosomal RNA gene rrnD of Escherichia coli has the opposite orientation fro rrnB and rrnE and is separated from these genes by roughly 20% of the chromosome. Starting with a derivative of Cavalli Hfr, we have constructed mutants that have an inversion of the segment between rrnD and either rrnB or rrnE. These mutants are generally quite viable but do exhibit a slight reduction in growth rate relative to the parental strain. A major line of laboratory E. coli, W3110 and its derivatives, also has an inversion between rrnD and rrnE, probably created directly by a recombinational event between these highly homologous genes. Images PMID:6273909

  9. SILVER NANOPARTICLES-DISK DIFFUSION TEST AGAINST Escherichia coli ISOLATES.

    PubMed

    Cunha, Francisco Afrânio; Maia, Kamila Rocha; Mallman, Eduardo José Jucá; Cunha, Maria da Conceição Dos Santos Oliveira; Maciel, Antonio Auberson Martins; Souza, Ieda Pereira de; Menezes, Everardo Albuquerque; Fechine, Pierre Basílio Almeida

    2016-09-22

    Nanotechnology can be a valuable ally in the treatment of infections. Silver nanoparticles (AgNPs) are structures that have antimicrobial activity. The aim of this study was to produce AgNPs by green methods, characterize these structures, and assess their antimicrobial activity against Escherichia coli associated with the antibiotic ciprofloxacin. AgNPs were characterized by spectroscopic and microscopic techniques. Antimicrobial activity was evaluated by the disk diffusion method against 10 strains of E. coli. The synthesized AgNPs showed a spherical shape and a size of 85.07 ± 12.86 nm (mean ± SD). AgNPs increased the activity of ciprofloxacin by 40% and may represent a new therapeutic option for the treatment of bacterial infections.

  10. Continuous-sterilization system that uses photosemiconductor powders. [Escherichia coli

    SciTech Connect

    Matsunaga, T.; Tomoda, R.; Nakajima, T.; Nakamura, N.; Komine, T.

    1988-06-01

    We report a novel photochemical sterilization system in which Escherichia coli cells were sterilized with photosemiconductor powders (titanium oxide). For sterilization that could be used in practice, it was necessary to separate the TiO/sub 2/ powders from the cell suspension. Therefore, semiconductor powders were immobilized on acetylcellulose membranes. We constructed a continuous-sterilization system consisting of TiO/sub 2/-immobilized acetylcellulose membrane reactor, a mercury lamp, and a masterflex pump. As a result, under the various sterilization conditions examined, E.coli (10/sup 2/ cells per ml) was sterilized to < 1% survival when the cell suspension flowed in this system at a mean residence time of 16.0 min under irradiation (1800 microeinsteins/m/sup 2/ per s). We found that this system was reusable.

  11. Detecting the Significant Flux Backbone of Escherichia coli metabolism.

    PubMed

    Güell, Oriol; Sagués, Francesc; Serrano, M Ángeles

    2017-04-09

    The heterogeneity of computationally predicted reaction fluxes in metabolic networks within a single flux state can be exploited to detect their significant flux backbone. Here, we disclose the backbone of Escherichia coli, and compare it with the backbones of other bacteria. We find that, in general, the core of the backbones is mainly composed of reactions in energy metabolism corresponding to ancient pathways. In E. coli, the synthesis of nucleotides and the metabolism of lipids form smaller cores which rely critically on energy metabolism. Moreover, the consideration of different media leads to the identification of pathways sensitive to environmental changes. The metabolic backbone of an organism is thus useful for tracing, simultaneously, both its evolution and adaptation fingerprints. This article is protected by copyright. All rights reserved.

  12. The SIGNAL experiment in BIORACK: Escherichia coli in microgravity.

    PubMed

    Thévenet, D; D'Ari, R; Bouloc, P

    1996-06-27

    Microgravity affects certain physical properties of fluids, such as convection movement and surface tension. As a consequence, cells and living organisms may exhibit different behaviour in space, which may result from differences in the immediate environment of the cell or changes in the structure of the membrane in microgravity. Two experiments to examine the effects of microgravity on cell microenvironment and signal transduction through membranes were performed using a well-characterized system with different strains of the non-pathogenic Gram-negative bacterium Escherichia coli. Our results indicate that (i) microgravity appears to reduce the lag period of a non-motile culture of E. coli, and (ii) the ompC gene, regulated by the two-component system EnvZ-OmpR, is induced as well or better in microgravity than in ground controls.

  13. Mounting of Escherichia coli spheroplasts for AFM imaging.

    SciTech Connect

    Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P; Doktycz, Mitchel John

    2005-11-01

    The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.

  14. The action of beta-galactosidase (Escherichia coli) on allolactose.

    PubMed

    Huber, R E; Wallenfels, K; Kurz, G

    1975-09-01

    The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.

  15. In Vivo study of naturally deformed Escherichia coli bacteria.

    PubMed

    Tavaddod, Sharareh; Naderi-Manesh, Hossein

    2016-06-01

    A combination of light-microscopy and image processing has been applied to study naturally deformed Escherichia coli under in vivo condition and at the order of sub-pixel high-resolution accuracy. To classify deflagellated non-dividing E. coli cells to the rod-shape and bent-shape, a geometrical approach has been applied. From the analysis of the geometrical data which were obtained of image processing, we estimated the required effective energy for shaping a rod-shape to a bent-shape with the same size. We evaluated the energy of deformation in the naturally deformed bacteria with minimum cell manipulation, under in vivo condition, and with minimum influence of any external force, torque and pressure. Finally, we have also elaborated on the possible scenario to explain how naturally deformed bacteria are formed from initial to final-stage.

  16. SILVER NANOPARTICLES-DISK DIFFUSION TEST AGAINST Escherichia coli ISOLATES

    PubMed Central

    CUNHA, Francisco Afrânio; MAIA, Kamila Rocha; MALLMAN, Eduardo José Jucá; CUNHA, Maria da Conceição dos Santos Oliveira; MACIEL, Antonio Auberson Martins; de SOUZA, Ieda Pereira; MENEZES, Everardo Albuquerque; FECHINE, Pierre Basílio Almeida

    2016-01-01

    SUMMARY Nanotechnology can be a valuable ally in the treatment of infections. Silver nanoparticles (AgNPs) are structures that have antimicrobial activity. The aim of this study was to produce AgNPs by green methods, characterize these structures, and assess their antimicrobial activity against Escherichia coli associated with the antibiotic ciprofloxacin. AgNPs were characterized by spectroscopic and microscopic techniques. Antimicrobial activity was evaluated by the disk diffusion method against 10 strains of E. coli. The synthesized AgNPs showed a spherical shape and a size of 85.07 ± 12.86 nm (mean ± SD). AgNPs increased the activity of ciprofloxacin by 40% and may represent a new therapeutic option for the treatment of bacterial infections. PMID:27680178

  17. Mutational analysis of UMP kinase from Escherichia coli.

    PubMed

    Bucurenci, N; Serina, L; Zaharia, C; Landais, S; Danchin, A; Bârzu, O

    1998-02-01

    UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

  18. Identification, expression, and characterization of Escherichia coli guanine deaminase.

    PubMed

    Maynes, J T; Yuan, R G; Snyder, F F

    2000-08-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease.

  19. Identification, Expression, and Characterization of Escherichia coli Guanine Deaminase

    PubMed Central

    Maynes, Jason T.; Yuan, Richard G.; Snyder, Floyd F.

    2000-01-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a Km of 15 μM with guanine and a kcat of 3.2 s−1. The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3′ from an open reading frame which shows homology to a bacterial purine base permease. PMID:10913105

  20. 75 FR 6222 - Notice of Permit Applications Received Under the Antarctic Conservation Act of 1978 (Pub. L. 95-541)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-08

    ... Introduce non-indigenous species into Antarctica. The applicant plans to use Escherichia coli strain BL21DE3... obtain these proteins, they will express G. gibberifrons actin and tubulin substrates in E. coli in a medium supplemented with \\35\\S-methionine. The E. coli will not be released to the environment....

  1. Effect of various nonionic surfactants on growth of Escherichia coli.

    PubMed

    Rose, M J; Aron, S A; Janicki, B W

    1966-05-01

    Rose, Michael J., Jr. (Veterans Administration Hospital, Washington, D.C.), Stephen A. Aron, and Bernard W. Janicki. Effect of various nonionic surfactants on growth of Escherichia coli. J. Bacteriol. 91:1863-1868. 1966.-Escherichia coli cultivated in media containing 0.5, 1.0, 2.0, or 4.0% concentrations of surface-active polyoxyethylene derivatives of formaldehyde polymers of octyl phenol (Triton WR-1339; Macrocyclon) or of sorbitan mono-fatty acid esters (Tween 20, 40, 60, and 80) exhibited significantly retarded growth only at the highest concentration. To determine the mechanism of bacteriostasis, certain derivatives and compounds related to the surfactants were investigated. Experiments with compounds related to the Triton-type agents demonstrated that incorporation of monomeric substances (Triton X-205, X-305, Igepal CA-730, or Dowfax 9N20) into the medium at a concentration of 4.0% did not inhibit the growth of E. coli. It was concluded that the formaldehyde polymer was essential for growth inhibition by the polyoxyethylene derivatives of octyl phenol. The inhibitory activity of the Tween compounds, in contrast, appeared to result from the unesterified fatty acids which contaminate the commercial preparations. Polyol (60), the sorbitan polyoxyethylene derivative of Tween 60 and the basic structural unit of all the Tween-type compounds, and a Tween 80 preparation which was purified by extraction of the unesterified oleic acid, were not inhibitory. Moreover, the amount of free oleic acid present as a contaminant of Tween 80 was found to be sufficient to cause significant growth inhibition. These results and the observation that E. coli does not appear to hydrolyze the esterified fatty acid of Tween 80 led to the conclusion that growth inhibition obtained with various Tween compounds probaby is a function of their respective fatty acid contaminants.

  2. Adhesive Escherichia coli in inflammatory bowel disease and infective diarrhoea.

    PubMed Central

    Burke, D. A.; Axon, A. T.

    1988-01-01

    The clinical features of ulcerative colitis and Crohn's disease are similar to those of infections of the bowel, although their cause is uncertain. Many bacteria that cause intestinal diseases adhere to the gut mucosa, and adhesion of pathogenic Escherichia coli is resistant to D-mannose. The adhesive properties of isolates of E coli were assessed by assay of adhesion to buccal epithelial cells with mannose added. The isolates were obtained from patients with inflammatory bowel diseases (50 with a relapse of ulcerative colitis, nine with ulcerative colitis in remission, 13 with Crohn's disease, and 11 with infectious diarrhoea not due to E coli) and 22 controls. The median index of adhesion to buccal epithelial cells (the proportion of cells with more than 50 adherent bacteria) for E coli from patients with ulcerative colitis in relapse was significantly higher (43%) than that for controls (5%) and patients with infectious diarrhoea (14%). The index was not significantly different among isolates from patients with ulcerative colitis in relapse, Crohn's disease (53%), and ulcerative colitis in remission (30%). If an index of adhesion of greater than 25% is taken as indicating an adhesive strain 86% of isolates of E coli from patients with inflammatory bowel disease were adhesive compared with 27% from patients with infective diarrhoea and none from controls. The adhesive properties of the isolates from patients with inflammatory bowel disease were similar to those of pathogenic intestinal E coli, raising the possibility that they may have a role in the pathogenesis of the condition; the smaller proportion of adhesive isolates in patients with infective diarrhoea due to other bacteria suggests that the organism may be of primary importance rather than arising secondarily. Images a PMID:3044496

  3. Pathogenic Escherichia coli in rural household container waters.

    PubMed

    Jagals, P; Barnard, T G; Mokoena, M M; Ashbolt, N; Roser, D J

    2013-01-01

    Plastic containers in the range of 5-20 L are widely used - especially in rural African settings - to collect, transport and store water for domestic use, including drinking, bathing and hygiene. The pathogen content of the waters in these containers has not been adequately characterized as yet. This paper presents the primary findings of a synoptic survey of drinking water quality samples from these containers and involved collection of bacterial indicator and pathogenicity gene data. In total, 571 samples of a variety of waters were taken in rural communities in South Africa and the Escherichia coli numbers measured. Of the E. coli positive samples, 46% (n = 148) were screened for the presence of E. coli pathogen gene markers. Though synoptic, the survey provided many insights into the issues that drove the study. Container use markedly degraded water quality as judged by indicator counts, even where improved water supply services were in place. Household container use also appeared to promote regrowth or contamination of containers with pathogenic E. coli strains. Polymerase chain reaction (PCR) analysis also showed that the diversity of potential pathogenic E. coli carrying virulence genes was great. All seven genes screened for (Ial, Stx1, Stx2, EaeA, Eagg, ST, LT) were found in the waters, alone or as mixtures (number of different combinations = 31) including those characteristic of the more dangerous invasive and haemorrhagic E. coli strains. Given the central role of containers in the management of water supply to rural communities, it is clear the microbiology of these waters requires much further characterization.

  4. Dynamic regulation of extracellular ATP in Escherichia coli.

    PubMed

    Alvarez, Cora Lilia; Corradi, Gerardo; Lauri, Natalia; Marginedas-Freixa, Irene; Leal Denis, María Florencia; Enrique, Nicolás; Mate, Sabina María; Milesi, Verónica; Ostuni, Mariano Anibal; Herlax, Vanesa; Schwarzbaum, Pablo Julio

    2017-04-04

    We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [(32)P]Pi released from [γ-(32)P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-(32)P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

  5. Expression of a synthetic pertussis toxin operon in Escherichia coli.

    PubMed

    Pozza, T D; Yan, H; Walker, M J

    1997-06-01

    Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli beta-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

  6. A structural view of the dissociation of Escherichia coli tryptophanase.

    PubMed

    Green, Keren; Qasim, Nasrin; Gdaelvsky, Garik; Kogan, Anna; Goldgur, Yehuda; Parola, Abraham H; Lotan, Ofra; Almog, Orna

    2015-12-01

    Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.

  7. Genomic analysis of extra-intestinal pathogenic Escherichia coli urosepsis.

    PubMed

    McNally, A; Alhashash, F; Collins, M; Alqasim, A; Paszckiewicz, K; Weston, V; Diggle, M

    2013-08-01

    Urosepsis is a bacteraemia infection caused by an organism previously causing an infection in the urinary tract of a patient, a diagnosis which has been classically confirmed by culture of the same species of bacteria from both blood and urine samples. Given the new insights afforded by sequencing technologies into the complicated population structures of infectious agents affecting humans, we sought to investigate urosepsis by comparing the genome sequences of blood and urine isolates of Escherichia coli from five patients with urosepsis. The results confirm the classical urosepsis hypothesis in four of the five cases, but also show the complex nature of extra-intestinal E. coli infection in the fifth case, where three distinct strains caused two distinct infections. Additionally, we show there is little to no variation in the bacterial genome as it progressed from urine to blood, and also present a minimal set of virulence genes required for bacteraemia in E. coli based on gene association. These suggest that most E. coli have the genetic propensity to cause bacteraemia.

  8. Characterization of a second lysine decarboxylase isolated from Escherichia coli.

    PubMed Central

    Kikuchi, Y; Kojima, H; Tanaka, T; Takatsuka, Y; Kamio, Y

    1997-01-01

    We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli. PMID:9226257

  9. A second DNA methyltransferase repair enzyme in Escherichia coli.

    PubMed Central

    Rebeck, G W; Coons, S; Carroll, P; Samson, L

    1988-01-01

    The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains. Images PMID:3283737

  10. Redefining the requisite lipopolysaccharide structure in Escherichia coli.

    PubMed

    Meredith, Timothy C; Aggarwal, Parag; Mamat, Uwe; Lindner, Buko; Woodard, Ronald W

    2006-02-17

    Gram-negative bacteria possess an asymmetric lipid bilayer surrounding the cell wall, the outer membrane (OM). The OM inner leaflet is primarily composed of various glycerophospholipids, whereas the outer leaflet predominantly contains the unique amphiphilic macromolecule, lipopolysaccharide (LPS or endotoxin). The majority of all gram-negative bacteria elaborate LPS containing at least one 2-keto 3-deoxy-D-manno-octulosonate (Kdo) molecule. The minimal LPS structure required for growth of Escherichia coli has long been recognized as two Kdo residues attached to lipid A, inextricably linking viability to toxicity. Here we report the construction and characterization of the nonconditional E. coli K-12 suppressor strain KPM22 that lacks Kdo and is viable despite predominantly elaborating the endotoxically inactive LPS precursor lipid IV(A). Our results challenge the established E. coli Kdo2-lipid A dogma, indicating that the previously observed and well-documented dependence of cell viability on the synthesis of Kdo stems from a lethal pleiotropy precipitated after the depletion of the carbohydrate, rather than an inherent need for the Kdo molecule itself as an indispensable structural component of the OM LPS layer. Inclusion of the inner membrane LPS transporter MsbA on a multicopy plasmid partially suppresses the lethal deltaKdo phenotype directly in the auxotrophic parent strain, suggesting increased rates of nonglycosylated lipid A transport can, in part, compensate for Kdo depletion. The unprecedented nature of a lipid IV(A) OM redefines the requisite LPS structure for viability in E. coli.

  11. Resistance patterns of Escherichia coli causing urinary tract infection

    PubMed Central

    Ferdosi-Shahandashti, Elaheh; Javanian, Mostafa; Moradian-Kouchaksaraei, Masoomeh; Yeganeh, Babak; Bijani, Ali; Motevaseli, Elahe; Moradian- Kouchaksaraei, Fatemeh

    2015-01-01

    Background: Urinary tract infection (UTI) is one of the most prevalent infectious diseases and Escherichia coli is its common cause. The aim of this study was to assess the resistance patterns of E.coli in urinary tract infections and to determine the susceptibility of E.coli to commonly used antimicrobials and also to evaluate the options for empirical treatment of UTI. Methods: This study was conducted in the Ayatollah Rouhani Teaching Hospital of Babol Medical Sciences University in North of Iran. Between January of 2013 to December 2013, antimicrobial susceptibility tests were done by disk diffusion and microdilution method. Growth of >=105 cfu/ml was considered as positive urine test. Ten commonly used antibiotics were examined for susceptibility test. Data and the results were collected and analyzed. Results: E.coli grew in 57 urine samples. Imipenem, ofloxacin, ciprofloxacin were the most sensitive antibiotics at 87.7%, 87.7% and 78.9% respectively. Whereas, cotrimoxazole, cefexime, cefotaxcime and ceftriaxone were the most resistant antibiotics. Antibiotic sensitivity of disk diffusion compared minimum inhibitory concentration (MIC) detected by microdilution had the sensitivity, specificity, positive predictive value and negative predictive value of 82%, 98%, 99% and 74%, respectively. Conclusion: Imipenem, ofloxacin and ciprofloxacin should be used in empirical therapy of UTI. PMID:26644881

  12. Curli fimbria: an Escherichia coli adhesin associated with human cystitis.

    PubMed

    Cordeiro, Melina Aparecida; Werle, Catierine Hirsch; Milanez, Guilherme Paier; Yano, Tomomasa

    2016-01-01

    Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-d-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.

  13. Magnetically-Actuated Escherichia coli System for Micro Lithography

    NASA Astrophysics Data System (ADS)

    Lauback, S.; Brown, E.; Pérez-Guzman, L.; Peace, C.; Pierce, C.; Lower, B. H.; Lower, S. K.; Sooryakumar, R.

    2015-03-01

    Technologies that control matter at the nano- and micro-scale are crucial for developing new engineered materials and devices. While the more traditional approaches for such manipulations often depend on lithographic fabrication, they can be expanded upon by taking advantage of the biological systems within a living cell which also operate on the nano- and micro- scale. In this study, a system is being developed to functionalize a targeted location on the surface of a chip with the protein AmCyan from transformed Escherichia coli cells. Using established methods in molecular biology where a plasmid with the amcyan gene sequence is inserted into the cell, E. coli are engineered to express the AmCyan protein on their outer surface. In order to transport the cells to the targeted location, the transformed E. coli are labeled with superparamagnetic micro-beads which exert directed forces on the cells in an external field. Preliminary results of the protein expression on E. coli, the transport of the cell through weak magnetic fields to targeted locations and the potential to transfer protein from the cell to the chip surface will be presented.

  14. Extraintestinal Escherichia coli carrying virulence genes in coastal marine sediments.

    PubMed

    Luna, G M; Vignaroli, C; Rinaldi, C; Pusceddu, A; Nicoletti, L; Gabellini, M; Danovaro, R; Biavasco, F

    2010-09-01

    Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx(1), stx(2), aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological

  15. Enteroaggregative Escherichia coli an emergent pathogen with different virulence properties.

    PubMed

    Villaseca, J M; Hernández, U; Sainz-Espuñes, T R; Rosario, C; Eslava, C

    2005-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emergent bacterial pathogen. The first studies in developing countries with EAEC strains, showed that this bacterium was associated with persistent diarrhea. However, new studies showed that EAEC may be associated also with acute diarrhea, with both nosocomial and community outbreaks worldwide, and as an important pathogen of diarrheal disease in human immunodeficiency virus-infected adults. EAEC strains are recognized by their characteristic aggregative adherence or "stacked-brick" pattern to epithelial cells. Although the pathogenesis of EAEC infection is not well understood, cellular changes observed in animal models and in vitro assays, suggested that the alterations in the intestinal mucosa during EAEC infection are associated with adherence factors and toxins production. The damage has been associated with the release of inflammatory mediators, which may contribute also to the intestinal illness. The dissemination of the high pathogenicity island from Yersinia pestis evolutionary group to EAEC has been show; different studies suggest that it may contribute to the virulence of EAEC strains. Molecular methods to investigate the presence of plasmid and chromosomal EAEC-associated virulence markers, have been used for the characterization and epidemiological studies of EAEC strains. Although the clinical and epidemiological importance of EAEC have been demonstrated in different studies, Escherichia coli strains with adherent agreggative phenotype are commonly isolated from healthy children and environmental sources. This support the necessity to study virulence factors no related with the cells adherence pattern, that show the specific EAEC pathogenic clones associated whit intestinal disease.

  16. Protein turnover in the cell cycle of Escherichia coli.

    PubMed

    Nishi, A; Kogoma, T

    1965-10-01

    Nishi, Arasuke (University of Tokyo, Tokyo, Japan), and Tokio Kogoma. Protein turnover in the cell cycle of Escherichia coli. J. Bacteriol. 90:884-890. 1965.-Protein metabolism and enzyme formation throughout the cell cycle were investigated in synchronized cultures of Escherichia coli. The cells showed a temporary cessation of the net increase of bulk protein and of constitutive beta-galactosidase activity during the division period. By contrast, when tested by short-term experiments performed with cells at different growth stages, the bacteria displayed a constant incorporation of labeled protein precursors into the protein fraction, even during the fission period. Similar results were obtained with respect to the capacities for induced enzyme formation. On the other hand, when the cells were previously labeled and then subjected to synchronization in a nonradioactive medium, the radioactivity of the protein fraction decreased temporarily by nearly 10% during the fission period and then regained its previous level at the beginning of the ensuing phase of growth. This indicates that the products of partial degradation of protein were again utilized for protein synthesis in the next cell cycle. It was concluded that the temporary lagging of net increase of bulk protein may be due to the partial breakdown of protein occurring during the fission period.

  17. DNA-damaging activity of patulin in Escherichia coli.

    PubMed Central

    Lee, K S; Röschenthaler, R J

    1986-01-01

    At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity. PMID:2431653

  18. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI I.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. I. Effect of tryptophan and related compounds. J. Bacteriol. 84:979–987. 1962.—The effect of tryptophan and related compounds on tryptophanase and tryptophan synthetase formation in Escherichia coli was determined. Several of these compounds stimulated the formation of tryptophanase while concomitantly decreasing the production of synthetase. A number of tryptophan analogues were found to inhibit growth. The possible mode of action of these substances was examined further. 5-Hydroxytryptophan greatly inhibited the formation of synthetase and also reduced growth. Its inhibitory action on growth was attributed, at least partially, to the false feedback inhibition of anthranilic acid formation. Tryptamine was found to be a potent inhibitor of the activity of synthetase, as well as of the enzyme(s) involved in the synthesis of anthranilic acid from shikimic acid. However, growth reduction was only partially reversed by tryptophan. Indole-3-acetic acid and indole-3-propionic acid decreased growth and increased the formation of synthetase six- to eightfold. The action of these compounds was ascribed to their ability to block the endogenous formation of tryptophan. PMID:13959621

  19. Translocation and thermal inactivation of Shiga-toxin producing Escherichia coli in non-intact beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We compared translocation of genetically-marked strains of serotype O157:H7 Escherichia coli (ECOH) to non-O157:H7 Shiga-Toxin producing Escherichia coli (STEC) following blade tenderization of beef subprimals and the subsequent lethality of these pathogens following cooking of steaks prepared from ...

  20. High-Efficiency Expression of TAT-bFGF Fusion Protein in Escherichia coli and the Effect on Hypertrophic Scar Tissue

    PubMed Central

    Jia, Xuechao; Tian, Haishan; Tang, Lu; Zheng, Long; Zheng, Lulu; Yang, Ting; Yu, Bingjie; Wang, Zhitao; Lin, Peng; Li, Xiaokun; Wang, Xiaojie

    2015-01-01

    Background Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family that has effects on wounding healing and neuro-protection. However, it is difficult to use bFGF to treat diseases that are separated by physiological barriers, such as the dermal barrier and blood brain barrier. Methodology/Principal Findings To improve bFGF’s penetration ability, we fused the recombinant human fibroblast growth factor (rhbFGF) gene with TAT. We constructed a pET3c vector that contained the recombinant bFGF gene and successfully expressed this gene in the E. coli strain BL21 (DE3) pLsS. The fusion protein was purified using CM Sepharose FF and heparin affinity chromatography. The purity of the TAT-rhbFGF was greater than 95%, as detected by SDS-PAGE. An in vitro MTT trial revealed that the modified bFGF significantly promoted the proliferation of NIH3T3 cells. The cell penetration trial and the mouse skin penetration trial demonstrated that the fusion protein had certain penetration abilities. The animal experiments confirmed that TAT-rhbFGF was effective in the treatment of the hypertrophic scars. Conclusions/Significance We have successfully expressed and purified a TAT-rhbFGF fusion protein in this study. Our results have shown that the fusion protein had a greater ability to penetrate the dermal skin layer. TAT-rhbFGF improved the physical appearance of hypertrophic scars. TAT-rhbFGF may be a potential fusion protein in the treatment of dermal disorders, including hypertrophic scar. PMID:25706539

  1. Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter.

    PubMed

    Chao, Yun-Peng; Chiang, Chung-Jen; Hung, Wen-Bin

    2002-01-01

    The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.

  2. [Expression of GST-3B fusion protein of Escherichia coli of Ee strain producing SLT-IIe toxin and study on its biological activities and immunogenicity].

    PubMed

    Liu, Guo-ping; Wu, Bin; Lin, Yi-yuan; Jin, Mei-lin; Chen, Huan-chun

    2007-08-01

    Three copies of DNA fragment encoding the truncated SLT-IIeB of Ee strain which was responsible for the edema disease in piglets in Hubei province were fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pK3 B. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-3B fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against diseases of edema of swine. The fusion protein was further purified and used as an antigen for receptor-binding inhibition assay. The receptor-binding inhibition assay showed GST-3B fusion protein had more strong biological activities than GST-B. The fusion protein of GST-3B or GST-B was purified and emulsified with Freund' s incomplete adjuvant in equal volumes to get subunit bacterin. Groups of SPF KM mice were vaccinated subcutaneously at 0 week with 25 micrograms and at 2 weeks with 25 micrograms of purified GST-3B or GST-B and challenged intraperitoneally with volume of 5 x OD50 Ee strain. Serological tests were performed one week interval with ELISA. The IgG titres against SLT-IIeB in the sera collected at the same period from the Group GST-3B were higher than in the Group GST-B and the immune protection rate against Ee strain was respectively 60% and 40%. These results show the fusion protein GST-3B had more strong biological activities, immunogenicity and better protection against Ee strain, which built a good foundation for the further research of high efficacy vaccine against porcine edema disease.

  3. F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

    PubMed Central

    Mergeay, M; Gerits, J

    1978-01-01

    Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4). PMID:97267

  4. Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli

    PubMed Central

    Blum, Shlomo E.; Heller, Elimelech D.; Sela, Shlomo; Elad, Daniel; Edery, Nir; Leitner, Gabriel

    2015-01-01

    Escherichia coli is a major etiological agent of intra-mammary infections (IMI) in cows, leading to acute mastitis and causing great economic losses in dairy production worldwide. Particular strains cause persistent IMI, leading to recurrent mastitis. Virulence factors of mammary pathogenic E. coli (MPEC) involved pathogenesis of mastitis as well as those differentiating strains causing acute or persistent mastitis are largely unknown. This study aimed to identify virulence markers in MPEC through whole genome and phenome comparative analysis. MPEC strains causing acute (VL2874 and P4) or persistent (VL2732) mastitis were compared to an environmental strain (K71) and to the genomes of strains representing different E. coli pathotypes. Intra-mammary challenge in mice confirmed experimentally that the strains studied here have different pathogenic potential, and that the environmental strain K71 is non-pathogenic in the mammary gland. Analysis of whole genome sequences and predicted proteomes revealed high similarity among MPEC, whereas MPEC significantly differed from the non-mammary pathogenic strain K71, and from E. coli genomes from other pathotypes. Functional features identified in MPEC genomes and lacking in the non-mammary pathogenic strain were associated with synthesis of lipopolysaccharide and other membrane antigens, ferric-dicitrate iron acquisition and sugars metabolism. Features associated with cytotoxicity or intra-cellular survival were found specifically in the genomes of strains from severe and acute (VL2874) or persistent (VL2732) mastitis, respectively. MPEC genomes were relatively similar to strain K-12, which was subsequently shown here to be possibly pathogenic in the mammary gland. Phenome analysis showed that the persistent MPEC was the most versatile in terms of nutrients metabolized and acute MPEC the least. Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism. This study

  5. Nucleotide sequence of an Escherichia coli chromosomal hemolysin.

    PubMed Central

    Felmlee, T; Pellett, S; Welch, R A

    1985-01-01

    We determined the DNA sequence of an 8,211-base-pair region encompassing the chromosomal hemolysin, molecularly cloned from an O4 serotype strain of Escherichia coli. All four hemolysin cistrons (transcriptional order, C, A, B, and D) were encoded on the same DNA strand, and their predicted molecular masses were, respectively, 19.7, 109.8, 79.9, and 54.6 kilodaltons. The identification of pSF4000-encoded polypeptides in E. coli minicells corroborated the assignment of the predicted polypeptides for hlyC, hlyA, and hlyD. However, based on the minicell results, two polypeptides appeared to be encoded on the hlyB region, one similar in size to the predicted molecular mass of 79.9 kilodaltons, and the other a smaller 46-kilodalton polypeptide. The four hemolysin gene displayed similar codon usage, which is atypical for E. coli. This reflects the low guanine-plus-cytosine content (40.2%) of the hemolysin DNA sequence and suggests the non-E. coli origin of the hemolysin determinant. In vitro-derived deletions of the hemolysin recombinant plasmid pSF4000 indicated that a region between 433 and 301 base pairs upstream of the putative start of hlyC is necessary for hemolysin synthesis. Based on the DNA sequence, a stem-loop transcription terminator-like structure (a 16-base-pair stem followed by seven uridylates) in the mRNA was predicted distal to the C-terminal end of hlyA. A model for the general transcriptional organization of the E. coli hemolysin determinant is presented. Images PMID:3891743

  6. Discovery of Escherichia coli CRISPR sequences in an undergraduate laboratory.

    PubMed

    Militello, Kevin T; Lazatin, Justine C

    2016-09-28

    Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers. Students started the project by isolating genomic DNA from E. coli and amplifying the iap CRISPR locus using the polymerase chain reaction (PCR). The PCR products were analyzed by Sanger DNA sequencing, and the sequences were examined for the presence of CRISPR repeat sequences. The regions between the repeats, the spacers, were extracted and analyzed with BLASTN searches. Overall, CRISPR loci were sequenced from several previously uncharacterized E. coli strains and one E. coli K-12 strain. Sanger DNA sequencing resulted in the discovery of 36 spacer sequences and their corresponding surrounding repeat sequences. Five of the spacers were homologous to foreign (non-E. coli) DNA. Assessment of the laboratory indicates that improvements were made in the ability of students to answer questions relating to the structure and function of CRISPRs. Future directions of the laboratory are presented and discussed. © 2016 by The International Union of Biochemistry and Molecular Biology, 2016.

  7. Anaerobic respiration of Escherichia coli in the mouse intestine.

    PubMed

    Jones, Shari A; Gibson, Terri; Maltby, Rosalie C; Chowdhury, Fatema Z; Stewart, Valley; Cohen, Paul S; Conway, Tyrrell

    2011-10-01

    The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in

  8. A Novel Putrescine Exporter SapBCDF of Escherichia coli.

    PubMed

    Sugiyama, Yuta; Nakamura, Atsuo; Matsumoto, Mitsuharu; Kanbe, Ayaka; Sakanaka, Mikiyasu; Higashi, Kyohei; Igarashi, Kazuei; Katayama, Takane; Suzuki, Hideyuki; Kurihara, Shin

    2016-12-16

    Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.

  9. Genetic determinants of heat resistance in Escherichia coli

    PubMed Central

    Mercer, Ryan G.; Zheng, Jinshui; Garcia-Hernandez, Rigoberto; Ruan, Lifang; Gänzle, Michael G.; McMullen, Lynn M.

    2015-01-01

    Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of β- and γ-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7ΔpHR1 and DH5α. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the β- and γ-proteobacteria and is a reliable indicator of high heat resistance in E. coli. PMID:26441869

  10. FILAMENT FORMATION BY ESCHERICHIA COLI AT INCREASED HYDROSTATIC PRESSURES1

    PubMed Central

    Zobell, Claude E.; Cobet, Andre B.

    1964-01-01

    ZoBell, Claude E. (University of California, La Jolla), and Andre B. Cobet. Filament formation by Escherichia coli at increased hydrostatic pressures. J. Bacteriol. 87:710–719. 1964.—The reproduction as well as the growth of Escherichia coli is retarded by hydrostatic pressures ranging from 200 to 500 atm. Reproduction was indicated by an increase in the number of cells determined by plating on EMB Agar as well as by direct microscopic counts. Growth, which is not necessarily synonymous with reproduction, was indicated by increase in dry weight and protein content of the bacterial biomass. At increased pressures, cells of three different strains of E. coli tended to form long filaments. Whereas most normal cells of E. coli that developed at 1 atm were only about 2 μ long, the mean length of those that developed at 475 atm was 2.93 μ for strain R4, 3.99 μ for strain S, and 5.82 μ for strain B cells. Nearly 90% of the bacterial biomass produced at 475 atm by strain B was found in filaments exceeding 5 μ in length; 74.7 and 16.4% of the biomass produced at 475 atm by strains S and R4, respectively, occurred in such filaments. Strain R4 formed fewer and shorter (5 to 35 μ) filaments than did the other two strains, whose filaments ranged in length from 5 to >100 μ. The bacterial biomass produced at all pressures had approximately the same content of protein and nucleic acids. But at increased pressures appreciably more ribonucleic acid (RNA) and proportionately less deoxyribonucleic acid (DNA) was found per unit of biomass. Whereas the RNA content per cell increased with cell length, the amount of DNA was nearly the same in long filaments formed at increased pressure as in cells of normal length formed at 1 atm. The inverse relationship between the concentration of DNA and cell length in all three strains of E. coli suggests that the failure of DNA to replicate at increased pressure may be responsible for a repression of cell division and consequent filament

  11. Metabolic self-organization of bioluminescent Escherichia coli.

    PubMed

    Simkus, Remigijus; Baronas, Romas

    2011-01-01

    A possible reason for the complexity of the signals produced by bioluminescent biosensors might be self-organization of the cells. In order to verify this possibility, bioluminescence images of cultures of lux gene reporter Escherichia coli were recorded for several hours after being placed into 8-10 mm diameter cylindrical containers. It was found that luminous cells distribute near the three-phase contact line, forming irregular azimuthal waves. As we show, space-time plots of quasi-one-dimensional bioluminescence measured along the contact line can be simulated by reaction-diffusion-chemotaxis equations, in which the reaction term for the cells is a logistic (autocatalytic) growth function. It was found that the growth rate of the luminous cells (~0.02 s(-1)) is >100 times higher than the growth rate of E. coli. We provide an explanation for this result by assuming that E. coli exhibits considerable respiratory flexibility (the ability of oxygen-induced switching from one metabolic pathway to another). According to the simple two-state model presented here, the number of oxic (luminous) cells grows at the expense of anoxic (dark) cells, whereas the total number of (oxic and anoxic) cells remains unchanged. It is conjectured that the corresponding reaction-diffusion-chemotaxis model for bioluminescence pattern formation can be considered as a model for the energy-taxis and metabolic self-organization in the population of the metabolically flexible bacteria under hypoxic conditions.

  12. Properties of a Clostridium thermocellum Endoglucanase Produced in Escherichia coli

    PubMed Central

    Schwarz, Wolfgang H.; Gräbnitz, Folke; Staudenbauer, Walter L.

    1986-01-01

    A cellulase gene of Clostridium thermocellum was transferred to Escherichia coli by molecular cloning with bacteriophage lambda and plasmid vectors and shown to be indentical with the celA gene. The celA gene product was purified from extracts of plasmid-bearing E. coli cells by heat treatment and chromatography on DEAE-Trisacryl. It was characterized as a thermophilic endo-β-1,4-glucanase, the properties of which closely resemble those of endoglucanase A previously isolated from C. thermocellum supernatants. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme purified from E. coli exhibited two protein bands with molecular weights of 49,000 and 52,000. It had a temperature optimum at 75°C and was stable for several hours at 60°C. Endoglucanase activity was optimal between pH 5.5 and 6.5. The enzyme was insensitive against end product inhibition by glucose and cellobiose and remarkably resistant to the denaturing effects of detergents and organic solvents. It was capable of degrading, in addition to cellulosic substrates, glucans with alternating β-1,4 and β-1,3 linkages such as barley β-glucan and lichenan. PMID:16347088

  13. Comprehensive Mapping of the Escherichia coli Flagellar Regulatory Network

    PubMed Central

    Fitzgerald, Devon M.; Bonocora, Richard P.; Wade, Joseph T.

    2014-01-01

    Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process. PMID:25275371

  14. Global regulation of gene expression in Escherichia coli.

    PubMed Central

    Chuang, S E; Daniels, D L; Blattner, F R

    1993-01-01

    Global transcription responses of Escherichia coli to various stimuli or genetic defects were studied by measuring mRNA levels in about 400 segments of the genome. Measuring mRNA levels was done by analyzing hybridization to DNA dot blots made with overlapping lambda clones spanning the genome of E. coli K-12. Conditions examined included isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, heat shock, osmotic shock, starvation for various nutrients, entrance of cells into the stationary phase of growth, anaerobic growth in a tube, growth in the gnotobiotic mouse gut, and effects of pleiotropic mutations rpoH, himA, topA, and crp. Most mapped genes known to be regulated by a particular situation were successfully detected. In addition, many chromosomal regions containing no previously known regulated genes were discovered that responded to various stimuli. This new method for studying globally regulated genetic systems in E. coli combines detection, cloning, and physical mapping of a battery of coregulated genes in one step. Images PMID:8458845

  15. Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

    PubMed Central

    Garrett, Teresa A.; Raetz, Christian R. H.; Richardson, Travis; Kordestani, Reza; Son, Jennifer D.; Rose, Rebecca L.

    2009-01-01

    Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655. PMID:19096047

  16. Improving alkane synthesis in Escherichia coli via metabolic engineering.

    PubMed

    Song, Xuejiao; Yu, Haiying; Zhu, Kun

    2016-01-01

    Concerns about energy security and global petroleum supply have made the production of renewable biofuels an industrial imperative. The ideal biofuels are n-alkanes in that they are chemically and structurally identical to the fossil fuels and can "drop in" to the transportation infrastructure. In this work, an Escherichia coli strain that produces n-alkanes was constructed by heterologous expression of acyl-acyl carrier protein (ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) from Synechococcus elongatus PCC7942. The accumulation of alkanes ranged from 3.1 to 24.0 mg/L using different expressing strategies. Deletion of yqhD, an inherent aldehyde reductase in E. coli, or overexpression of fadR, an activator for fatty acid biosynthesis, exhibited a nearly twofold increase in alkane titers, respectively. Combining yqhD deletion and fadR overexpression resulted in a production titer of 255.6 mg/L in E. coli, and heptadecene was the most abundant product.

  17. Production of 2-methyl-1-butanol in engineered Escherichia coli.

    PubMed

    Cann, Anthony F; Liao, James C

    2008-11-01

    Recent progress has been made in the production of higher alcohols by harnessing the power of natural amino acid biosynthetic pathways. Here, we describe the first strain of Escherichia coli developed to produce the higher alcohol and potential new biofuel 2-methyl-1-butanol (2MB). To accomplish this, we explored the biodiversity of enzymes catalyzing key parts of the isoleucine biosynthetic pathway, finding that AHAS II (ilvGM) from Salmonella typhimurium and threonine deaminase (ilvA) from Corynebacterium glutamicum improve 2MB production the most. Overexpression of the native threonine biosynthetic operon (thrABC) on plasmid without the native transcription regulation also improved 2MB production in E. coli. Finally, we knocked out competing pathways upstream of threonine production (DeltametA, Deltatdh) to increase its availability for further improvement of 2MB production. This work led to a strain of E. coli that produces 1.25 g/L 2MB in 24 h, a total alcohol content of 3 g/L, and with yields of up to 0.17 g 2MB/g glucose.

  18. Reductive transformation of TNT by Escherichia coli: pathway description.

    PubMed

    Yin, Hong; Wood, Thomas K; Smets, Barth F

    2005-05-01

    The reductive transformation of 2,4,6-trinitrotoluene (TNT) was studied using aerobically grown Escherichia coli cultures. In the absence of an external carbon or energy source, E. coli resting cells transformed TNT to hydroxylaminodinitrotoluenes (2HADNT, 4HADNT, with 4HADNT as the dominant isomer), aminodinitrotoluenes (4ADNT, with sporadic detection of 2ADNT), 2,4-di(hydroxylamino)-6-nitrotoluene (24D(HA)6NT), 2,4-diamino-6-nitrotoluene (24DA6NT), and an additional compound which was tentatively identified as a (hydroxylamino)aminonitrotoluene isomer via gas chromatography/mass spectroscopy and spectral analysis. The resting cell assay, performed in an oxygen-free atmosphere, avoided formation of azoxy dimers and provided good mass balances. Significant preference for reduction in the para versus ortho position was detected. The formation of 24D(HA)6NT, but not ADNT, appeared inhibited by the presence of TNT. The rate and extent of TNT reduction were significantly enhanced at higher cell densities, or by supplying an exogenous reducing power source, revealing the importance of enzyme concentration and reducing power. Whether the oxygen-insensitive E. coli nitroreductases, encoded by nfsA and nfsB, directly catalyze the TNT reduction or account for the complete TNT transformation pathway, remains to be determined.

  19. Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase.

    PubMed

    Romão, Célia V; Vicente, João B; Borges, Patrícia T; Victor, Bruno L; Lamosa, Pedro; Silva, Elísio; Pereira, Luís; Bandeiras, Tiago M; Soares, Cláudio M; Carrondo, Maria A; Turner, David; Teixeira, Miguel; Frazão, Carlos

    2016-11-20

    Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2 and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-β-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a μ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2 or NO permeation were found. The present data represent the first structure for a NO-selective FDP.

  20. Identification of diarrheagenic Escherichia coli strains from avian organic fertilizers.

    PubMed

    Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P; Nishio, Erick K; Kobayashi, Renata K T; Nakazato, Gerson

    2014-08-28

    The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

  1. Heterologous biosynthesis and manipulation of alkanes in Escherichia coli.

    PubMed

    Cao, Ying-Xiu; Xiao, Wen-Hai; Zhang, Jin-Lai; Xie, Ze-Xiong; Ding, Ming-Zhu; Yuan, Ying-Jin

    2016-11-01

    Biosynthesis of alkanes in microbial foundries offers a sustainable and green supplement to traditional fossil fuels. The dynamic equilibrium of fatty aldehydes, key intermediates, played a critical role in microbial alkanes production, due to the poor catalytic capability of aldehyde deformylating oxygenase (ADO). In our study, exploration of competitive pathway together with multi-modular optimization was utilized to improve fatty aldehydes balance and consequently enhance alkanes formation in Escherichia coli. Endogenous fatty alcohol formation was supposed to be competitive with alkane production, since both of the two routes consumed the same intermediate-fatty aldehyde. Nevertheless, in our case, alkanes production in E. coli was enhanced from trace amount to 58.8mg/L by the facilitation of moderate fatty alcohol biosynthesis, which was validated by deletion of endogenous aldehyde reductase (AHR), overexpression of fatty alcohol oxidase (FAO) and consequent transcriptional assay of aar, ado and adhP genes. Moreover, alkanes production was further improved to 81.8mg/L, 86.6mg/L or 101.7mg/L by manipulation of fatty acid biosynthesis, lipids degradation or electron transfer system modules, which directly referenced to fatty aldehydes dynamic pools. A titer of 1.31g/L alkanes was achieved in 2.5L fed-batch fermentation, which was the highest reported titer in E. coli. Our research has offered a reference for chemical overproduction in microbial cell factories facilitated by exploring competitive pathway.

  2. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli

    PubMed Central

    Kay, Emily J.; Yates, Laura E.; Terra, Vanessa S.; Cuccui, Jon; Wren, Brendan W.

    2016-01-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  3. Characterization of the YdeO regulon in Escherichia coli.

    PubMed

    Yamanaka, Yuki; Oshima, Taku; Ishihama, Akira; Yamamoto, Kaneyoshi

    2014-01-01

    Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD) system plays a major role in acid resistance in Escherichia coli, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip to interrogate the E. coli genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of E. coli survival in both acidic and anaerobic conditions.

  4. Baicalin Protects Mice from Lethal Infection by Enterohemorrhagic Escherichia coli.

    PubMed

    Zhang, Yong; Qi, Zhimin; Liu, Yan; He, Wenqi; Yang, Cheng; Wang, Quan; Dong, Jing; Deng, Xuming

    2017-01-01

    Shiga-like toxin-producing Escherichia coli (STEC) O157:H7 poses grave challenges to public health by its ability to cause severe colonic diseases and renal failure in both human and animals. Shiga-like toxins are the major pathogenic factor for some highly virulent E. coli expecially Shiga-like toxin 2. Conventional treatments such as antibiotics can facilitate the release of the toxin thus potentially exacerbate the diseases. Small molecule inhibitors and antibodies capable of neutralizing the toxins are the two major venues for the development of therapeutics against enterohemorrhagic serotype E. coli infection. While promising and potentially effective at clinical settings, these approaches need to overcome obstacles such as the limited routes of administration, responses from the host immune system, which are known to differ greatly among individuals. Our previous studies demonstrate that Baicalin (BAI), a flavonoid compound isolated from Scutellaria baicalensis protects against rStx2-induced cell cytotoxicity and also protects mice from lethal rStx2 challenges by inducing Stx2 to form inactive oligomers. In this manuscript, we present some exciting work showing that baicalin is an effective agent for therapeutic treatment of STEC O157:H7 infection.

  5. Microaerobic conversion of glycerol to ethanol in Escherichia coli.

    PubMed

    Wong, Matthew S; Li, Mai; Black, Ryan W; Le, Thao Q; Puthli, Sharon; Campbell, Paul; Monticello, Daniel J

    2014-05-01

    Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.

  6. Escherichia coli Chromosomal Loci Segregate from Midcell with Universal Dynamics.

    PubMed

    Cass, Julie A; Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2016-06-21

    The structure of the Escherichia coli chromosome is inherently dynamic over the duration of the cell cycle. Genetic loci undergo both stochastic motion around their initial positions and directed motion to opposite poles of the rod-shaped cell during segregation. We developed a quantitative method to characterize cell-cycle dynamics of the E. coli chromosome to probe the chromosomal steady-state mobility and segregation process. By tracking fluorescently labeled chromosomal loci in thousands of cells throughout the entire cell cycle, our method allows for the statistical analysis of locus position and motion, the step-size distribution for movement during segregation, and the locus drift velocity. The robust statistics of our detailed analysis of the wild-type E. coli nucleoid allow us to observe loci moving toward midcell before segregation occurs, consistent with a replication factory model. Then, as segregation initiates, we perform a detailed characterization of the average segregation velocity of loci. Contrary to origin-centric models of segregation, which predict distinct dynamics for oriC-proximal versus oriC-distal loci, we find that the dynamics of loci were universal and independent of genetic position.

  7. The D-allose operon of Escherichia coli K-12.

    PubMed Central

    Kim, C; Song, S; Park, C

    1997-01-01

    Escherichia coli K-12 can utilize D-allose, an all-cis hexose, as a sole carbon source. The operon responsible for D-allose metabolism was localized at 92.8 min of the E. coli linkage map. It consists of six genes, alsRBACEK, which are inducible by D-allose and are under the control of the repressor gene alsR. This operon is also subject to catabolite repression. Three genes, alsB, alsA, and alsC, appear to be necessary for transport of D-allose. D-Allose-binding protein, encoded by alsB, is a periplasmic protein that has an affinity for D-allose, with a Kd of 0.33 microM. As was found for other binding-protein-mediated ABC transporters, the allose transport system includes an ATP-binding component (AlsA) and a transmembrane protein (AlsC). It was found that AlsE (a putative D-allulose-6-phosphate 3-epimerase), but not AlsK (a putative D-allose kinase), is necessary for allose metabolism. During this study, we observed that the D-allose transporter is partially responsible for the low-affinity transport of D-ribose and that strain W3110, an E. coli prototroph, has a defect in the transport of D-allose mediated by the allose permease. PMID:9401019

  8. Escherichia coli bacteria detection by using graphene-based biosensor.

    PubMed

    Akbari, Elnaz; Buntat, Zolkafle; Afroozeh, Abdolkarim; Zeinalinezhad, Alireza; Nikoukar, Ali

    2015-10-01

    Graphene is an allotrope of carbon with two-dimensional (2D) monolayer honeycombs. A larger detection area and higher sensitivity can be provided by graphene-based nanosenor because of its 2D structure. In addition, owing to its special characteristics, including electrical, optical and physical properties, graphene is known as a more suitable candidate compared to other materials used in the sensor application. A novel model employing a field-effect transistor structure using graphene is proposed and the current-voltage (I-V) characteristics of graphene are employed to model the sensing mechanism. This biosensor can detect Escherichia coli (E. coli) bacteria, providing high levels of sensitivity. It is observed that the graphene device experiences a drastic increase in conductance when exposed to E. coli bacteria at 0-10(5) cfu/ml concentration. The simple, fast response and high sensitivity of this nanoelectronic biosensor make it a suitable device in screening and functional studies of antibacterial drugs and an ideal high-throughput platform which can detect any pathogenic bacteria. Artificial neural network and support vector regression algorithms have also been used to provide other models for the I-V characteristic. A satisfactory agreement has been presented by comparison between the proposed models with the experimental data.

  9. Butyrate production under aerobic growth conditions by engineered Escherichia coli.

    PubMed

    Kataoka, Naoya; Vangnai, Alisa S; Pongtharangkul, Thunyarat; Yakushi, Toshiharu; Matsushita, Kazunobu

    2017-01-11

    Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.

  10. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  11. Rotational tumbling of Escherichia coli aggregates under shear

    NASA Astrophysics Data System (ADS)

    Portela, R.; Patrício, P.; Almeida, P. L.; Sobral, R. G.; Franco, J. M.; Leal, C. R.

    2016-12-01

    Growing living cultures of Escherichia coli bacteria are investigated using real-time in situ rheology and rheoimaging measurements. In the early stages of growth (lag phase) and when subjected to a constant stationary shear, the viscosity slowly increases with the cell's population. As the bacteria reach the exponential phase of growth, the viscosity increases rapidly, with sudden and temporary abrupt decreases and recoveries. At a certain stage, corresponding grossly to the late phase of growth, when the population stabilizes, the viscosity also keeps its maximum constant value, with drops and recoveries, for a long period of time. This complex rheological behavior, which is observed to be shear strain dependent, is a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties. Particular attention is given to the late phase of growth of E. coli populations under shear. Rheoimaging measurements reveal, near the static plate, a rotational motion of E. coli aggregates, collectively tumbling and flowing in the shear direction. This behavior is interpreted in the light of a simple theoretical approach based on simple rigid body mechanics.

  12. Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

    PubMed Central

    Wong, Matthew S.; Li, Mai; Black, Ryan W.; Le, Thao Q.; Puthli, Sharon; Campbell, Paul

    2014-01-01

    Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process. PMID:24584248

  13. Engineering Escherichia coli for Microbial Production of Butanone

    PubMed Central

    Srirangan, Kajan; Liu, Xuejia; Akawi, Lamees; Bruder, Mark; Moo-Young, Murray

    2016-01-01

    To expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway in Escherichia coli for heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producing E. coli host strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohologenesis under selected fermentative culture conditions were investigated. Using the engineered E. coli strain for batch cultivation with 30 g liter−1 glycerol as the carbon source, we achieved coproduction of 1.3 g liter−1 butanone and 2.9 g liter−1 acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform. PMID:26896132

  14. Baicalin Protects Mice from Lethal Infection by Enterohemorrhagic Escherichia coli

    PubMed Central

    Zhang, Yong; Qi, Zhimin; Liu, Yan; He, Wenqi; Yang, Cheng; Wang, Quan; Dong, Jing; Deng, Xuming

    2017-01-01

    Shiga-like toxin-producing Escherichia coli (STEC) O157:H7 poses grave challenges to public health by its ability to cause severe colonic diseases and renal failure in both human and animals. Shiga-like toxins are the major pathogenic factor for some highly virulent E. coli expecially Shiga-like toxin 2. Conventional treatments such as antibiotics can facilitate the release of the toxin thus potentially exacerbate the diseases. Small molecule inhibitors and antibodies capable of neutralizing the toxins are the two major venues for the development of therapeutics against enterohemorrhagic serotype E. coli infection. While promising and potentially effective at clinical settings, these approaches need to overcome obstacles such as the limited routes of administration, responses from the host immune system, which are known to differ greatly among individuals. Our previous studies demonstrate that Baicalin (BAI), a flavonoid compound isolated from Scutellaria baicalensis protects against rStx2-induced cell cytotoxicity and also protects mice from lethal rStx2 challenges by inducing Stx2 to form inactive oligomers. In this manuscript, we present some exciting work showing that baicalin is an effective agent for therapeutic treatment of STEC O157:H7 infection. PMID:28337193

  15. Characterization of Enterohemorrhagic Escherichia coli on Veal Hides and Carcasses.

    PubMed

    Bosilevac, Joseph M; Wang, Rong; Luedtke, Brandon E; Hinkley, Susanne; Wheeler, Tommy L; Koohmaraie, Mohammad

    2017-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are Shiga toxin-producing E. coli associated with the most severe forms of foodborne illnesses. The U.S. Department of Agriculture, Food Safety and Inspection Service has identified a higher percentage of non-O157 EHEC compared with E. coli O157:H7-positive samples collected from veal trimmings than from products produced from other cattle slaughter classes. Therefore samples were collected from hides and preevisceration carcasses at five veal processors to assess E. coli O157:H7 and non-O157 EHEC contamination during bob veal and formula-fed veal dressing procedures. E. coli O157:H7 prevalence was measured by culture isolation and found to be on 20.3% of hides and 6.7% of carcasses. In contrast, a non-O157 EHEC molecular screening assay identified 90.3% of hides and 68.2% of carcasses as positive. Only carcass samples were taken forward to culture confirmation and 38.7% yielded one or more non-O157 EHEC isolates. The recovery of an EHEC varied by plant and sample collection date; values ranged from 2.1 to 87.8% among plants and from 4.2 to 64.2% within the same plant. Three plants were resampled after changes were made to sanitary dressing procedures. Between the two collection times at the three plants, hide-to-carcass transfer of E. coli O157:H7 and non-O157 EHEC was significantly reduced. All adulterant EHEC serogroups (O26, O45, O103, O111, O121, and O145) were isolated from veal carcasses as well as four other potentially pathogenic serogroups (O5, O84, O118, and O177). Bob veal was found to have a greater culture prevalence of E. coli O157:H7 and greater positive molecular screens for non-O157 EHEC than formula-fed veal (P < 0.05), but the percentage of culture-confirmed non-O157 EHEC was not different (P > 0.05) between the two types of calves. EHEC-O26, -O111, and -O121 were found more often in bob veal (P < 0.05), whereas EHEC-O103 was found more often in formula-fed veal (P < 0.05).

  16. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  17. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  18. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  19. 40 CFR 180.1301 - Escherichia coli O157:H7 specific bacteriophages; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Escherichia coli O157:H7 specific... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1301 Escherichia coli O157:H7 specific... Escherichia coli O157:H7, sequence negative for shiga toxins I and II, and grown on atoxigenic host...

  20. Modification of emodin and aloe-emodin by glycosylation in engineered Escherihia coli.

    PubMed

    Ghimire, Gopal Prasad; Koirala, Niranjan; Pandey, Ramesh Prasad; Jung, Hye Jin; Sohng, Jae Kyung

    2015-04-01

    Glycosyltransferase from Bacillus licheniformis DSM13 (YjiC) was used for enzymatic modification of emodin and aloe-emodin in vitro and in vivo. In order to increase the availability of UDP-glucose, three genes involved in the production of precursors of NDP-sugar in Escherichia coli BL21 (DE3) viz. D-glucose phosphate isomerase (pgi), D-glucose-6-phosphate dehydrogenase (zwf), and UDP-sugar hydrolase (ushA) were deleted and glucose-1-phosphate urididyltransferase (galU) gene was over expressed. To improve the yield of the products; substrate, time and media parameters were optimized, and the production was scaled up using a 3 L fermentor. The maximum yield of glycosylated products of emodin (emodin-O-β-D-glucoside) and aloe-emodin (aloe-emodin-O-β-D-glucoside) were approximately 144 µM (38 mg/L) and 168 µM (45 mg/L) respectively, representing almost 72 % and 84 % bioconversion of emodin and aloe-emodin when 200 µM of emodin and aloe-emodin were supplemented in the culture. Additionally, the emodin and aloe emodin major glycosylated products exhibited the highest stability at pH 8.0 and the stability of products was up to 70 °C and 60 °C respectively. Furthermore, the biological activities of emodin and its major glucoside (P1) were compared and their anti-cancer activities were assayed in several cancer cell lines. The results demonstrate that YjiC has the capacity to catalyze the glycosylation of these aromatic compounds and that glycosylation of anthraquinones enhances their aqueous solubility while retaining their biological activities.

  1. Production of bioactive chicken follistatin315 in Escherichia coli.

    PubMed

    Lee, Sang Beum; Choi, Rocky; Park, Sung Kwon; Kim, Yong Soo

    2014-12-01

    Follistatin (FST) binds to myostatin (MSTN), a potent negative regulator of skeletal muscle growth. Inhibition of MSTN activity by FST treatment has shown to enhance muscle growth as well as ameliorate symptoms of muscular dystrophy in animal models, illustrating the potential of FST as an agent to enhance muscle growth in animal agriculture or to treat muscle wasting conditions or disease in humans. Therefore, we designed a study to produce biologically active recombinant chicken FST315 (chFST315) in an Escherichia coli host. Since FST contains multiple intramolecular disulfide bonds, we expressed chFST315 protein in either a system that utilizes a periplasmic expression strategy, or a genetically modified E. coli system (SHuffle strain) that is capable of disulfide bond formation in the cytoplasm. Periplasmic expression of chFST315 using the pMAL-p5x vector system, which was designed to express maltose-binding protein (MBP) fusion protein, failed to produce a soluble recombinant protein. However, cytoplasmic expression of chFST315 using pMAL-c5x vector in SHuffle E. coli strain resulted in a soluble expression of the recombinant protein (MBP-chFST315). Combination of heparin and amylose resin affinity chromatography yielded about 6 mg/L purified MBP-chFST315. The purified MBP-chFST315 showed binding affinity to MSTN and activin in a pull-down assay, as well as inhibited MSTN and activin activity in an in vitro reporter gene assay. In conclusion, results of the study demonstrate that for the first time a recombinant, biologically active FST molecule can be produced in a soluble form in E. coli. The ability to produce FST in a cost-effective system is expected to allow us to investigate the potentials of FST as an agent to improve skeletal muscle growth of meat producing animals via suppression of MSTN.

  2. Characterization of three novel mechanosensitive channel activities in Escherichia coli.

    PubMed

    Edwards, Michelle D; Black, Susan; Rasmussen, Tim; Rasmussen, Akiko; Stokes, Neil R; Stephen, Terri-Leigh; Miller, Samantha; Booth, Ian R

    2012-01-01

    Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions.

  3. Azorean wild rabbits as reservoirs of antimicrobial resistant Escherichia coli.

    PubMed

    Marinho, Catarina; Igrejas, Gilberto; Gonçalves, Alexandre; Silva, Nuno; Santos, Tiago; Monteiro, Ricardo; Gonçalves, David; Rodrigues, Tiago; Poeta, Patrícia

    2014-12-01

    Antibiotic resistance in bacteria is an increasing problem that is not only constrained to the clinical setting but also to other environments that can lodge antibiotic resistant bacteria and therefore they may serve as reservoirs of genetic determinants of antibiotic resistance. One hundred and thirty-six faecal samples from European wild rabbits (Oryctolagus cuniculus algirus) were collected on São Jorge Island in Azores Archipelago, and analysed for Escherichia coli isolates. Seventy-seven isolates (56.6%) were recovered and studied for antimicrobial resistance, one isolate per positive sample. Thirteen (16.9%), 19 (24.7%), 25 (32.4%) and 20 (26%) isolates were ascribed to A, B1, B2 and D phylogenetic groups, respectively, by specific primer polymerase chain reaction. Different E. coli isolates were found to be resistant to ampicillin (16.9%), tetracycline (1.3%), streptomycin (42.9%), sulfamethoxazole-trimethoprim (1.3%), amikacin (1.3%), tobramycin (2.6%) and nalidixic acid (1.3%). Additionally, the blaTEM, tetA, strA/strB, aadA, sul1, intI, intI2 and qacEΔ+sul1 genes were found in most resistant isolates. This study showed that E. coli from the intestinal tract of wild rabbits from Azores Archipelago are resistant to widely prescribed antibiotics in medicine and they constitute a reservoir of antimicrobial resistant genes, which may play a significant role in the spread of antimicrobial resistance. Therefore, antibiotic resistant E. coli from Azorean wild rabbits may represent an ecological and public health problem.

  4. Characterization of fimbriae produced by enteropathogenic Escherichia coli.

    PubMed Central

    Girón, J A; Ho, A S; Schoolnik, G K

    1993-01-01

    Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC. Images PMID:7901197

  5. Inactivation kinetics of Escherichia coli by pulsed electron beam.

    PubMed

    Chalise, P R; Hotta, E; Matak, K E; Jaczynski, J

    2007-09-01

    A novel and compact low-energy (keV) high-power pulsed electron beam (e-beam) that utilizes a secondary emission electron gun (SEEG) was designed and constructed. Escherichia coli JM 109 at a concentration of 10(6) CFU/mL was spread-plated on Luria-Bertani (LB) medium and subjected to the SEEG e-beam. The e-beam was administered as 1 or 5 pulses. The duration of a single pulse was constant at 5 micros, e-beam current density was constant at 25 mA/cm2, and e-beam energy varied between 60 and 82.5 keV. Following treatment with the SEEG e-beam, survivors of the irradiated E. coli samples were enumerated by a standard 10-fold dilution and spread-plated. The survivor curves were plotted on logarithmic scale as a function of e-beam dose. The D10-values were calculated as a negative reciprocal of the slope of the survivor curves. The D10-values for E. coli inactivated with 1- and 5-pulse SEEG e-beam were 0.0026 and 0.0217 Gy, respectively. These D10-values were considerably lower than published D10-values for E. coli inactivated with conventional high-energy continuous e-beam, likely due to shorter exposure time (t), greater current density (J), and a pulse mode of the SEEG e-beam. The SEEG e-beam showed promising results for microbial inactivation in a nonthermal manner; however, due to low energy of the SEEG e-beam, current applications are limited to surface decontamination. The SEEG e-beam may be an efficient processing step for surface inactivation of food-borne pathogens on ready-to-eat products, including fresh and leafy vegetables.

  6. [Sensitivity to drugs of Escherichia coli strains isolated from poultry with coli septicemia].

    PubMed

    Giurov, B

    1985-01-01

    Investigations were carried out into the susceptibility of a total of 223 strains of Escherichia coli to therapeutic agents with the employment of the disk diffusion method. The organisms were isolated from internal organs and bone marrow of birds died of coli septicaemia. The serologic classification of the strains was defined with the use of 88 anti-group OK-agglutinating sera obtained through hyperimmunization of rabbits with the following Escherichia coli serotypes: 01-063, 068, 071, 073, 075, 078, 086, 0101, 0103, 0111-0114, 0119, 0124, 0129, 0135-0141, 0146, 0147, and 0149. It was found that serologically the strains referred as follows: 01-41 strains, 02-70 strains, 04-2 strains, 08-3 strains, 026-1 strain, 078-70 strains, 0111-2 strains, 0103-1 strain, 0141-1 strain. The number of untypable strains amounted to 32. Highest number of strains proved sensitive to colistin--96.06%, the remaining drugs following in a descending order: flumequine--95.65%, apramycin - 95.5%, gentamycin--93.72%, amoxicillin--93,8%, amikacin--88.57%, carbenicillin--86.88%, furazolidone--83,13%, and kanamycin--79.36%. High was the percent of strains resistant to tetracycline--66.17%, spectinomycin--61.67%, ampicillin--51.12%, chloramphenicol--50.23%, and streptomycin--44.84%.

  7. Uropathogenic Escherichia coli are less likely than paired fecal E. coli to have CRISPR loci.

    PubMed

    Dang, Trang Nguyen Doan; Zhang, Lixin; Zöllner, Sebastian; Srinivasan, Usha; Abbas, Khadija; Marrs, Carl F; Foxman, Betsy

    2013-10-01

    CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are short fragments of DNA that act as an adaptive immune system protecting bacteria against invasion by phages, plasmids or other forms of foreign DNA. Bacteria without a CRISPR locus may more readily adapt to environmental changes by acquiring foreign genetic material. Uropathogenic Escherichia coli (UPEC) live in a number of environments suggesting an ability to rapidly adapt to new environments. If UPEC are more adaptive than commensal E. coli we would expect that UPEC would have fewer CRISPR loci, and--if loci are present--that they would harbor fewer spacers than CRISPR loci in fecal E. coli. We tested this in vivo by comparing the number of CRISPR loci and spacers, and sensitivity to antibiotics (resistance is often obtained via plasmids) among 81 pairs of UPEC and fecal E. coli isolated from women with urinary tract infection. Each pair included one uropathogen and one commensal (fecal) sample from the same female patient. Fecal isolates had more repeats (p=0.009) and more unique spacers (p<0.0001) at four CRISPR loci than uropathogens. By contrast, uropathogens were more likely than fecal E. coli to be resistant to ampicillin, cefazolin and trimethoprim/sulfamethoxazole. However, no consistent association between CRISPRs and antibiotic resistance was identified. To our knowledge, this is the first study to compare fecal E. coli and pathogenic E. coli from the same individuals, and to test the association of CRISPR loci with antibiotic resistance. Our results suggest that the absence of CRISPR loci may make UPEC more susceptible to infection by phages or plasmids and allow them to adapt more quickly to various environments.

  8. Colonization with extraintestinal pathogenic Escherichia coli among nursing home residents and its relationship to fluoroquinolone resistance.

    PubMed

    Maslow, Joel N; Lautenbach, Ebbing; Glaze, Thomas; Bilker, Warren; Johnson, James R

    2004-09-01

    In a cross-sectional fecal prevalence survey involving 49 residents of a Veterans Affairs nursing home, 59% of subjects were colonized with extraintestinal pathogenic Escherichia coli (ExPEC), 22% were colonized with adhesin-positive E. coli, and 51% were colonized with fluoroquinolone-resistant E. coli. Among 80 unique isolates, adhesins correlated negatively and aerobactin correlated positively with fluoroquinolone resistance.

  9. A glimpse of Escherichia coli O157:H7 survival in soils from eastern China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7 (E. coli O157:H7) is an important food-borne pathogen, which continues to be a major public health concern worldwide. It is known that E. coli O157:H7 survive in soil environment might result in the contamination of fresh produce or water source. To investigate how the soils...

  10. Escherichia coli strain diversity: Selecting isolates for use as pathogen surrogates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Escherichia coli (E. coli) is commonly used as a surrogate for pathogens in research to identify sources of agricultural contamination and to characterize how pathogens persist on plant surfaces. However, E. coli strains are highly diverse, exhibiting differences in physical, chemical and...

  11. Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli (E. coli) food contaminations pose serious health and food safety concerns, and have been the subject of massive food recalls. Shiga toxin 2 (Stx2)-producing E. coli has been identified as the major cause of hemorrhagic colitis and hemolytic uremic syndrome (HUS), the most severe di...

  12. Detection of Escherichia Coli O157:H7 in Fecal Samples in Meat Goats

    ERIC Educational Resources Information Center

    Mobley, Ray; Madden, Uford; Brooks-Walter, Alexis

    2004-01-01

    Studies have reported the isolation of Escherichia coli (E. coli)O157:H7 from pork, lamb and poultry products, and from other animals including deer, horses, dogs, birds and humans. There is limited or no information on the presence of the organism in goats. The objectives of this study were to determine if E. coli O157:H7 was naturally occurring…

  13. Resistance of various shiga toxin-producing Escherichia coli to electrolyzed oxidizing water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The resistance of thirty two strains of Escherichia coli O157:H7 and six major serotypes of non-O157 Shiga toxin- producing E. coli (STEC) plus E. coli O104 was tested against Electrolyzed oxidizing (EO) water using two different methods; modified AOAC 955.16 sequential inoculation method and minim...

  14. Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. Canine E. coli represents a good experimental model useful to study this pathology. Moreover, as des...

  15. A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae

    NASA Technical Reports Server (NTRS)

    Hopkins, C. M.; White, F. F.; Heaton, L. A.; Guikema, J. A.; Leach, J. E.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.

  16. Comparison of whole genome sequences from human and non-human Escherichia coli O26 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) O26 is the second leading E. coli serogroup responsible for human illness outbreaks behind E. coli O157:H7. Recent outbreaks have been linked to emerging pathogenic O26:H11 strains harboring stx2 only. Cattle have been recognized as an important reserv...

  17. SURVIVAL OF ESCHERICHIA COLI 0157:H7 IN DAIRY CATTLE FEED WATER

    EPA Science Inventory

    Cattle feed waters from two dairy farms were used in a study to determine the survival characteristics of the bacterial pathogen Escherichia coli )157:H7 and wild-type E. coli. The E. coli 0157:H7 inoculum consisted of a consortium of isolates obtained from dairy cattle. Fresh ma...

  18. Proteomic differences between Escherichia coli strains that cause transient versus persistent intramammary infections [abstract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature and lasts 2-3 days. However, in a minority of cases, E. coli can cause a persistent intramammary infection. The mechanisms that enable certain strains of E. coli to cause a p...

  19. SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

    PubMed

    Boysen, Anders; Borch, Jonas; Krogh, Thøger Jensen; Hjernø, Karin; Møller-Jensen, Jakob

    2015-09-01

    Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the

  20. Engineering Escherichia coli to synthesize free fatty acids

    PubMed Central

    Lennen, Rebecca M.; Pfleger, Brian F.

    2013-01-01

    Fatty acid metabolism has received significant attention as a route for producing high-energy density, liquid transportation fuels and high-value oleochemicals from renewable feedstocks. If microbes can be engineered to produce these compounds at yields that approach the theoretical limits of 0.3–0.4 g/g glucose, then processes can be developed to replace current petrochemical technologies. Here, we review recent metabolic engineering efforts to maximize production of free fatty acids (FFA) in Escherichia coli, the first step towards production of downstream products. To date, metabolic engineers have succeeded in achieving higher yields of FFA than any downstream products. Regulation of fatty acid metabolism and the physiological effects of fatty acid production will also be reviewed from the perspective of identifying future engineering targets. PMID:23102412

  1. Phenotypic bistability in Escherichia coli's central carbon metabolism

    PubMed Central

    Kotte, Oliver; Volkmer, Benjamin; Radzikowski, Jakub L; Heinemann, Matthias

    2014-01-01

    Fluctuations in intracellular molecule abundance can lead to distinct, coexisting phenotypes in isogenic populations. Although metabolism continuously adapts to unpredictable environmental changes, and although bistability was found in certain substrate-uptake pathways, central carbon metabolism is thought to operate deterministically. Here, we combine experiment and theory to demonstrate that a clonal Escherichia coli population splits into two stochastically generated phenotypic subpopulations after glucose-gluconeogenic substrate shifts. Most cells refrain from growth, entering a dormant persister state that manifests as a lag phase in the population growth curve. The subpopulation-generating mechanism resides at the metabolic core, overarches the metabolic and transcriptional networks, and only allows the growth of cells initially achieving sufficiently high gluconeogenic flux. Thus, central metabolism does not ensure the gluconeogenic growth of individual cells, but uses a population-level adaptation resulting in responsive diversification upon nutrient changes. PMID:24987115

  2. Composition of cardiolipin molecular species in Escherichia coli.

    PubMed Central

    Yokota, K; Kanamoto, R; Kito, M

    1980-01-01

    The composition of the molecular species of acidic phospholipids in Escherichia coli B during the late exponential growth phase at 37 degrees C was determined. Two phosphatidyl groups of cardiolipin, the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of cardiolipin, were isolated by limited hydrolysis with phospholipase C. No significant difference in the composition of the molecular species was found between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties. On the other hand, the composition of the molecular species of phosphatidylglycerol was different from that of cardiolipin. Phosphatidylglycerol contained more of the 1-palmitoyl 2-cis-9,10-methylenehexadecanoyl and 1-palmitoyl 2-cis-11,12-methyleneoctadecanoyl species than did cardiolipin. The difference in the composition of the molecular species between cardiolipin and phosphatidylglycerol may depend on the difference in the turnover rates of both phospholipids. PMID:6988400

  3. Low Ubiquinone Content in Escherichia coli Causes Thiol Hypersensitivity

    PubMed Central

    Zeng, H.; Snavely, I.; Zamorano, P.; Javor, G. T.

    1998-01-01

    Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb− phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery. PMID:9658014

  4. Electron Microscopy of Chloramphenicol-treated Escherichia coli

    PubMed Central

    Morgan, Councilman; Rosenkranz, Herbert S.; Carr, Howard S.; Rose, Harry M.

    1967-01-01

    Thin sections of Escherichia coli were examined by electron microscopy at sequential intervals after addition and then removal of chloramphenicol. The first changes, occurring at 1 hr after exposure to the drug, were disappearance of the ribosomes and aggregation of the nuclear material toward the center of the bacteria. At 2 hr, aggregates of abnormal cytoplasmic granules first appeared and subsequently increased in size. By 23 hr, amorphous, electron-dense material had accumulated within, and at the periphery of, the nuclear matrix. With the removal of chloramphenicol, the bacteria became normal in appearance, passing through a series of stages that were sequential but not synchronous. At 145 min after removal of chloramphenicol, bacteria were encountered in the process of abnormal division. The influence of deoxyribonucleic acid and ribonucleic acid synthesis, and of energy metabolism, upon the changes seen electron microscopically in chloramphenicol-treated cells, was investigated by selectively inhibiting these functions with hydroxyurea, azauracil, and sodium azide, respectively. Images PMID:5337775

  5. Membrane protein production in Escherichia coli cell-free lysates.

    PubMed

    Henrich, Erik; Hein, Christopher; Dötsch, Volker; Bernhard, Frank

    2015-07-08

    Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.

  6. Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli*

    PubMed Central

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-01-01

    Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters. PMID:20525689

  7. Combinatorial method for overexpression of membrane proteins in Escherichia coli.

    PubMed

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-07-30

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

  8. Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli

    PubMed Central

    Lai, YuShuan; Rosenshine, Ilan; Leong, John M.; Frankel, Gad

    2013-01-01

    Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions. PMID:23927593

  9. Filling holes in peptidoglycan biogenesis of Escherichia coli.

    PubMed

    Ruiz, Natividad

    2016-12-01

    The peptidoglycan cell wall is an essential mesh-like structure in most bacteria. It is built outside the cytoplasmic membrane by polymerizing a disaccharide-pentapeptide into glycan chains that are crosslinked by peptides. The disaccharide-pentapeptide is synthetized as a lipid-linked precursor called lipid II, which is exported across the cytoplasmic membrane so that synthases can make new glycan chains. Growth of the peptidoglycan wall requires careful balancing of synthesis of glycan chains and hydrolysis of the preexisting structure to allow incorporation of new material. Recent studies in Escherichia coli have advanced our understanding of lipid II translocation across the membrane and how synthases are regulated to ensure proper envelope growth.

  10. Activity of murein hydrolases in synchronized cultures of Escherichia coli.

    PubMed Central

    Hakenbeck, R; Messer, W

    1977-01-01

    Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited. PMID:321419

  11. High-resolution structure of the Escherichia coli ribosome

    DOE PAGES

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

    2015-03-16

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

  12. Programming a Pavlovian-like conditioning circuit in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Zhang, Haoqian; Lin, Min; Shi, Handuo; Ji, Weiyue; Huang, Longwen; Zhang, Xiaomeng; Shen, Shan; Gao, Rencheng; Wu, Shuke; Tian, Chengzhe; Yang, Zhenglin; Zhang, Guosheng; He, Siheng; Wang, Hao; Saw, Tiffany; Chen, Yiwei; Ouyang, Qi

    2014-01-01

    Synthetic genetic circuits are programmed in living cells to perform predetermined cellular functions. However, designing higher-order genetic circuits for sophisticated cellular activities remains a substantial challenge. Here we program a genetic circuit that executes Pavlovian-like conditioning, an archetypical sequential-logic function, in Escherichia coli. The circuit design is first specified by the subfunctions that are necessary for the single simultaneous conditioning, and is further genetically implemented using four function modules. During this process, quantitative analysis is applied to the optimization of the modules and fine-tuning of the interconnections. Analogous to classical Pavlovian conditioning, the resultant circuit enables the cells to respond to a certain stimulus only after a conditioning process. We show that, although the conditioning is digital in single cells, a dynamically progressive conditioning process emerges at the population level. This circuit, together with its rational design strategy, is a key step towards the implementation of more sophisticated cellular computing.

  13. De novo biosynthesis of Gastrodin in Escherichia coli.

    PubMed

    Bai, Yanfen; Yin, Hua; Bi, Huiping; Zhuang, Yibin; Liu, Tao; Ma, Yanhe

    2016-05-01

    Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

  14. Collective motion in an active suspension of Escherichia coli bacteria

    NASA Astrophysics Data System (ADS)

    Gachelin, J.; Rousselet, A.; Lindner, A.; Clement, E.

    2014-02-01

    We investigate experimentally the emergence of collective motion in the bulk of an active suspension of Escherichia coli bacteria. When increasing the concentration from a dilute to a semi-dilute regime, we observe a continuous crossover from a dynamical cluster regime to a regime of ‘bio-turbulence’ convection patterns. We measure a length scale characterizing the collective motion as a function of the bacteria concentration. For bacteria fully supplied with oxygen, the increase of the correlation length is almost linear with concentration and at the largest concentrations tested, the correlation length could be as large as 24 bacterial body sizes (or 7-8 when including the flagella bundle). In contrast, under conditions of oxygen shortage the correlation length saturates at a value of around 7 body lengths.

  15. A new Escherichia coli cell division gene, ftsK.

    PubMed Central

    Begg, K J; Dewar, S J; Donachie, W D

    1995-01-01

    A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer. PMID:7592387

  16. Curing of an R Factor from Escherichia coli by Trimethoprim

    PubMed Central

    Pinney, R. J.; Smith, J. T.

    1973-01-01

    R factor 1818, which we have shown previously to be eliminated by thymine starvation, was cured from three strains of Escherichia coli K-12 by overnight exposure to trimethoprim. Elimination was abolished in the presence of added thymine or thymidine, which suggests that curing is the result of the induction of thymineless conditions by trimethoprim. Starvation of the required amino acids proline and histidine had little effect on elimination, whereas methionine deprivation enhanced it. R factor curing was abolished by the presence of chloramphenicol, and it is concluded that protein synthesis is required for elimination to occur. It is suggested that elimination may result from the activity of a nuclease which is synthesized or induced during both direct thymine starvation and by trimethoprim treatment. PMID:4597737

  17. K99 surface antigen of Escherichia coli: antigenic characterization.

    PubMed Central

    Isaacson, R E

    1978-01-01

    K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99. Images PMID:83300

  18. Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy.

    PubMed

    Tang, Yulong; Li, Fengna; Tan, Bie; Liu, Gang; Kong, Xiangfeng; Hardwidge, Philip R; Yin, Yulong

    2014-06-25

    The morbidity and mortality in piglets caused by enterotoxigenic Escherichia coli (ETEC) results in large economic losses to the swine industry, but the precise pathogenesis of ETEC-associated diseases remains unknown. Intestinal epithelial cell autophagy serves as a host defense against pathogens. We found that ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC. ETEC triggered autophagy in IPEC-1 cells through a pathway involving the mammalian target of rapamycin (mTOR), the extracellular signal-regulated kinases 1/2 (ERK1/2), and the AMP-activated protein kinase (AMPK).

  19. [Avian Escherichia coli virulence factors associated with coli septicemia in broiler chickens].

    PubMed

    Ramirez Santoyo, R M; Moreno Sala, A; Almanza Marquez, Y

    2001-01-01

    In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility. Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae and 80% were positive for motility. None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied. These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia.

  20. Improvements In Ethanologenic Escherichia Coli and Klebsiella Oxytoca

    SciTech Connect

    Dr. David Nunn

    2010-09-30

    The current Verenium cellulosic ethanol process is based on the dilute-acid pretreatment of a biomass feedstock, followed by a two-stage fermentation of the pentose sugar-containing hydrolysate by a genetically modified ethanologenic Escherichia coli strain and a separate simultaneous saccharification-fermentation (SSF) of the cellulosic fraction by a genetically modified ethanologenic Klebsiella oxytoca strain and a fungal enzyme cocktail. In order to reduce unit operations and produce a fermentation beer with higher ethanol concentrations to reduce distillation costs, we have proposed to develop a simultaneous saccharification co-fermentation (SScF) process, where the fermentation of the pentose-containing hydrolysate and cellulosic fraction occurs within the same fermentation vessel. In order to accomplish this goal, improvements in the ethanologens must be made to address a number of issues that arise, including improved hydrolysate tolerance, co-fermentation of the pentose and hexose sugars and increased ethanol tolerance. Using a variety of approaches, including transcriptomics, strain adaptation, metagenomics and directed evolution, this work describes the efforts of a team of scientists from Verenium, University of Florida, Massachusetts Institute of Technology and Genomatica to improve the E. coli and K. oxytoca ethanologens to meet these requirements.