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Sample records for high-copy t7 escherichia

  1. Detection of Escherichia coli in drinking water using T7 bacteriophage-conjugated magnetic probe.

    PubMed

    Chen, Juhong; Alcaine, Samuel D; Jiang, Ziwen; Rotello, Vincent M; Nugen, Sam R

    2015-09-01

    In this study, we demonstrate a bacteriophage (phage)-based magnetic separation scheme for the rapid detection of Escherichia coli (E. coli) in drinking water. T7 phage is a lytic phage with a broad host range specificity for E. coli. Our scheme was as follows: (1) T7 bacteriophage-conjugated magnetic beads were used to capture and separate E. coli BL21 from drinking water; (2) subsequent phage-mediated lysis was used to release endemic β-galactosidase (β-gal) from the bound bacterial cells; (3) the release of β-gal was detected using chlorophenol red-β-d-galactopyranoside (CRPG), a colorimetric substrate which changes from yellow to red in the presence of β-gal. Using this strategy, we were able to detect E. coli at a concentration of 1 × 10(4) CFU·mL(-1) within 2.5 h. The specificity of the proposed magnetic probes toward E. coli was demonstrated against a background of competing bacteria. By incorporating a pre-enrichment step in Luria-Bertani (LB) broth supplemented with isopropyl β-d-thiogalactopyranoside (IPTG), we were able to detect 10 CFU·mL(-1) in drinking water after 6 h of pre-enrichment. The colorimetric change can be determined either by visual observation or with a reader, allowing for a simple, rapid quantification of E. coli in resource-limited settings.

  2. Gene 5. 5 protein of bacteriophaze T7 inhibits the nucleoid protein H-NS of Escherichia coli

    SciTech Connect

    Liu, Q.; Richardson, C.C. )

    1993-03-01

    Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection. Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps. A fusion protein of gene 5.5 protein and maltose binding protein also purifies with H-NS. The fusion protein binds to the DNA-H-NS complex and abolishes H-NS-mediated inhibition of transcription by Escherichia coli and T7 RNA polymerases in vitro. Expression of gene 5.5 also relieves the repression of the Escherichia coli proU promoter by H-NS in vivo. The change of leucine to proline at residue 30 of gene 5.5 protein abolishes the interaction between gene 5.5 protein and H-NS. 30 refs., 4 figs., 1 tab.

  3. Conformational Dynamics of Bacteriophage T7 DNA Polymerase and its Processivity Factor, Escherichia coli thioredoxin

    SciTech Connect

    Akabayov, B.; Akabayov, S; Lee , S; Tabor, S; Kulczyk , A; Richardson, C

    2010-01-01

    Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.

  4. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed Central

    DeWyngaert, M A; Hinkle, D C

    1980-01-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein. Images PMID:6997508

  5. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed

    DeWyngaert, M A; Hinkle, D C

    1980-02-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

  6. Transformation of Escherichia coli K-12 with a high-copy plasmid encoding the green fluorescent protein reduces growth: implications for predictive microbiology.

    PubMed

    Oscar, T P; Dulal, K; Boucaud, D

    2006-02-01

    The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (mumax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40 degrees C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40 degrees C, mumax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

  7. Biological toxicity of cellulose nanocrystals (CNCs) against the luxCDABE-based bioluminescent bioreporter Escherichia coli 652T7.

    PubMed

    Du, Liyu; Arnholt, Kelly; Ripp, Steven; Sayler, Gary; Wang, Siqun; Liang, Chenghua; Wang, Jingkuan; Zhuang, Jie

    2015-12-01

    The aim of this study was to evaluate the biological toxicity of cellulose nanocrystals (CNCs) using the constitutively bioluminescent luxCDABE-based bioreporter Escherichia coli 652T7. The effects of CNCs on E. c oli 652T7 biotoxicity were investigated at different CNC concentrations, reaction times, and IC50 values. CNC toxicity was also compared with and without ultrasonic dispersion to establish dispersibility effects. The results demonstrated that CNCs were not significantly toxic at concentrations at or below 250 mg/L. At concentrations higher than 300 mg/L, toxicity increased linearly as CNC concentrations increased up to 2000 mg/L. IC50 calculations demonstrated an increase in cytotoxicity as CNC exposure times increased, and elevated dispersibility of the CNCs were shown to increase cytotoxicity effects. These results suggest that CNCs can impact microbial populations if elevated concentration thresholds are met.

  8. High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system.

    PubMed

    Chao, Y-P; Law, W; Chen, P T; Hung, W-B

    2002-03-01

    The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.

  9. Structural and Mechanistic Basis for the Inhibition of Escherichia coli RNA Polymerase by T7 Gp2

    PubMed Central

    James, Ellen; Liu, Minhao; Sheppard, Carol; Mekler, Vladimir; Cámara, Beatriz; Liu, Bing; Simpson, Pete; Cota, Ernesto; Severinov, Konstantin; Matthews, Steve; Wigneshweraraj, Sivaramesh

    2012-01-01

    Summary The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) β′ subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec β′ jaw domain complex and show that Gp2 and DNA compete for binding to the β′ jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ70 domain region 1.1 (R1.1), and that Gp2 antagonizes the obligatory movement of R1.1 during RPo formation. We demonstrate that Gp2 inhibits RPo formation not just by steric occlusion of the RNAp-DNA interaction but also through long-range antagonistic effects on RNAp-promoter interactions around the RNAp active center that likely occur due to repositioning of R1.1 by Gp2. The inhibition of Ec RNAp by Gp2 thus defines a previously uncharacterized mechanism by which bacterial transcription is regulated by a viral factor. PMID:22819324

  10. A series of medium and high copy number arabinose-inducible Escherichia coli expression vectors compatible with pBR322 and pACYC184.

    PubMed

    Chakravartty, Vandana; Cronan, John E

    2015-09-01

    The original pBAD24 plasmid and the derived lower copy number (the pBAD322 series) expression vectors have been widely used in Escherichia coli, Salmonella enterica, and related bacteria. However, a flexible pBAD expression system has been available only in pMB1 (ColE1) vectors. We report a series of pBAD vectors that replicate using the origin of plasmid RSF1030 that are compatible with pMB1 (ColE1) and p15A (pACYC) vectors. Both high (≥pBAD24) and medium (~pBAD322) copy number plasmids encoding resistance to ampicillin, chloramphenicol, kanamycin, tetracycline, spectinomycin/streptomycin, gentamycin, or trimethoprim are available.

  11. T7 Early RNAs and Escherichia coli Ribosomal RNAs are Cut from Large Precursor RNAs In Vivo by Ribonuclease III

    PubMed Central

    Dunn, John J.; Studier, F. William

    1973-01-01

    The early region of T7 DNA is transcribed as a single unit in a Ribonuclease III-deficient E. coli strain to produce large molecules essentially identical to those produced in vitro by E. coli RNA polymerase. As with the in vitro RNAs, these molecules are cut by purified RNase III in vitro to produce the messenger RNAs normally observed in vivo. Thus, the normal pathway for producing the T7 early messenger RNAs in vivo appears to involve endonucleolytic cleavage by RNase III. The uninfected RNase III-deficient strain contains several RNAs not observed in the parent strain. Patterns of labeling in vivo suggest that the largest of these RNAs, about 1.8 × 106 daltons, may be a precursor to the 16S and 23S ribosomal RNAs. When this large molecule is treated in vitro with purified RNase III, molecules the size of precursor 16S and 23S ribosomal RNAs are released; hybridization competition experiments also indicate that the 1.8 × 106 dalton RNA does indeed represent ribosomal RNA. Thus, RNase III cleavage seems to be part of the normal pathway for producing at least the 16S and 23S ribosomal RNAs in vivo. Several smaller molecules are also released from the 1.8 × 106 dalton RNA by RNase III, but it is not yet established whether any of these contain 5S RNA sequences. Images PMID:4587248

  12. Rapid quantification of Escherichia coli in food and media using bacteriophage T7 amplification and liquid chromatography-multiple reaction monitoring tandem mass spectrometry.

    PubMed

    Banu, Mazlina; Ng, Daniel; Zheng, Lu; Goh, Lin-Tang; Bi, Xuezhi; Ow, Dave Siak-Wei

    2014-12-20

    Conventional microbiological assays have been a valuable tool for specific enumeration of indicative bacteria of relevance to food and public health, but these culture-based methods are time-consuming and require tedious biochemical and morphological identification. In this work, we exploit the ability of bacteriophage T7 to specifically infect Escherichia coli and amplify nearly a 100-fold in 1–2 h. Bacteriophage amplification is integrated with liquid chromatography-multiple reaction monitoring tandem mass spectrometry (LC-MRM–MS/MS) for quantitation of phage-specific peptides. Heavy isotopic 15N labeled T7 is introduced as the inoculum phage and internal standard. Quantification is performed by determining the ratio of phage-specific peptides over the internal standard which value is proportional to E. coli numbers. A broad dynamic range of 6-log orders ranging from 3.0 × 10(3) to 3.0 × 10(9) CFU/ml is attained in LB, while between 4.1 × 10(4)–2.7 × 10(9) CFU/ml and 1.9 × 10(3)–3.0 × 10(7) CFU/ml was enumerated respectively in coconut water and apple juice. With this method, viable E. coli are quantified in 4 h with a detection limit of 3.0 × 10(3) CFU/ml, 4.1 × 10(4) CFU/ml and 1.9 × 10(3) CFU/ml in LB, coconut water and apple juice, respectively. This method has potential as a rapid tool for detection of fecal contamination during food bioprocessing and distribution to safeguard public health.

  13. Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli

    PubMed Central

    Cress, Brady F.; Jones, J. Andrew; Kim, Daniel C.; Leitz, Quentin D.; Englaender, Jacob A.; Collins, Shannon M.; Linhardt, Robert J.; Koffas, Mattheos A. G.

    2016-01-01

    Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli. We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering. PMID:27079979

  14. 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.

  15. Molecular mechanism of promoter selection in gene transcription. I. Development of a rapid mixing-photocrosslinking technique to study the kinetics of Escherichia coli RNA polymerase binding to T7 DNA.

    PubMed

    Park, C S; Hillel, Z; Wu, C W

    1982-06-25

    A combined rapid mixing-photocrosslinking technique has been developed to investigate the kinetics of the interaction between Escherichia coli RNA polymerase and T7 DNA. The reactants were rapidly mixed in a modified Durrum stopped-flow apparatus, and the intermediates formed at different stages of the binding process were "frozen" by photocrosslinking with a UV light pulse of 10-mus duration at various times after mixing. The results indicate that the initial binding between RNA polymerase and T7 DNA is a diffusion-controlled reaction. Furthermore, the extents of initial contracts with DNA made with the beta, beta', and sigma subunits of RNA polymerase are roughly proportional to the sizes of these subunits, suggesting that complex formation occurs through random collision between the two reactants. After the initial complex formation, the rate of transfer of polymerase between individual DNA molecules is slow, implying that the polymerase molecules are undergoing predominantly intramolecular transfer during the promoter search. From the kinetic studies of subunit-DNA contacts during RNA polymerase binding to T7 DNA, it can be inferred that the beta, beta', and sigma subunits are directly participating in the promoter search process.

  16. Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised.

    PubMed

    Shadrin, Andrey; Sheppard, Carol; Severinov, Konstantin; Matthews, Steve; Wigneshweraraj, Sivaramesh

    2012-11-01

    The Escherichia coli-infecting bacteriophage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 phage development. The interaction site for Gp2 on the E. coli RNAp is the β' jaw domain, which is part of the DNA binding channel. The binding of Gp2 to the β' jaw antagonizes several steps associated with interactions between the RNAp and promoter DNA, leading to inhibition of transcription at the open promoter complex formation step. In the structure of the complex formed between Gp2 and a fragment of the β' jaw, amino acid residues in the β3 strand of Gp2 contribute to the primary interaction interface with the β' jaw. The 7009 E. coli strain is resistant to T7 because it carries a charge reversal point mutation in the β' jaw that prevents Gp2 binding. However, a T7 phage encoding a mutant form of Gp2, called Gp2(β), which carries triple amino acid substitutions E24K, F27Y and R56C, can productively infect this strain. By studying the molecular basis of inhibition of RNAp from the 7009 strain by Gp2(β), we provide several lines of evidence that the E24K and F27Y substitutions facilitate an interaction with RNAp when the primary interaction interface with the β' jaw is compromised. The proposed additional interaction interface between RNAp and Gp2 may contribute to the multipronged mechanism of transcription inhibition by Gp2.

  17. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  18. Cloning and expression of the tumstatin active peptides-T(7) and its derivant-T(7)-NGR.

    PubMed

    Naling, Song; Xin, He; Qiren, Zhao; Tingdong, Yan; Lei, Wen

    2009-06-01

    To enhance the role targeting, design to link NGR sequence with tumstatin active peptides-T(7)'s C-terminal, the derivant called T(7)-NGR. The cloning vector pMD-T(7) and pMD-T(7) N were constructed by PCR and gene synthesis methods, respectively, identified by digestion and DNA sequencing. After the digested plasmids were isolated by the low melting point agarose electrophoresis, the target-fragment was cut off and mixed with the recovery of the digested vector pET28a. Expression vector pET-T(7) and pET-T(7) N were constructed in low melting point agarose, identified by digestion and DNA sequencing, transformed into competent Escherichia coli BL21 (DE3), induced by IPTG. Identification result shows that pET-T(7) and pET-T(7) N were correct. Tricine-SDS-PAGE results showed that IPTG concentration of 1 mM, after the induction of 25 degrees C, 8 h, T(7) peptides and T(7)-NGR peptides have achieved the optimum conditions of expression. In conclusion, the expression vectors of the two peptides has been successfully constructed, and got product, no coverage at home and abroad, laid the foundation for further activity experiments.

  19. Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus.

    PubMed

    Umelo-Njaka, E; Nomellini, J F; Yim, H; Smit, J

    2001-07-01

    Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host. The vectors are based on a narrow-host-range colE1-replicon-based plasmid commonly used in Escherichia coli, to which was added the replication origin of the IncQ plasmid RSF1010. C. crescentus strains were modified to enable plasmid replication by introduction of the RSF1010 repBAC genes at the recA locus. The small (4.0-4.5 kb) plasmids were in high copy numbers in both C. crescentus and E. coli and amenable to rapid methods for plasmid isolation and DNA sequencing. The method for introducing repBAC is suitable for other C. crescentus strains or any bacterium with an adequately homologous recA gene. Application of the vector for protein expression, based on the type I secretion system of the S-layer protein, when compared to constructs in broad-host-range plasmids, resulted in reduced time and steps required from clone construction to recombinant protein recovery and increased protein yield.

  20. A T3 and T7 Recombinant Phage Acquires Efficient Adsorption and a Broader Host Range

    PubMed Central

    Lin, Tiao-Yin; Lo, Yi-Haw; Tseng, Pin-Wei; Chang, Shun-Fu; Lin, Yann-Tsyr; Chen, Ton-Seng

    2012-01-01

    It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress. PMID:22347414

  1. A T3 and T7 recombinant phage acquires efficient adsorption and a broader host range.

    PubMed

    Lin, Tiao-Yin; Lo, Yi-Haw; Tseng, Pin-Wei; Chang, Shun-Fu; Lin, Yann-Tsyr; Chen, Ton-Seng

    2012-01-01

    It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress.

  2. T7 replisome directly overcomes DNA damage

    NASA Astrophysics Data System (ADS)

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-12-01

    Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

  3. Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

    PubMed Central

    Shis, David L.; Bennett, Matthew R.

    2013-01-01

    The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host’s native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits. PMID:23479654

  4. Choreography of bacteriophage T7 DNA replication.

    PubMed

    Lee, Seung-Joo; Richardson, Charles C

    2011-10-01

    The replication system of phage T7 provides a model for DNA replication. Biochemical, structural, and single-molecule analyses together provide insight into replisome mechanics. A complex of polymerase, a processivity factor, and helicase mediates leading strand synthesis. Establishment of the complex requires an interaction of the C-terminal tail of the helicase with the polymerase. During synthesis the complex is stabilized by other interactions to provide for a processivity of 5 kilobase (kb). The C-terminal tail also interacts with a distinct region of the polymerase to captures dissociating polymerase to increase the processivity to >17kb. The lagging strand is synthesized discontinuously within a loop that forms and resolves during each cycle of Okazaki fragment synthesis. The synthesis of a primer as well as the termination of a fragment signal loop resolution.

  5. Effects of space environment on T-7 bacteriophage and spores of Bacillus subtilis 168

    NASA Technical Reports Server (NTRS)

    Spizizen, J.; Isherwood, J. E.

    1973-01-01

    Two strains of Bacillus subtilis were exposed to components of the ultraviolet spectrum in space. Both strains possess multiple genetic markers, and one of the strains is defective in the ability to repair ultraviolet damage. The T-7 bacteriophage of Escherichia coli was also exposed to selected wavelengths and energy levels of ultraviolet light in space. Preliminary findings do not reveal anomalies in survival rates. Data are not yet available on detailed genetic analyses.

  6. Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

    PubMed Central

    Biebricher, C K; Luce, R

    1996-01-01

    A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase. Images PMID:8670848

  7. Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro.

    PubMed

    Sun, M; Louie, D; Serwer, P

    1999-09-01

    Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (+/-3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (+/-6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer-associated T7 genomes is cooperative.

  8. T7 ejectosome assembly: A story unfolds

    PubMed Central

    Leptihn, Sebastian; Gottschalk, Julia; Kuhn, Andreas

    2016-01-01

    ABSTRACT T7 phage DNA is transported from the capsid into the host cytoplasm across the cell wall by an ejectosome comprised of the viral proteins gp14, gp15 and gp16. Prior to infection, these proteins form the so-called internal core in the mature virion. Gp16 was shown to associate with pure phospholipid bilayers while gp15 bound to DNA. A complex of both proteins appears as spiral-like rods in electron micrographs. It was also shown that the proteins gp15 and gp16 have the propensity to regain their full structure after thermal unfolding. From these observations it was concluded that (partial) unfolding of the proteins occurs during the translocation through the narrow portal of the phage capsid. After leaving the phage head, the proteins refold to form the ejectosome channel across the periplasm of the host. In this work, we analyzed the structure of gp15 and gp16 in presence of lipids and their stability toward chemical denaturants. A model to explain how the ejectosome might assemble in the host cell is discussed. PMID:27144087

  9. Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

    PubMed Central

    Stroud, R M; Serwer, P; Ross, M J

    1981-01-01

    The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV. Images FIGURE 2 FIGURE 3 PMID:7326332

  10. Characterization of the initial steps in the T7 DNA ejection process

    PubMed Central

    González-García, Verónica A; Bocanegra, Rebeca; Pulido-Cid, Mar; Martín-Benito, Jaime; Cuervo, Ana; Carrascosa, José L

    2015-01-01

    A specialized complex, the tail, is the most common strategy employed by bacterial viruses to deliver their genome without disrupting cell integrity. T7 has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Recent studies showed that incubation of the virus with Escherichia coli lipopolysaccharides (LPS) resulted in complete delivery of the viral genome, demonstrating for the first time that LPS are the T7 receptor. Further screening of the bacterial envelope for proteinaceous compounds that affect T7 ejection showed that porins OmpA and OmpF affect viral particle adsorption and infection kinetics, suggesting that these proteins play a role in the first steps of virus-host interaction. Comparison of the structures before and after ejection showed the conformational changes needed in the tail for genome delivery. Structural similarities between T7 and other viruses belonging to the Podoviridae family suggests that they could also follow a similar DNA ejection mechanism. PMID:26458390

  11. Complete genome sequence of 285P, a novel T7-like polyvalent E. coli bacteriophage.

    PubMed

    Xu, Bin; Ma, Xiangyu; Xiong, Hongyan; Li, Yafei

    2014-06-01

    Bacteriophages are considered potential biological agents for the control of infectious diseases and environmental disinfection. Here, we describe a novel T7-like polyvalent Escherichia coli bacteriophage, designated "285P," which can lyse several strains of E. coli. The genome, which consists of 39,270 base pairs with a G+C content of 48.73 %, was sequenced and annotated. Forty-three potential open reading frames were identified using bioinformatics tools. Based on whole-genome sequence comparison, phage 285P was identified as a novel strain of subgroup T7. It showed strongest sequence similarity to Kluyvera phage Kvp1. The phylogenetic analyses of both non-structural proteins (endonuclease gp3, amidase gp3.5, DNA primase/helicase gp4, DNA polymerase gp5, and exonuclease gp6) and structural protein (tail fiber protein gp17) led to the identification of 285P as T7-like phage. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses verified the annotation of the structural proteins (major capsid protein gp10a, tail protein gp12, and tail fiber protein gp17).

  12. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  13. [Antirestriction and antimodification activities of the T7 Ocr protein: effect of mutations in interface].

    PubMed

    Zavil'gel'skiĭ, G B; Kotova, V Iu; Rastorguev, S M

    2009-01-01

    Antirestriction protein Ocr (bacteriophage T7) is specific inhibitor of the type I restriction-modification enzymes. The bacteriophage T7 0.3 (ocr) gene is cloned in pUC18 vector. It was shown that T7 Ocr protein inhibits both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The mutation form of Ocr-Ocr F53D A57E, which inhibits only the restriction activity of EcoKI-enzyme, was constructed. The T7 0.3 (ocr) and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltet0-1) promoter which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltet0-1) promoter can be regulated over and up to 5000 fold range by supplying anhydrotetracycline (aTc) to the E. coli MG1655Z1 tetR+ cells. It was determined the dependence of the effectiveness of the antirestriction activity of the Ocr and Ocr F53D A57E proteins on the intracellular concentration. It was shown that the values of the dissociation constants K(d) for Ocr and Ocr F53D A57E proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, K(d) (Ocr F53D A57E) = 10(-7) M.

  14. Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

    PubMed

    Wild, Jadwiga; Hradecna, Zdenka; Szybalski, Waclaw

    2002-09-01

    The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

  15. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  16. Mapping IS6110 in high-copy number Mycobacterium tuberculosis strains shows specific insertion points in the Beijing genotype

    PubMed Central

    2013-01-01

    Background Mycobacterium tuberculosis Beijing strains are characterized by a large number of IS6110 copies, suggesting the potential implication of this element in the virulence and capacity for rapid dissemination characteristic of this family. This work studies the insetion points of IS6110 in high-copy clinical isolates specifically focusing on the Beijing genotype. Results In the present work we mapped the insertion points of IS6110 in all the Beijing strains available in the literature and in the DNA sequence databases. We generated a representative primer collection of the IS6110 locations, which was used to analyse 61 high-copy clinical isolates. A total of 440 points of insertion were identified and analysis of their flanking regions determined the exact location, the direct repeats (DRs), the orientation and the distance to neighboring genes of each copy of IS6110. We identified specific points of insertion in Beijing strains that enabled us to obtain a dendrogram that groups the Beijing genotype. Conclusions This work presents a detailed analysis of locations of IS6110 in high-copy clinical isolates, showing points of insertion present with high frequency in the Beijing family and absent in other strains. PMID:23800083

  17. THE MASS OF CoRoT-7b

    SciTech Connect

    Hatzes, Artie P.; Wuchterl, Guenther; Fridlund, Malcolm; Gandolfi, Davide; Nachmani, Gil; Mazeh, Tsevi; Valencia, Diana; Hebrard, Guillaume; Borde, Pascal; Carone, Ludmila; Paetzold, Martin; Udry, Stephane; Bouchy, Francois; Deleuil, Magali; Moutou, Claire; Barge, Pierre; Deeg, Hans; Tingley, Brandon; Dvorak, Rudolf; Ferraz-Mello, Sylvio E-mail: malcolm.fridlund@esa.int; and others

    2011-12-10

    The mass of CoRoT-7b, the first transiting super-Earth exoplanet, is still a subject of debate. A wide range of masses have been reported in the literature ranging from as high as 8 M{sub Circled-Plus} to as low as 2.3 M{sub Circled-Plus }. This range in mass is largely due to the activity level of the star that contributes a significant amount of radial velocity (RV) 'jitter' and how the various methods correct this jitter. Although most mass determinations give a density consistent with a rocky planet, the lower value permits a bulk composition that can be up to 50% water. We present an analysis of the CoRoT-7b RV measurements that uses very few and simple assumptions in treating the activity signal. By analyzing those RV data for which multiple measurements were made in a given night, we remove the activity related RV contribution without any a priori model. We argue that the contribution of activity to the final RV curve is negligible and that the K-amplitude due to the planet is well constrained. This yields a mass of 7.42 {+-} 1.21 M{sub Circled-Plus} and a mean density of {rho} = 10.4 {+-} 1.8 gm cm{sup -3}. CoRoT-7b is similar in mass and radius to the second rocky planet to be discovered, Kepler-10b, and within the errors they have identical bulk densities-they are virtual twins. These bulk densities lie close to the density-radius relationship for terrestrial planets similar to what is seen for Mercury. CoRoT-7b and Kepler-10b may have an internal structure more like Mercury than the Earth.

  18. The 5.5 Protein of Phage T7 Inhibits H-NS through Interactions with the Central Oligomerization Domain▿†

    PubMed Central

    Ali, Sabrina S.; Beckett, Emily; Bae, Sandy Jeehoon; Navarre, William Wiley

    2011-01-01

    The 5.5 protein (T7p32) of coliphage T7 (5.5T7) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5T7 protein is insoluble when expressed in Escherichia coli, but we find that 5.5T7 can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5T7 binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5T7 can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5T7 protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5T7 protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression. PMID:21764926

  19. Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

    PubMed Central

    Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

    2015-01-01

    Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

  20. DNA damage under simulated extraterrestrial conditions in bacteriophage T7

    NASA Astrophysics Data System (ADS)

    Fekete, A.; Kovács, G.; Hegedüs, M.; Módos, K.; Rontó, Gy.; Lammer, H.; Panitz, C.

    The experiment ``Phage and uracil response'' (PUR) will be accommodated in the EXPOSE facility of the ISS aiming to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. To achieve this new method was elaborated for the preparation of DNA and nucleoprotein thin films (1). During the EXPOSE Experiment Verification Tests (EVT) the samples were exposed to vacuum (10 -6 Pa), to monochromatic (254 nm) and polychromatic (200-400 nm) UV radiation in air as well in simulated space vacuum. Using neutral density (ND) filters dose-effect curves were performed in order to define the maximum doses tolerated, and we also studied the effect of temperature in vacuum as well as the influence of temperature fluctuations. We obtained substantial evidence that DNA lesions (e.g. strand breaks, DNA-protein cross-links, DNA-DNA cross-links) accumulate throughout exposure. DNA damage was determined by quantitative PCR using 555 bp and 3826 bp fragments of T7 DNA (2) and by neutral and alkaline agarose gel electrophoresis; the structural/chemical effects were analyzed by spectroscopic and microscopical methods. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of the PCR products have been detected indicating the damage of isolated and intraphage DNA. Preliminary results suggest a synergistic action of space vacuum and UV radiation with DNA being the critical target. Fekete et al. J. Luminescence 102-103, 469-475, 2003 Hegedüs et al. Photochem. Photobiol. 78, 213-219, 2003

  1. Optimized T7 amplification system for microarray analysis.

    PubMed

    Pabón, C; Modrusan, Z; Ruvolo, M V; Coleman, I M; Daniel, S; Yue, H; Arnold, L J

    2001-10-01

    Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

  2. A novel fluorescent probe: europium complex hybridized T7 phage.

    PubMed

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  3. T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.

    PubMed Central

    Pinkham, J L; Dudley, A M; Mason, T L

    1994-01-01

    An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression. Images PMID:8007968

  4. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-11-03

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  5. Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  6. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F.W.; Dubendorff, J.W.

    1998-10-20

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods. 12 figs.

  7. Cloning and expression of autogenes encoding RNA polymerases of T7-like bacteriophages

    DOEpatents

    Studier, F. William; Dubendorff, John W.

    1998-01-01

    This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.

  8. Comet C/2002 T7 (LINEAR): Polarimetric and photometric studies

    NASA Astrophysics Data System (ADS)

    Rosenbush, V. K.; Velichko, F. P.; Kiselev, N. N.; Velichko, S. F.; Shakhovskoy, N. M.; Efimov, Yu. S.; Antonyuk, K. A.; Kolesnikov, S. V.; Shakhovskoy, D. N.

    2006-05-01

    We present the results of polarimetric and photometric observations of dynamically new comet C/2002 T7 (LINEAR) at phase angles from 6° to 26°. During the observations, the comet was at a distance of 2.7 1.3 AU from the Sun and 1.7 2.0 AU from the Earth. The aperture polarimetry was made with the 2.6-m Shain telescope and the 1.25-m AZT-11 telescope of the Crimean Astrophysical Observatory and with the 0.7-m telescope of the Astronomical Institute of the Kharkiv National University during the period from November 21, 2003, to February 21, 2004. The wideband UBVRI and WRC (λ7228/1142 Å) filters and the narrowband GC (λ5260/56 Å) filter were used. The photometric observations of the comet were carried out on February 21, 2004, with narrowband filters isolated the BC (λ4845/65 Å) and RC (λ6840/90 Å) continuum and the C2 emission (λ5140/90 Å). The phase-angle dependence of linear polarization of the comet has been obtained, and its parameters, such as the minimal polarization P min = -1.63%, the phase angle of the minimal polarization αmin = 10.6°, the inversion angle αinv = 22.7°, and the slope of the phase curve at the inversion angle h = 0.24% per degree, were found. From the photometric observations, the following quantities have been obtained: the column density of molecules C2 in the line of sight logN (C2) = -9.15 mol/cm2 and their production rate log Q (C2) = 27.11 mol/s, the spectral gradient of reflectivity for the dust S‧(BC, RC) ≈ 3%/1000 Å, and the dust production parameter Afρ equal to 371 and 273 cm for the blue and red continuum ranges, respectively. According to these results, the physical parameters of comet C/2002 T7 are close to the average characteristics of typical dusty comets.

  9. Modular control of multiple pathways using engineered orthogonal T7 polymerases

    PubMed Central

    Temme, Karsten; Hill, Rena; Segall-Shapiro, Thomas H.; Moser, Felix; Voigt, Christopher A.

    2012-01-01

    Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure. PMID:22743271

  10. T7 RNA polymerase elongation complex structure and movement.

    PubMed

    Huang, J; Sousa, R

    2000-10-27

    We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, lambda exonuclease, RNAse T1, and treatment with KMnO(4). Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5' to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand.

  11. A High Copy Suppressor Screen for Autophagy Defects in Saccharomyces arl1Δ and ypt6Δ Strains

    PubMed Central

    Yang, Shu; Rosenwald, Anne

    2016-01-01

    In Saccharomyces cerevisiae, Arl1 and Ypt6, two small GTP-binding proteins that regulate membrane traffic in the secretory and endocytic pathways, are also necessary for autophagy. To gain information about potential partners of Arl1 and Ypt6 specifically in autophagy, we carried out a high copy number suppressor screen to identify genes that when overexpressed suppress the rapamycin sensitivity phenotype of arl1Δ and ypt6Δ strains at 37°. From the screen results, we selected COG4, SNX4, TAX4, IVY1, PEP3, SLT2, and ATG5, either membrane traffic or autophagy regulators, to further test whether they can suppress the specific autophagy defects of arl1Δ and ypt6Δ strains. As a result, we identified COG4, SNX4, and TAX4 to be specific suppressors for the arl1Δ strain, and IVY1 and ATG5 for the ypt6Δ strain. Through this screen, we were able to confirm several membrane traffic and autophagy regulators that have novel relationships with Arl1 and Ypt6 during autophagy. PMID:27974437

  12. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1990-01-01

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  13. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1999-02-09

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  14. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F. William; Davanloo, Parichehre; Rosenberg, Alan H.; Moffatt, Barbara A.; Dunn, John J.

    1997-12-02

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells.

  15. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

    1999-02-09

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

  16. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.; Moffatt, B.A.; Dunn, J.J.

    1997-12-02

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the R7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties. T7 RNA polymerase is also used in a system for selective, high-level synthesis of RNAs and proteins in suitable host cells. 10 figs.

  17. Cloning and expression of the gene for bacteriophage T7 RNA polymerase

    DOEpatents

    Studier, F.W.; Davanloo, P.; Rosenberg, A.H.

    1984-03-30

    This application describes a means to clone a functional gene for bacteriophage T7 RNA polymerase. Active T7 RNA polymerase is produced from the cloned gene, and a plasmid has been constructed that can produce the active enzyme in large amounts. T7 RNA polymerase transcribes DNA very efficiently and is highly selective for a relatively long promoter sequence. This enzyme is useful for synthesizing large amounts of RNA in vivo or in vitro, and is capable of producing a single RNA selectively from a complex mixture of DNAs. The procedure used to obtain a clone of the T7 RNA polymerase gene can be applied to other T7-like phages to obtain clones that produce RNA polymerases having different promoter specificities, different bacterial hosts, or other desirable properties.

  18. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level

    PubMed Central

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-01-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698

  19. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.

    PubMed

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-03-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg(-1). It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

  20. Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli

    SciTech Connect

    Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy; Qasba, Pradman K.

    2010-04-09

    The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.

  1. Multi-input regulation and logic with T7 promoters in cells and cell free systems

    SciTech Connect

    Iyer, Sukanya; Karig, David K; Norred, Sarah E; Simpson, Michael L; Doktycz, Mitchel John

    2014-01-01

    Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on host E. coli promoters for the construction of circuit elements, such as logic gates, makes implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in a commercially available E. coli cell-free protein expression system. Together, our results contribute to the first demonstration of multi-input regulation of T7 promoters and expand the utility of T7 promoters in cell based as well as cell-free gene circuits.

  2. Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation.

    PubMed

    Ross, C; Samuel, M; Broitman, S L

    1992-12-30

    We have identified a purine-rich triplex binding sequence overlapping a -35 transcriptional early promoter region of the bacteriophage T7. Triplex-forming oligonucleotide designed to bind this target was annealed to T7 templates and introduced into in vitro transcription systems under conditions favoring specific initiation from this promoter. These templates demonstrated significant transcriptional inhibition relative to naked genomic templates and templates mixed with non-triplex-forming oligonucleotide. It is suggested that triplex formation along this target interferes with transcriptional initiation, and this mechanism may hold potential to disrupt bacteriophage T7 early transcription in vivo.

  3. A method for quantifying the force dependence of initiation by T7 RNA polymerase

    NASA Astrophysics Data System (ADS)

    Kalafut, Bennett S.; Skinner, Gary M.; Visscher, Koen

    2009-08-01

    To access the genetic code to be transcribed to RNA, RNA polymerases must first open a "transcription bubble" in the DNA. Structural studies suggest that the minimal model of initiation by T7 bacterophage RNA polymerase (T7 RNAP) consists of two distinct steps: initial binding, in which the T7 RNAP binds to and bends the DNA, and opening, achieved by "scrunching" of the DNA. Since both steps involve mechanical deformation of the DNA, both may be affected by downstream DNA tension. Using an oscillating two-bead optical tweezers assay, we have measured the lifetime of single T7 RNAP-DNA initation complexes under tension. Global maximumlikelihood fitting of force-dependent and non-force-dependent versions of this minimal model shows that there is no conclusively discernible force-dependence of initiation in the measured 0-2 pN DNA tension range.

  4. Comparative analysis of anti-restriction activities of ArdA (ColIb-P9) and Ocr (T7) proteins.

    PubMed

    Zavilgelsky, G B; Kotova, V Yu; Rastorguev, S M

    2008-08-01

    Anti-restriction proteins ArdA and Ocr are specific inhibitors of type I restriction-modification enzymes. The IncI1 transmissible plasmid ColIb-P9 ardA and bacteriophage T7 0.3(ocr) genes were cloned in pUC18 vector. Both ArdA (ColIb-P9) and Ocr (T7) proteins inhibit both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. ColIb-P9 ardA, T7 0.3(ocr), and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltetO-1) promoter, which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltetO-1) promoter can be regulated over an up to 5000-fold range by supplying anhydrotetracycline to the E. coli MG1655Z1 tetR(+) cells. Effectiveness of the anti-restriction activity of the ArdA and Ocr proteins depended on the intracellular concentration. It is shown that the dissociation constants K(d) for ArdA and Ocr proteins with EcoKI enzyme differ 1700-fold: K(d) (Ocr) = 10(-10) M, K(d) (ArdA) = 1.7.10(-7) M.

  5. UV irradiation experiments under simulated martian surface conditions: Bio-effects on glycine, phage T7 and isolated T7 DNA

    NASA Astrophysics Data System (ADS)

    Bérces, Attila; ten Kate, I. L.; Fekete, A.; Hegedus, M.; Garry, J. R. C.; Lammer, Helmut; Ehrenfreund, Pascale; Peeters, Zan; Kovacs, G.; Ronto, G.

    Mars is considered as a main target for astrobiologically relevant exploration programmes. In order to explain the non-detection of organic material to a detection level of several parts per billion (ppb) by the Viking landers, several hypotheses have been suggested, including degradation processes occurring on the martian surface and in the martian soil and subsurface. UV exposure experiments have been performed in which thin layers of glycine ( 300 nm), and aqueous suspensions of phage T7 and isolated T7 DNA were irradiated with a Deuterium lamp and for comparison with a Xenon arc lamp, modified to simulate the solar irradiation on the surface of Mars (MarsUV). The glycine sample was subjected to 24 hours of irradiation with MarsUV. The results of this glycine experiment show a destruction rate comparable to the results of previous experiments in which thin layers of glycine were irradiated with a deuterium lamp (ten Kate et al., 2005, 2006). After exposure of different doses of simulated Martian UV radiation a decrease of the biological activity of phages and characteristic changes in the UV absorption spectrum have been detected, indicating the UV damage of isolated and intraphage T7 DNA. The results of our experiments show that intraphage DNA is 4 times more sensitive to simulated martian UV and deuterium lamp radiation than isolated T7 DNA. This result indicates the significant role that phage proteins play in the UV damage. The effect of simulated martian radiation is smaller than the biological defects observed after the exposure with a deuterium lamp for both cases, in intraphage and isolated DNA, despite of the 100 times larger intensity of the MarsUV lamp. The detected spectral differences are about ten times smaller; the biological activity is about 3 - 4 times smaller, indicating that the shorter wavelength UV radiation from the deuterium lamp is more effective in inducing DNA damage, irrespective of being intraphage or isolated.

  6. Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Joyce, Gerald F.

    2001-01-01

    A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the non-template and template strands of the promoter. These residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and P-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing its promoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that employ T7 RNA polymerase for in vitro transcription.

  7. Exploration of peptide T7 and its derivative as integrin αvβ3-targeted imaging agents

    PubMed Central

    He, Xin; Hao, Yumei; Long, Wei; Song, Naling; Fan, Saijun; Meng, Aimin

    2015-01-01

    Objective The aim of the present study was to develop potential candidates of integrin αvβ3-targeted imaging agent, which can facilitate the diagnosis and treatment of malignant solid tumors. Methods Peptides derived from tumstatin, named T7 and T7-6H, were derivatized to contain histidine in the C-terminus of their sequence and were labeled with 99mTc via nitrido and carbonyl precursors. The radiochemical purity and stability of 99mTc-labeled T7 and T7-6H were characterized by thin-layer chromatography. The whole body biodistribution was studied in NCI-H157-bearing BALB/c nude mice. Results The 99mTc-labeled T7 and T7-6H showed adequate in vitro stability, with a high radiochemical purity of over 90%. The dissociation constant (Kd) value of the 99mTc-labeled T7 and T7-6H ranged from 68.5 nM to 140.8 nM in U251 and NCI-H157 cell lines. 99mTc-labeled T7 and T7-6H showed no significant difference of biodistribution in mice. Furthermore, both T7 and T7-6H exhibited a poor blood–brain barrier penetration and a transient accumulation in lung; the uptake in tumor tissues was significantly higher than in muscle tissue, with a ratio of 5.8. Conclusion 99mTc-labeled T7 and T7-6H can be regarded as promising single-photon emission computed tomography probes for imaging integrin αvβ3, and need to be further studied for noninvasive detection of tumors. PMID:26109872

  8. Planets and Stellar Activity: Hide and Seek in the CoRoT-7 system

    NASA Astrophysics Data System (ADS)

    Haywood, R. D.; Cameron, A. C.; Queloz, D.; Barros, S. C. C.; Deleuil, M.; Fares, R.; Gillon, M.; Hatzes, A.; Lanza, A. F.; Lovis, C.; Moutou, C.; Pepe, F.; Pollacco, D.; Santerne, A.; Ségransan, D.; Unruh, Y.

    2014-01-01

    Since the discovery of the transiting Super-Earth CoRoT-7b, several investigations have been made of the number and precise masses of planets present in the system, but they all yield different results, owing to the star's high level of activity. Radial velocity (RV) variations induced by stellar activity therefore need to be modelled and removed to allow a reliable detection of all planets in the system. We re-observed CoRoT-7 in January 2012 with both HARPS and the CoRoT satellite, so that we now have the benefit of simultaneous RV and photometric data. We fitted the off-transit variations in the CoRoT lightcurve using a harmonic decomposition similar to that implemented in Queloz et al. (2009). This fit was then used to model the stellar RV contribution, according to the methods described by Aigrain et al. (2011). This model was incorporated into a Monte Carlo Markov Chain in order to make a precise determination of the orbits of CoRoT-7b and CoRoT-7c. We also assess the evidence for the presence of one or two additional planetary companions.

  9. Importance of steric effects on the efficiency and fidelity of transcription by T7 RNA polymerase.

    PubMed

    Ulrich, Sébastien; Kool, Eric T

    2011-11-29

    DNA-dependent RNA polymerases such as T7 RNA polymerase (T7 RNAP) perform the transcription of DNA into mRNA with high efficiency and high fidelity. Although structural studies have provided a detailed account of the molecular basis of transcription, the relative importance of factors like hydrogen bonds and steric effects remains poorly understood. We report herein the first study aimed at systematically probing the importance of steric and electrostatic effects on the efficiency and fidelity of DNA transcription by T7 RNAP. We used synthetic nonpolar analogues of thymine with sizes varying in subangstrom increments to probe the steric requirements of T7 RNAP during the elongation mode of transcription. Enzymatic assays with internal radiolabeling were performed to compare the efficiency of transcription of modified DNA templates with a natural template containing thymine as a reference. Furthermore, we analyzed effects on the fidelity by measuring the composition of RNA transcripts by enzymatic digestion followed by two-dimensional thin layer chromatography separation. Our results demonstrate that hydrogen bonds play an important role in the efficiency of transcription but, interestingly, do not appear to be required for faithful transcription. Steric effects (size and shape variations) are found to be significant both in insertion of a new RNA base and in extension beyond it.

  10. Uncovering the planets and stellar activity of CoRoT-7 using only radial velocities

    NASA Astrophysics Data System (ADS)

    Faria, J. P.; Haywood, R. D.; Brewer, B. J.; Figueira, P.; Oshagh, M.; Santerne, A.; Santos, N. C.

    2016-04-01

    Stellar activity can induce signals in the radial velocities of stars, complicating the detection of orbiting low-mass planets. We present a method to determine the number of planetary signals present in radial-velocity datasets of active stars, using only radial-velocity observations. Instead of considering separate fits with different number of planets, we use a birth-death Markov chain Monte Carlo algorithm to infer the posterior distribution for the number of planets in a single run. In a natural way, the marginal distributions for the orbital parameters of all planets are also inferred. This method is applied to HARPS data of CoRoT-7. We confidently recover the orbits of both CoRoT-7b and CoRoT-7c although the data show evidence for the presence of additional signals. All data and software presented in this article are available online at http://https://github.com/j-faria/exoBD-CoRoT7

  11. Genome Sequence of Streptomyces viridosporus Strain T7A ATCC 39115, a Lignin-Degrading Actinomycete

    PubMed Central

    Davis, Jennifer R.; Goodwin, Lynne; Teshima, Hazuki; Detter, Chris; Tapia, Roxanne; Han, Cliff; Huntemann, Marcel; Wei, Chia-Lin; Han, James; Chen, Amy; Kyrpides, Nikos; Mavrommatis, Kostas; Szeto, Ernest; Markowitz, Victor; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Pagani, Ioanna; Pati, Amrita; Woyke, Tanja; Pitluck, Sam; Peters, Lin; Nolan, Matt; Land, Miriam

    2013-01-01

    We announce the availability of the genome sequence of Streptomyces viridosporus strain T7A ATCC 39115, a plant biomass-degrading actinomycete. This bacterium is of special interest because of its capacity to degrade lignin, an underutilized component of plants in the context of bioenergy. It has a full complement of genes for plant biomass catabolism. PMID:23833133

  12. Expression of zinc transporter ZnT7 in mouse superior cervical ganglion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The superior cervical ganglion (SCG) neurons contain a considerable amount of zinc ions, but little is known about zinc homeostasis in the SCG. It is known that zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 ZnT family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi...

  13. High density growth of T7 expression strains with auto-induction option

    DOEpatents

    Studier, F. William

    2013-03-19

    A method for promoting and suppressing auto-induction of transcription of a cloned gene 1 of bacteriophage T7 in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a promoter whose activity can be induced by an exogenous inducer whose ability to induce said promoter is dependent on the metabolic state of said bacterial cells.

  14. Genome Sequence of Streptomyces viridosporus Strain T7A ATCC 39115, a Lignin-Degrading Actinomycete

    SciTech Connect

    Davis, Jennifer R.; Goodwin, Lynne A.; Teshima, Hazuki; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Huntemann, Marcel; Wei, Chia-Lin; Han, James; Chen, Amy; Kyrpides, Nikos C; Mavromatis, K; Szeto, Ernest; Markowitz, Victor; Ivanova, N; Mikhailova, Natalia; Ovchinnikova, Galina; Pagani, Ioanna; Pati, Amrita; Woyke, Tanja; Pitluck, Sam; Peters, Lin; Nolan, Matt; Land, Miriam L; Sello, Jason K.

    2013-01-01

    We announce the availability of the genome sequence of Streptomyces viridosporus strain T7A ATCC 39115, a plant biomass- degrading actinomycete. This bacterium is of special interest because of its capacity to degrade lignin, an underutilized compo- nent of plants in the context of bioenergy. It has a full complement of genes for plant biomass catabolism.

  15. Qualitative and quantitative detection of T7 bacteriophages using paper based sandwich ELISA.

    PubMed

    Khan, Mohidus Samad; Pande, Tripti; van de Ven, Theo G M

    2015-08-01

    Viruses cause many infectious diseases and consequently epidemic health threats. Paper based diagnostics and filters can offer attractive options for detecting and deactivating pathogens. However, due to their infectious characteristics, virus detection using paper diagnostics is more challenging compared to the detection of bacteria, enzymes, DNA or antigens. The major objective of this study was to prepare reliable, degradable and low cost paper diagnostics to detect viruses, without using sophisticated optical or microfluidic analytical instruments. T7 bacteriophage was used as a model virus. A paper based sandwich ELISA technique was developed to detect and quantify the T7 phages in solution. The paper based sandwich ELISA detected T7 phage concentrations as low as 100 pfu/mL to as high as 10(9) pfu/mL. The compatibility of paper based sandwich ELISA with the conventional titre count was tested using T7 phage solutions of unknown concentrations. The paper based sandwich ELISA technique is faster and economical compared to the traditional detection techniques. Therefore, with proper calibration and right reagents, and by following the biosafety regulations, the paper based technique can be said to be compatible and economical to the sophisticated laboratory diagnostic techniques applied to detect pathogenic viruses and other microorganisms.

  16. Use of endogenous host plasmids for generation of Escherichia coli O157:H7 and Shigella sonnei strains that stably express the green fluorescent protein.

    PubMed

    Monday, Steven R; Weagant, Stephen D; Feng, Peter

    2003-09-01

    The gfp gene was manipulated from a commercially available, high copy vector into endogenous plasmids of Escherichia coli O157:H7 and Shigella sonnei to yield stable GFP strains that required neither high copy number for visualization nor antibiotics for stable maintenance of the phenotype. The GFP phenotype of these strains remained stable after repeated passages in media and conditions that enhance plasmid instability and loss from bacterial cells. These results demonstrate the utility of the endogenous plasmids in selectively marking bacteria without altering host cellular function or biochemical properties.

  17. Inactivation of E. coli, B. subtilis spores, and MS2, T4, and T7 phage using UV/H2O2 advanced oxidation.

    PubMed

    Mamane, Hadas; Shemer, Hilla; Linden, Karl G

    2007-07-31

    The goal of this study was to evaluate the potential of an advanced oxidation process (AOP) for microbiocidal and virucidal inactivation. The viruses chosen for this study were bacteriophage MS2, T4, and T7. In addition, Bacillus subtilis spores and Escherichia coli were studied. By using H(2)O(2) in the presence of filtered ultraviolet (UV) irradiation (UV/H(2)O(2)) to generate wavelengths above 295nm, the direct UV photolysis disinfection mechanism was minimized, while disinfection by H(2)O(2) was also negligible. Virus T4 and E. coli in phosphate buffered saline (PBS) were sensitive to >295nm filtered UV irradiation (without H(2)O(2)), while MS2 was very resistant. Addition of H(2)O(2) at 25mg/l in the presence of filtered UV irradiation over a 15min reaction time did not result in any additional disinfection of virus T4, while an additional one log inactivation for T7 and 2.5 logs for MS2 were obtained. With E. coli, only a slight additional effect was observed when H(2)O(2) was added. B. subtilis spores did not show any inactivation at any of the conditions used in this study. The OH radical exposure (CT value) was calculated to present the relationship between the hydroxyl radical dose and microbial inactivation.

  18. 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…

  19. Effects of solar ultraviolet radiations on Bacillus subtilis spores and T-7 bacteriophage

    NASA Technical Reports Server (NTRS)

    Spizizen, J.; Isherwood, J. E.; Taylor, G. R.

    1975-01-01

    Spores of Bacillus subtilis HA 101 and the DNA polymerase I-defective mutant HA 101 (59)F were exposed to selected wavelengths of solar ultraviolet light and space vacuum during the return of Apollo 16. In addition, coliphage T-7 suspensions were exposed to solar ultraviolet radiation as part of the Microbial Response to Space Environment Experiment. Optical filters were employed to provide different energy levels at wavelengths 254 nm and 280 nm. Dose-response curves for lethal and mutagenic effects were compared with ground-based data. A close parallel was observed between the results of solar radiation and ground tests with spores of the two strains. However, significantly greater inactivation of T-7 bacteriophage was observed after exposure to solar ultraviolet radiation.

  20. Control sites in the sequence at the beginning of T7 gene 1.

    PubMed Central

    McConnell, D J

    1979-01-01

    The DNA sequence of the fragment Hind.30, 378 bases long, from the beginning of gene 1 of T7 is presented. It contains the C promoter, two in vitro transcriptional terminator sites and a sequence of 171 bases which probably codes for the N terminus of the T7 RNA polymerase. The sequence also codes for the RNase III cleavage site before gene 1. The overlaps with the transcriptional terminators, The RNA transcript of the sequence about the terminators can be arranged in a set of alternative double-stranded hairpin structures. It is suggested that conversion between these structures may have a role in termination; this may be influenced by interactions with ribosomes and RNase III. The region of the C promoter between genes 0.7 and 1 thus contains several sites which may be involved in the control of transcription and translation. Images PMID:493111

  1. INFLUENCE OF URETHANE AND OF HYDROSTATIC PRESSURE ON THE GROWTH OF BACTERIOPHAGES T2, T5, T6, AND T7

    PubMed Central

    Foster, Ruth A. C.; Johnson, Frank H.

    1951-01-01

    In 0.5 per cent NaCl, nutrient broth at 35°C., urethane in a concentration of 0.4 M stops the reproduction of Escherichia coli, strain B. On dilution with 20 volumes of sterile medium, growth is resumed at its former rate after a short lag. In the one-step growth of T2, 15, T6, or T7, in the same medium at the same temperature, 0.4 M urethane, when added at the time of infection, had no apparent effect on adsorption and caused no decrease in titer throughout the latent period of the control, but completely prevented a rise in titer. If diluted 1:20 with sterile medium prior to a certain critical time in the latent period, however, bacteriophage was liberated at the same time, and in the same amount as in the control. The initial stage of apparent insensitivity to the drug lasts from the time of infection until the approximate critical times of 7 minutes with T7, T2, or T6, or 13 minutes with T5. Under the conditions described, the normal latent periods were 14, 23, 30, and 44 minutes for T7, T2, T6, and T5, respectively. At the critical times referred to above, there begins a stage characterized by complete sensitivity, rather than complete insensitivity, to 0.4 M urethane, in the sense that no active phage is subsequently liberated in continued presence of the drug. The length of this completely sensitive stage, as judged by addition of the drug at successive intervals during the latent period, extends from approximately 7 until 9 minutes after infection with T7, 7 until 15 minutes with T2 or T6, or 13 until 25 minutes with T5. When the urethane is added late in this stage of T2, a decrease in initial titer takes place as judged by assays made 40 minutes after infection, the maximum effect occurring when the drug is added between 14 and 15 minutes after infection. When added subsequently to the completely sensitive stage of each type, i.e. subsequently to 9 minutes after infection with T7, 15 minutes with T2 or T6, or 25 minutes with T5, liberation of the

  2. Characterisation data of simple sequence repeats of phages closely related to T7M.

    PubMed

    Lin, Tiao-Yin

    2016-09-01

    Coliphages T7M and T3, Yersinia phage ϕYeO3-12, and Salmonella phage ϕSG-JL2 share high homology in genomic sequences. Simple sequence repeats (SSRs) are found in their genomes and variations of SSRs among these phages are observed. Analyses on regions of sequences in T7M and T3 genomes that are likely derived from phage recombination, as well as the counterparts in ϕYeO3-12 and ϕSG-JL2, have been discussed by Lin in "Simple sequence repeat variations expedite phage divergence: mechanisms of indels and gene mutations" [1]. These regions are referred to as recombinant regions. The focus here is on SSRs in the whole genome and regions of sequences outside the recombinant regions, referred to as non-recombinant regions. This article provides SSR counts, relative abundance, relative density, and GC contents in the complete genome and non-recombinant regions of these phages. SSR period sizes and motifs in the non-recombinant regions of phage genomes are plotted. Genomic sequence changes between T7M and T3 due to insertions, deletions, and substitutions are also illustrated. SSRs and nearby sequences of T7M in the non-recombinant regions are compared to the sequences of ϕYeO3-12 and ϕSG-JL2 in the corresponding positions. The sequence variations of SSRs due to vertical evolution are classified into four categories and tabulated: (1) insertion/deletion of SSR units, (2) expansion/contraction of SSRs without alteration of genome length, (3) changes of repeat motifs, and (4) generation/loss of repeats.

  3. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    SciTech Connect

    Not Available

    1992-01-01

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3' to 5' exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  4. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    SciTech Connect

    Not Available

    1992-12-31

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  5. Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7.

    PubMed

    Guo, Fei; Liu, Zheng; Vago, Frank; Ren, Yue; Wu, Weimin; Wright, Elena T; Serwer, Philip; Jiang, Wen

    2013-04-23

    Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.

  6. [Construction of T7 phage display library from the anther of Honglian hybrid line of rice].

    PubMed

    Hu, Chao-Feng; Peng, Xiao-Jue; Zhou, Yang-Yong; Tan, Yan-Ping; Li, Shao-Qing; Zhu, Ying-Guo

    2008-06-01

    Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.

  7. An analysis of sequences stimulating frameshifting in the decoding of gene 10 of bacteriophage T7.

    PubMed Central

    Condron, B G; Gesteland, R F; Atkins, J F

    1991-01-01

    The signals necessary for the translational frameshift in the gene 10 message of bacteriophage T7 include the previously identified frameshift site and the 3' non-coding region, over 200 bases downstream. The functional components of the frameshift site are identified in this study and show that the site most probably operates by the retroviral type two site mechanism. However, the base pairing requirements for the first tRNA are much more relaxed after the slip than is seen in other examples. The element at the 3' end of the gene, also necessary for frameshifting, is examined but only the extreme 5' side of the transcriptional terminator stem-loop structure in the 3' non-coding region seems to be required. No simple secondary structural model can explain the involvement of this sequence. The T7 frameshift site can be replaced with either a T3 site or a E. coli dnaX site. Both show higher levels of frameshifting than with the T7 site. Images PMID:1945837

  8. The Structure of a Transcribing T7 RNA Polymerase in Transition from Initiation to Elongation

    SciTech Connect

    Durniak, K.; Bailey, S; Steitz, T

    2008-01-01

    Structural studies of the T7 bacteriophage DNA-dependent RNA polymerase (T7 RNAP) have shown that the conformation of the amino-terminal domain changes substantially between the initiation and elongation phases of transcription, but how this transition is achieved remains unclear. We report crystal structures of T7 RNAP bound to promoter DNA containing either a 7- or an 8-nucleotide (nt) RNA transcript that illuminate intermediate states along the transition pathway. The amino-terminal domain comprises the C-helix subdomain and the promoter binding domain (PBD), which consists of two segments separated by subdomain H. The structures of the intermediate complex reveal that the PBD and the bound promoter rotate by 45 degrees upon synthesis of an 8-nt RNA transcript. This allows the promoter contacts to be maintained while the active site is expanded to accommodate a growing heteroduplex. The C-helix subdomain moves modestly toward its elongation conformation, whereas subdomain H remains in its initiation- rather than its elongation-phase location, more than 70 angstroms away.

  9. T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis.

    PubMed

    Sequeira, Ana Filipa; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

    2016-09-01

    Efficacy of de novo gene synthesis largely depends on the quality of overlapping oligonucleotides used as template for PCR assembly. The error rate associated with current gene synthesis protocols limits the efficient and accurate production of synthetic genes, both in the small and large scales. Here, we analysed the ability of different endonuclease enzymes, which specifically recognize and cleave DNA mismatches resulting from incorrect impairments between DNA strands, to remove mutations accumulated in synthetic genes. The gfp gene, which encodes the green fluorescent protein, was artificially synthesized using an integrated protocol including an enzymatic mismatch cleavage step (EMC) following gene assembly. Functional and sequence analysis of resulting artificial genes revealed that number of deletions, insertions and substitutions was strongly reduced when T7 endonuclease I was used for mutation removal. This method diminished mutation frequency by eightfold relative to gene synthesis not incorporating an error correction step. Overall, EMC using T7 endonuclease I improved the population of error-free synthetic genes, resulting in an error frequency of 0.43 errors per 1 kb. Taken together, data presented here reveal that incorporation of a mutation-removal step including T7 endonuclease I can effectively improve the fidelity of artificial gene synthesis.

  10. RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes

    PubMed Central

    Wons, Ewa; Furmanek-Blaszk, Beata; Sektas, Marian

    2015-01-01

    DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential. PMID:25824942

  11. High-copy bacterial plasmids diffuse in the nucleoid-free space, replicate stochastically and are randomly partitioned at cell division.

    PubMed

    Reyes-Lamothe, Rodrigo; Tran, Tung; Meas, Diane; Lee, Laura; Li, Alice M; Sherratt, David J; Tolmasky, Marcelo E

    2014-01-01

    Bacterial plasmids play important roles in the metabolism, pathogenesis and bacterial evolution and are highly versatile biotechnological tools. Stable inheritance of plasmids depends on their autonomous replication and efficient partition to daughter cells at cell division. Active partition systems have not been identified for high-copy number plasmids, and it has been generally believed that they are partitioned randomly at cell division. Nevertheless, direct evidence for the cellular location of replicating and nonreplicating plasmids, and the partition mechanism has been lacking. We used as model pJHCMW1, a plasmid isolated from Klebsiella pneumoniae that includes two β-lactamase and two aminoglycoside resistance genes. Here we report that individual ColE1-type plasmid molecules are mobile and tend to be excluded from the nucleoid, mainly localizing at the cell poles but occasionally moving between poles along the long axis of the cell. As a consequence, at the moment of cell division, most plasmid molecules are located at the poles, resulting in efficient random partition to the daughter cells. Complete replication of individual molecules occurred stochastically and independently in the nucleoid-free space throughout the cell cycle, with a constant probability of initiation per plasmid.

  12. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

    PubMed

    Zyprian, E; Kado, C I

    1990-08-01

    A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

  13. Characterization of high-copy-number retrotransposons from the large genomes of the louisiana iris species and their use as molecular markers.

    PubMed Central

    Kentner, Edward K; Arnold, Michael L; Wessler, Susan R

    2003-01-01

    The Louisiana iris species Iris brevicaulis and I. fulva are morphologically and karyotypically distinct yet frequently hybridize in nature. A group of high-copy-number TY3/gypsy-like retrotransposons was characterized from these species and used to develop molecular markers that take advantage of the abundance and distribution of these elements in the large iris genome. The copy number of these IRRE elements (for iris retroelement), is approximately 1 x 10(5), accounting for approximately 6-10% of the approximately 10,000-Mb haploid Louisiana iris genome. IRRE elements are transcriptionally active in I. brevicaulis and I. fulva and their F(1) and backcross hybrids. The LTRs of the elements are more variable than the coding domains and can be used to define several distinct IRRE subfamilies. Transposon display or S-SAP markers specific to two of these subfamilies have been developed and are highly polymorphic among wild-collected individuals of each species. As IRRE elements are present in each of 11 iris species tested, the marker system has the potential to provide valuable comparative data on the dynamics of retrotransposition in large plant genomes. PMID:12807789

  14. A Genetic Screen for High Copy Number Suppressors of the Synthetic Lethality Between elg1Δ and srs2Δ in Yeast

    PubMed Central

    Gazy, Inbal; Liefshitz, Batia; Bronstein, Alex; Parnas, Oren; Atias, Nir; Sharan, Roded; Kupiec, Martin

    2013-01-01

    Elg1 and Srs2 are two proteins involved in maintaining genome stability in yeast. After DNA damage, the homotrimeric clamp PCNA, which provides stability and processivity to DNA polymerases and serves as a docking platform for DNA repair enzymes, undergoes modification by the ubiquitin-like molecule SUMO. PCNA SUMOylation helps recruit Srs2 and Elg1 to the replication fork. In the absence of Elg1, both SUMOylated PCNA and Srs2 accumulate at the chromatin fraction, indicating that Elg1 is required for removing SUMOylated PCNA and Srs2 from DNA. Despite this interaction, which suggests that the two proteins work together, double mutants elg1Δ srs2Δ have severely impaired growth as haploids and exhibit synergistic sensitivity to DNA damage and a synergistic increase in gene conversion. In addition, diploid elg1Δ srs2Δ double mutants are dead, which implies that an essential function in the cell requires at least one of the two gene products for survival. To gain information about this essential function, we have carried out a high copy number suppressor screen to search for genes that, when overexpressed, suppress the synthetic lethality between elg1Δ and srs2Δ. We report the identification of 36 such genes, which are enriched for functions related to DNA- and chromatin-binding, chromatin packaging and modification, and mRNA export from the nucleus. PMID:23704284

  15. Infrared observations of oxidized carbon in comet C/2002 t7 (LINEAR)

    NASA Astrophysics Data System (ADS)

    Anderson, William Michael, Jr.

    2010-11-01

    Cometary nuclei are generally recognized as the most primitive remnants of the early Solar System. Their physical and chemical attributes allow a glimpse into the conditions under which icy bodies formed. Parent volatiles in comets are now routinely studied, and a significant diversity in composition among the comets sampled to date has been demonstrated. This forms the foundation of an emerging cometary taxonomy based on chemical composition. In spring 2004, comet C/2002 T7 (LINEAR) was observed using the facility echelle spectrometer (CSHELL) at the NASA Infrared Telescope Facility on Mauna Kea, Hawaii. CSHELL offers seeing-limited spatial resolution and sufficiently high spectral resolving power (R = lambda/Deltalambda ˜ 2.5 x 10 4) to permit line-by-line intensities to be measured along its 30 arc-second slit. Its small pixels favor measurement of molecules released from ices housed in cometary nuclei ("native" ices) over those released from spatially extended sources in the coma. Emission lines from multiple molecular species were targeted in the 3 to 5 mum wavelength region. The observations revealed an extremely rich volatile chemistry in C/2002 T7. I present the chemical composition of oxidized carbon in C/2002 T7 (LINEAR). Carbon monoxide (CO), formaldehyde (H2CO), and methyl alcohol (CH 3OH) were detected simultaneously or nearly simultaneously with H 2O on multiple UT dates spanning 2004 May 3-9 (heliocentric distance Rh = 0.66 -- 0.71 AU) and May 30 - June 2 (R h = 0.99 -- 1.03 AU). I will discuss native production rates, rotational temperatures, and mixing ratios (abundances relative to H2O) for oxidized carbon. My results illustrate that C/2002 T7 (LINEAR) is enriched in CH3OH, while CO is borderline depleted compared to other Oort cloud comets that have been measured. I tested for chemical heterogeneity in C/2002 T7 (LINEAR), both diurnal, presumably associated with rotation of the nucleus, and serial (i.e., over a range in Rh). However, no evidence

  16. Mechanical Property Data on P/M Aluminum X7091-T7E69 Extrusion.

    DTIC Science & Technology

    1982-10-01

    block number) Powder-Metallurgy Notched Fatigue 7091-T7E69 Fracture Extrusion Fatigue Crack Growth Alyinum Stress Corrosion 20. AT RACT (Continue an...fatigue crack growth, and stress corrosion cracking. For notched fatigue investigations, stress concentration factors as high as 10 were examined...sensitivity to stress corrosion cracking under such conditions. UNCLASSIFIED SAZCu~rI1 V C.. Alit FC kVI0% 00~i u* £2(’hu Oee £e PREFACE This interim

  17. Characterization of the FAD2 Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number

    PubMed Central

    Lakhssassi, Naoufal; Zhou, Zhou; Liu, Shiming; Colantonio, Vincent; AbuGhazaleh, Amer; Meksem, Khalid

    2017-01-01

    Soybean seed oil typically contains 18–20% oleic acid. Increasing the content of oleic acid is beneficial for health and biodiesel production. Mutations in FAD2-1 genes have been reported to increase seed oleic acid content. A subset of 1,037 mutant families from a mutagenized soybean cultivar (cv.) Forrest population was screened using reverse genetics (TILLING) to identify mutations within FAD2 genes. Although no fad2 mutants were identified using gel-based TILLING, four fad2-1A and one fad2-1B mutants were identified to have high seed oleic acid content using forward genetic screening and subsequent target sequencing. TILLING has been successfully used as a non-transgenic reverse genetic approach to identify mutations in genes controlling important agronomic traits. However, this technique presents limitations in traits such as oil composition due to gene copy number and similarities within the soybean genome. In soybean, FAD2 are present as two copies, FAD2-1 and FAD2-2. Two FAD2-1 members: FAD2-1A and FAD2-1B; and three FAD2-2 members: FAD2-2A, FAD2-2B, and FAD2-2C have been reported. Syntenic, phylogenetic, and in silico analysis revealed two additional members constituting the FAD2 gene family: GmFAD2-2D and GmFAD2-2E, located on chromosomes 09 and 15, respectively. They are presumed to have diverged from other FAD2-2 members localized on chromosomes 19 (GmFAD2-2A and GmFAD2-2B) and 03 (GmFAD2-2C). This work discusses alternative solutions to the limitations of gel-based TILLING in functional genomics due to high copy number and multiple paralogs of the FAD2 gene family in soybean. PMID:28348573

  18. Characterization of the FAD2 Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number.

    PubMed

    Lakhssassi, Naoufal; Zhou, Zhou; Liu, Shiming; Colantonio, Vincent; AbuGhazaleh, Amer; Meksem, Khalid

    2017-01-01

    Soybean seed oil typically contains 18-20% oleic acid. Increasing the content of oleic acid is beneficial for health and biodiesel production. Mutations in FAD2-1 genes have been reported to increase seed oleic acid content. A subset of 1,037 mutant families from a mutagenized soybean cultivar (cv.) Forrest population was screened using reverse genetics (TILLING) to identify mutations within FAD2 genes. Although no fad2 mutants were identified using gel-based TILLING, four fad2-1A and one fad2-1B mutants were identified to have high seed oleic acid content using forward genetic screening and subsequent target sequencing. TILLING has been successfully used as a non-transgenic reverse genetic approach to identify mutations in genes controlling important agronomic traits. However, this technique presents limitations in traits such as oil composition due to gene copy number and similarities within the soybean genome. In soybean, FAD2 are present as two copies, FAD2-1 and FAD2-2. Two FAD2-1 members: FAD2-1A and FAD2-1B; and three FAD2-2 members: FAD2-2A, FAD2-2B, and FAD2-2C have been reported. Syntenic, phylogenetic, and in silico analysis revealed two additional members constituting the FAD2 gene family: GmFAD2-2D and GmFAD2-2E, located on chromosomes 09 and 15, respectively. They are presumed to have diverged from other FAD2-2 members localized on chromosomes 19 (GmFAD2-2A and GmFAD2-2B) and 03 (GmFAD2-2C). This work discusses alternative solutions to the limitations of gel-based TILLING in functional genomics due to high copy number and multiple paralogs of the FAD2 gene family in soybean.

  19. Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7*

    PubMed Central

    Daudén, María I.; Martín-Benito, Jaime; Sánchez-Ferrero, Juan C.; Pulido-Cid, Mar; Valpuesta, José M.; Carrascosa, José L.

    2013-01-01

    During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7. PMID:23632014

  20. Reassessing the radial-velocity evidence for planets around CoRoT-7

    NASA Astrophysics Data System (ADS)

    Pont, Frédéric; Aigrain, Suzanne; Zucker, Shay

    2011-03-01

    CoRoT-7 is an 11 th magnitude K-star whose light curve shows transits with a depth of 0.3 mmag and a period of 0.854 d, superimposed on variability at the 1 per cent level, due to the modulation of evolving active regions with the star's 23-d rotation period. In this paper, we revisit the published HARPS radial-velocity (RV) measurements of the object, which were previously used to estimate the companion mass, but have been the subject of ongoing debate. We build a realistic model of the star's activity during the HARPS observations, by fitting simultaneously the linewidth (as measured by the width of the cross-correlation function) and the line bisector, and use it to evaluate the contribution of activity to the RV variations. The data show clear evidence of errors above the level of the formal uncertainties, which are accounted for neither by activity nor by any plausible planet model and which increase rapidly with a decreasing signal-to-noise ratio (S/N) of the spectra. We cite evidence of similar systematics in mid-S/N spectra of other targets obtained with HARPS and other high-precision RV spectrographs, and discuss possible sources. Allowing for these, we re-evaluate the semi-amplitude of the CoRoT-7b signal, finding Kb= 1.6 ± 1.3 m s-1, a tentative detection with a much reduced significance (1.2σ) compared to previous estimates. We also argue that the combined presence of activity and additional errors precludes a meaningful search for additional low-mass companions, despite previous claims to the contrary. Taken at face value, our analysis points to a lower density for CoRoT-7b, the 1σ mass range spanning 1-4 M ⊕ and allowing for a wide range of bulk compositions. In particular, an ice-rich composition is compatible with the RV constraints. More generally, this study highlights the importance of a realistic treatment of both activity and uncertainties, particularly in the medium S/N regime, which applies to most small planet candidates from CoRoT and

  1. T7 RNA polymerase cannot transcribe through a highly knotted DNA template.

    PubMed Central

    Portugal, J; Rodríguez-Campos, A

    1996-01-01

    The ability of T7 RNA polymerase to transcribe a plasmid DNA in vitro in its linear, supercoiled, relaxed and knotted forms was analysed. Similar levels of transcription were found on each template with the exception of plasmids showing varying degrees of knotting (obtained using stoichiometric amounts of yeast topoisomerase II). A purified fraction of knotted DNA with a high number of nodes (crosses) was found to be refractory to transcription. The unknotting of the knotted plasmids, using catalytic amounts of topoisomerase II, restored their capacity as templates for transcription to levels similar to those obtained for the other topological forms. These results demonstrate that highly knotted DNA is the only topological form of DNA that is not a template for transcription. We suggest that the regulation of transcription, which depends on the topological state of the template, might be related to the presence of knotted DNA with different number of nodes. PMID:9016657

  2. [Antirestriction activity of T7 Ocr protein in monomeric and dimeric forms].

    PubMed

    Zavil'gelskiĭ, G B; Kotova, V Iu

    2014-01-01

    The Ocr protein, encoded by 0.3 (ocr) gene of bacteriophage T7, belongs to the family of antirestriction proteins that specifically inhibit the type I restriction-modification systems. Native Ocr forms homodimer (Ocr)2 both in solution and in the crystalline state. The Ocr protein belongs to the family of mimicry proteins. F53D A57E and E53R V77D mutant proteins were obtained, which form monomers. It was shown that the values of the dissociation constants Kd for Ocr, Ocr F53D A57E and Ocr F53RV77D proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, Kd (Ocr F53D A57E and Ocr F53R V77D) = 10(-7) M. Antimodification activity of the Ocr monomeric forms is significantly reduced. We have shown, that Ocr dimeric form has fundamental importance for high inhibitory activity.

  3. Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA.

    PubMed

    Walkinshaw, M D; Taylor, P; Sturrock, S S; Atanasiu, C; Berge, T; Henderson, R M; Edwardson, J M; Dryden, D T F

    2002-01-01

    We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

  4. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17

    PubMed Central

    Garcia-Doval, Carmela; van Raaij, Mark J.

    2012-01-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371–553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P212121 (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C2221 (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress. PMID:22297990

  5. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17.

    PubMed

    Garcia-Doval, Carmela; van Raaij, Mark J

    2012-02-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371-553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P2(1)2(1)2(1) (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C222(1) (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress.

  6. High density growth of T7 expression strains with auto-induction option

    DOEpatents

    Studier, F. William

    2010-07-20

    A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

  7. Interaction Analysis of T7 RNA Polymerase with Heparin and Its Low Molecular Weight Derivatives – An In Silico Approach

    PubMed Central

    Borkotoky, Subhomoi; Meena, Chetan Kumar; Murali, Ayaluru

    2016-01-01

    The single subunit T7 RNA polymerase (T7RNAP) is a model enzyme for studying the transcription process and for various biochemical and biophysical studies. Heparin is a commonly used inhibitor against T7RNAP and other RNA polymerases. However, exact interaction between heparin and T7RNAP is still not completely understood. In this work, we analyzed the binding pattern of heparin by docking heparin and few of its low molecular weight derivatives to T7RNAP, which helps in better understanding of T7RNAP inhibition mechanism. The efficiency of the compounds was calculated by docking the selected compounds and post-docking molecular mechanics/generalized Born surface area analysis. Evaluation of the simulation trajectories and binding free energies of the complexes after simulation showed enoxaparin to be the best among low molecular weight heparins. Binding free energy analysis revealed that van der Waals interactions and polar solvation energy provided the substantial driving force for the binding process. Furthermore, per-residue free energy decomposition analysis revealed that the residues Asp 471, Asp 506, Asp 537, Tyr 571, Met 635, Asp 653, Pro 780, and Asp 812 are important for heparin interaction. Apart from these residues, most favorable contribution in all the three complexes came from Asp 506, Tyr 571, Met 635, Glu 652, and Asp 653, which can be essential for binding of heparin-like structures with T7RNAP. The results obtained from this study will be valuable for the future rational design of novel and potent inhibitors against T7RNAP and related proteins. PMID:27594785

  8. Exploiting Radiation Damage to Map Proteins in Nucleoprotein Complexes: The Internal Structure of Bacteriophage T7

    PubMed Central

    Cheng, Naiqian; Wu, Weimin; Watts, Norman R.; Steven, Alasdair C.

    2014-01-01

    In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique (“bubblegram imaging”) by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 sec) intervals between exposures of 17 electrons/Å2 each, bubbling starts in the third exposure, with 1 – 4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal. PMID:24345345

  9. Multi-input regulation and logic with T7 promoters in cells and cell-free systems.

    PubMed

    Iyer, Sukanya; Karig, David K; Norred, S Elizabeth; Simpson, Michael L; Doktycz, Mitchel J

    2013-01-01

    Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.

  10. Multi-Input Regulation and Logic with T7 Promoters in Cells and Cell-Free Systems

    PubMed Central

    Norred, S. Elizabeth; Simpson, Michael L.; Doktycz, Mitchel J.

    2013-01-01

    Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications. PMID:24194933

  11. Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

    PubMed

    Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

    2017-01-01

    A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

  12. Use of T7 RNA polymerase to direct expression of outer Surface Protein A (OspA) from the Lyme disease Spirochete, Borrelia burgdorferi

    NASA Technical Reports Server (NTRS)

    Dunn, John J.; Lade, Barbara N.

    1991-01-01

    The OspA gene from a North American strain of the Lyme disease Spirochete, Borrelia burgdorferi, was cloned under the control of transciption and translation signals from bacteriophage T7. Full-length OspA protein, a 273 amino acid (31kD) lipoprotein, is expressed poorly in Escherichia coli and is associated with the insoluble membrane fraction. In contrast, a truncated form of OspA lacking the amino-terminal signal sequence which normally would direct localization of the protein to the outer membrane is expressed at very high levels (less than or equal to 100 mg/liter) and is soluble. The truncated protein was purified to homogeneity and is being tested to see if it will be useful as an immunogen in a vaccine against Lyme disease. Circular dichroism and fluorescence spectroscopy was used to characterize the secondary structure and study conformational changes in the protein. Studies underway with other surface proteins from B burgdorferi and a related spirochete, B. hermsii, which causes relapsing fever, leads us to conclude that a strategy similar to that used to express the truncated OspA can provide a facile method for producing variations of Borrelia lipoproteins which are highly expressed in E. coli and soluble without exposure to detergents.

  13. Report of the Snowmass T7 working group on high performance computing

    SciTech Connect

    K. Ko; R. Ryne; P. Spentzouris

    2002-12-05

    The T7 Working Group on High Performance Computing (HPC) had more than 30 participants. During the three weeks at Snowmass there were about 30 presentations. This working group also had joint sessions with a number of other working groups, including E1 (Neutrino Factories and Muon Colliders), M1 (Muon Based Systems), M6 (High Intensity Proton Sources), T4 (Particle sources), T5 (Beam dynamics), and T8 (Advanced Accelerators). The topics that were discussed fall naturally into three areas: (1) HPC requirements for next-generation accelerator design, (2) state-of-the-art in HPC simulation of accelerator systems, and (3) applied mathematics and computer science activities related to the development of HPC tools that will be of use to the accelerator community (as well as other communities). This document summarizes the material mentioned above and includes recommendations for future HPC activities in the accelerator community. The relationship of those activities to the HENP/SciDAC project on 21st century accelerator simulation is also discussed.

  14. Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

    NASA Astrophysics Data System (ADS)

    Gottschalk, Leda M. F.; Bon, Elba P. S.; Nobrega, Ronaldo

    It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.

  15. Evidence of Icy Grains in Comet C/2002 T7 (LINEAR) at 3.52 AU

    NASA Astrophysics Data System (ADS)

    Kawakita, Hideyo; Watanabe, Jun-ichi; Ootsubo, Takafumi; Nakamura, Ryosuke; Fuse, Tetsuharu; Takato, Naruhisa; Sasaki, Sho; Sasaki, Takanori

    2004-02-01

    We present evidence of icy grains in the coma of comet C/2002 T7 (LINEAR) at 3.52 AU from the Sun. This comet will approach the Sun in the spring of 2004, and it is expected to be very bright near its perihelion passage. The comet was observed using the Subaru Telescope with the Cooled Infrared Spectrograph and Camera for OHS (CISCO) on 2003 September 14.6 (UT). The near-infrared (J, H, K) spectrum was extracted from the near-nucleus region (1250 km × 1250 km at the comet), and it showed clear absorption features at 1.5 and 2.05 μm that originated from water ice grains. The calculated reflectance spectrum, based on the intimate mixture model for water ice grains and astronomical silicate grains (the diameters are 5 and 0.5 μm, respectively), can reproduce the observed reflectance spectrum of the comet up to 2.1 μm. The poor fit for the wavelength region longer than 2.1 μm is probably indicative of other grain species to be included in the model. Furthermore, the absence of the 1.65 μm feature of crystalline water ice may indicate that the water ice was in an amorphous state during the observation.

  16. Exploring the DNA mimicry of the Ocr protein of phage T7.

    PubMed

    Roberts, Gareth A; Stephanou, Augoustinos S; Kanwar, Nisha; Dawson, Angela; Cooper, Laurie P; Chen, Kai; Nutley, Margaret; Cooper, Alan; Blakely, Garry W; Dryden, David T F

    2012-09-01

    DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.

  17. Dissection of the DNA mimicry of the bacteriophage T7 Ocr protein using chemical modification.

    PubMed

    Stephanou, Augoustinos S; Roberts, Gareth A; Cooper, Laurie P; Clarke, David J; Thomson, Andrew R; MacKay, C Logan; Nutley, Margaret; Cooper, Alan; Dryden, David T F

    2009-08-21

    The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an approximately 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove approximately 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to approximately 800-fold.

  18. Characterisation of the structure of ocr, the gene 0.3 protein of bacteriophage T7.

    PubMed

    Atanasiu, C; Byron, O; McMiken, H; Sturrock, S S; Dryden, D T

    2001-07-15

    The product of gene 0.3 of bacteriophage T7, ocr, is a potent inhibitor of type I DNA restriction and modification enzymes. We have used biophysical methods to examine the mass, stability, shape and surface charge distribution of ocr. Ocr is a dimeric protein with hydrodynamic behaviour equivalent to a prolate ellipsoid of axial ratio 4.3 +/- 0.7:1 and mass of 27 kDa. The protein is resistant to denaturation but removal of the C-terminal region reduces stability substantially. Six amino acids, N4, D25, N43, D62, S68 and W94, are all located on the surface of the protein and N4 and S68 are also located at the interface between the two 116 amino acid monomers. Negatively charged amino acid side chains surround W94 but these side chains are not part of the highly acidic C-terminus after W94. Ocr is able to displace a short DNA duplex from the binding site of a type I enzyme with a dissociation constant of the order of 100 pM or better. These results suggest that ocr is of a suitable size and shape to effectively block the DNA binding site of a type I enzyme and has a large negatively charged patch on its surface. This charge distribution may be complementary to the charge distribution within the DNA binding site of type I DNA restriction and modification enzymes.

  19. Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

    PubMed Central

    Geertsema, Hylkje J.; Kulczyk, Arkadiusz W.; Richardson, Charles C.; van Oijen, Antoine M.

    2014-01-01

    Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading- and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution. PMID:24591606

  20. MIRO Observation of Comet C/2002 T7 (LINEAR) Water Line Spectrum

    NASA Technical Reports Server (NTRS)

    Lee, Seungwon; Frerking, Margaret; Hofstadter, Mark; Gulkis, Samuel; von Allmen, Paul; Crovisier, Jaques; Biver, Nicholas; Bockelee-Morvan, Dominique

    2011-01-01

    Comet C/2002 T7 (LINEAR) was observed with the Microwave Instrument for Rosetta Orbiter (MIRO) on April 30, 2004, between 5 hr and 16 hr UT. The comet was 0.63AU distance from the Sun and 0.68AU distance from the MIRO telescope at the time of the observations. The water line involving the two lowest rotational levels at 556.936 GHz is observed at 557.070 GHz due to a large Doppler frequency shift. The detected water line spectrum is interpreted using a non local thermal equilibrium (Non-LTE) molecular excitation and radiative transfer model. Several synthetic spectra are calculated with various coma profiles that are plausible for the comet at the time of observations. The coma profile is modeled with three characteristic parameters: outgassing rate, a constant expansion velocity, and a constant gas temperature. The model calculation result shows that for the distant line observation where contributions from a large coma space is averaged, the combination of the outgassing rate and the gas expansion velocity determines the line shape while the gas temperature has a negligible effect. The comparison between the calculated spectra and the MIRO measured spectrum suggests that the outgassing rate of the comet is about 2.0x1029 molecules/second and its gas expansion velocity about 1.2 km/s at the time of the observations.

  1. [Point contacts of T7 RNA polymerase in the promotor complex, as determined with phosphate-activated oligonucleotide derivatives].

    PubMed

    Filippova, S E; Ivanovskaia, M G; Romanova, E A; Tunitskaia, V L; Kochetkov, S N

    2002-01-01

    The contacts between phosphate groups of promoter DNA an Lys or His of T7 RNA polmerase (Pol) in the Pol-promoter complex were studied with single- and double- stranded oligonucleotides, which corresponded to the T7 promoter consensus and contained activated phosphate groups at position +1, +2, or -14 relative to the transcription start. To obtain reactive groups, terminal phosphates were modified with N-oxybenzotriazole (HOBT), and internucleotide phosphates were repalced with a trisubstituted pyrophosphate (TSP). The resulting derivatives produced covalent complexes with T7 Pol. Covalent bonding involved His in the case of TSP at position +1 or HOBT at position +1 or -14, and Lys in the case of TSB at position -14.

  2. The T7-Related Pseudomonas putida Phage ϕ15 Displays Virion-Associated Biofilm Degradation Properties

    PubMed Central

    Cornelissen, Anneleen; Ceyssens, Pieter-Jan; T'Syen, Jeroen; Van Praet, Helena; Noben, Jean-Paul; Shaburova, Olga V.; Krylov, Victor N.; Volckaert, Guido; Lavigne, Rob

    2011-01-01

    Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy. PMID:21526174

  3. Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters.

    PubMed

    Martin, C T; Coleman, J E

    1987-05-19

    Specific interactions between T7 RNA polymerase and its promoter have been studied by a simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base message. A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and -12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate constant Kcat = 50 min-1 and a Michaelis constant Km = 0.02 microM, indicating that the consensus sequence to position -17 is sufficient for maximum promoter usage. Shortening the upstream region of the promoter to -14 substantially increases Km (0.3 microM) but does not significantly reduce the maximum velocity (kcat = 30 min-1). Finally, truncation of the promoter at position -12 results in extremely low levels of specific transcription. The coding and noncoding strands appear to make different contributions to promoter recognition. Although the double-stranded promoter of upstream length -12 is very poor as a transcription template, extension of only the noncoding strand to -17 very significantly improves both Kcat and Km. In contrast, extension of only the coding strand results in no significant improvement. Substitution of an AT base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a 10-fold increase in Km, with little effect on Kcat. Comparison of two promoters containing a base pair mismatch at this site (AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding strands.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7.

    PubMed

    Zhang, Huidong; Lee, Seung-Joo; Richardson, Charles C

    2012-07-06

    DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.

  5. Residues in the central beta-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding.

    PubMed

    Satapathy, Ajit K; Kochaniak, Anna B; Mukherjee, Sourav; Crampton, Donald J; van Oijen, Antoine; Richardson, Charles C

    2010-04-13

    The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic beta-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the beta-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.

  6. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.

    PubMed

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas

    2015-01-07

    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

  7. Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

    PubMed Central

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas

    2015-01-01

    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases. PMID:25566793

  8. Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis.

    PubMed

    Chen, Po Ting; Shaw, Jei-Fu; Chao, Yun-Peng; David Ho, Tuan-Hua; Yu, Su-May

    2010-05-12

    Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated T7 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the T7 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase E1 with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.

  9. Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

    PubMed Central

    Khan, S A; Griess, G A; Serwer, P

    1992-01-01

    To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s). Subsequent binding (phase 2) occurs with first-order kinetics. Only the phase 1 binding occurs for metrizamide high density capsid II. These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites. The number of these internal sites increases as the density of the packaged DNA decreases. The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer. Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid. Images FIGURE 5 PMID:1477280

  10. Lysogenic conversion and phage resistance development in phage exposed Escherichia coli biofilms.

    PubMed

    Moons, Pieter; Faster, David; Aertsen, Abram

    2013-01-11

    In this study, three-day old mature biofilms of Escherichia coli were exposed once to either a temperate Shiga-toxin encoding phage (H-19B) or an obligatory lytic phage (T7), after which further dynamics in the biofilm were monitored. As such, it was found that a single dose of H-19B could rapidly lead to a near complete lysogenization of the biofilm, with a subsequent continuous release of infectious H-19B particles. On the other hand, a single dose of T7 rapidly led to resistance development in the biofilm population. Together, our data indicates a profound impact of phages on the dynamics within structured bacterial populations.

  11. A glucose-insensitive T7 expression system for fully-induced expression of proteins at a subsaturating level of L-arabinose.

    PubMed

    Wang, Zei Wen; Lai, Cheng-Bon; Chang, Chih-Hsiang; Chiang, Chung-Jen; Chao, Yun-Peng

    2011-06-22

    The L-arabinose (Ara)-controlled T7 expression system was previously constructed by creation of an Escherichia coli BL21(BAD) strain. The production of recombinant proteins in this strain was stringently regulated and reached a high level upon induction with Ara. Nevertheless, this system is still associated with inherent problems of interference with glucose and of the all-or-nothing induction profile at a subsaturating level of Ara. In this study, these problems were circumvented by modifying the physiological traits of BL21(BAD) strain. This was followed by deletion of ptsG gene and the araFGH and araBAD operon. The former encodes the glucose transporter while the latter two gene operons produce proteins responsible for Ara uptake and catabolism. In addition, the expression of genomic araE (encodes the Ara transporter) was constitutively enhanced. The resulting strain was designated BAD-5. By expression of the faster degrader GFP(LAA) at a subsaturating level of Ara, 80% of BAD-5 strain was found visually bright in the presence or absence of glucose. A further analysis by flow cytometry showed a uniform distribution of GFP expression for BAD-5 strain. In marked contrast, BL21(BAD) strain exhibiting visual brightness was less than 10% of the cell population and remained dark in the presence of glucose. Moreover, a saturated level of luciferase from Renilla reniformis (Rluc) could be readily obtained in BAD-5 strain at 20 μM Ara regardless of glucose. Rluc in BL21(BAD) strain was produced in an Ara dose-dependent manner, and the protein production became arrested when glucose was present. Overall, it illustrates the usefulness of the improved system for overproduction of recombinant proteins in an efficient, homogeneous, and glucose-insensitive way.

  12. Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA.

    PubMed Central

    Bailey, J N; Dembinski, D R; McAllister, W T

    1980-01-01

    The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7. Images PMID:6251266

  13. A mutational analysis of DNA mimicry by ocr, the gene 0.3 antirestriction protein of bacteriophage T7.

    PubMed

    Stephanou, Augoustinos S; Roberts, Gareth A; Tock, Mark R; Pritchard, Emily H; Turkington, Rachel; Nutley, Margaret; Cooper, Alan; Dryden, David T F

    2009-01-02

    The ocr protein of bacteriophage T7 is a structural and electrostatic mimic of approximately 24 base pairs of double-stranded B-form DNA. As such, it inhibits all Type I restriction and modification (R/M) enzymes by blocking their DNA binding grooves and inactivates them. This allows the infection of the bacterial cell by T7 to proceed unhindered by the action of the R/M defence system. We have mutated aspartate and glutamate residues on the surface of ocr to investigate their contribution to the tight binding between the EcoKI Type I R/M enzyme and ocr. Contrary to expectations, all of the single and double site mutations of ocr constructed were active as anti-R/M proteins in vivo and in vitro indicating that the mimicry of DNA by ocr is very resistant to change.

  14. Quasi-atomic model of bacteriophage t7 procapsid shell: insights into the structure and evolution of a basic fold.

    PubMed

    Agirrezabala, Xabier; Velázquez-Muriel, Javier A; Gómez-Puertas, Paulino; Scheres, Sjors H W; Carazo, José M; Carrascosa, José L

    2007-04-01

    The existence of similar folds among major structural subunits of viral capsids has shown unexpected evolutionary relationships suggesting common origins irrespective of the capsids' host life domain. Tailed bacteriophages are emerging as one such family, and we have studied the possible existence of the HK97-like fold in bacteriophage T7. The procapsid structure at approximately 10 A resolution was used to obtain a quasi-atomic model by fitting a homology model of the T7 capsid protein gp10 that was based on the atomic structure of the HK97 capsid protein. A number of fold similarities, such as the fitting of domains A and P into the L-shaped procapsid subunit, are evident between both viral systems. A different feature is related to the presence of the amino-terminal domain of gp10 found at the inner surface of the capsid that might play an important role in the interaction of capsid and scaffolding proteins.

  15. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  16. Spectrum Fatigue Crack Growth Rate Characteristics of Cast Aluminum Alloys A201-T7 and A357-T6.

    DTIC Science & Technology

    The spectrum fatigue crack growth characteristics of cast aluminum alloys A201-T7 and A357 -T6 were evaluated and compared with wrought aluminum 7050...T76 data. For specimens tested at three different spectrum stress levels, A357 -T6 consistently possessed the longest fatigue life, nearly double that...Further analysis of the a vs. N record of each sample to obtain the crack growth rate (FCGR) vs. the corresponding stress intensity indicated that A357 -T6

  17. Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

    PubMed Central

    Hernandez, Alfredo J.; Lee, Seung-Joo; Richardson, Charles C.

    2016-01-01

    DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis. PMID:27162371

  18. Early gene expression in bacteriophage T7. I. In vivo synthesis, inactivation, and translational utilization of early mRNA's.

    PubMed Central

    Hercules, K; Jovanovich, S; Sauerbrier, W

    1976-01-01

    In vivo decay rates for the individual T7 early mRNA species were determined. The physical half-lives, measured at 37 C, range from 1.1 min for gene 0.7 RNA to 4.5 min for gene 0.3 RNA. Physical half-lives, as observed after rifampin inhibition of RNA synthesis and polyacylamide electrophoresis of RNAs, are approximately 30% longer than functional half-lives, as observed by 14C-labeled amino acid uptake into individual T7 early proteins. The different RNA species are synthesized at grossly different rates, 0.3 RNA at four times the rate of 1.0 RNA, 0.7 RNA at twice the rate, and 1.1 and 1.3 RNAs at about the same or a slightly lower rate than 1.0 RNA. Rho-factor-mediated termination of transcription behind genes 0.3, 0.7, and perhaps behind 1.0 is inferred from these data. The in vivo translational utilization of the individual T7 early-message species was found to vary by not more than a factor of 2. Images PMID:1255850

  19. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    SciTech Connect

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  20. [Combination of TLR7 agonist T7-ethacrynic acid conjugate with ROR1 has a stronger anti-breast cancer effect].

    PubMed

    Zhang, Na; Jin, Guangyi; Jin, Zhenchao; Liu, Bing; Peng, Boya; Gao, Ningning; Hu, Yunlong; Tang, Li

    2016-07-01

    Objective To investigate the synergistic anti-breast cancer effect of Toll-like receptor 7 agonist T7-ethacrynic acid conjugate (T7-EA) in combination with receptor-tyrosine-kinase-like orphan receptor 1 (ROR1). Methods ROR1 cytotoxic T lymphocyte (CTL) epitope was predicted using Syfpeithi online software. Mouse spleen lymphocytes and bone marrow dendritic cells (DCs) were separately stimulated with 4 μmol/L T7-EA and 4 μmol/L ROR1 alone or in combination. ELISA assay was used to measure the levels of interferon-γ (IFN-γ), interleukin 12 (IL-12) and tumor necrosis factor-α (TNF-α). Xenograft model was established via subcutaneous injection of mouse breast cancer 4T1 cells. The mice were weekly treated through intraperitoneal administration of 3 mg/kg T7-EA, 15 mg/kg ROR1 or the combination of T7-EA and ROR1. After four rounds of treatment, tumor tissues were weighed. Serum level of anti-4T1 tumor protein IgG was measured by ELISA. Specific CTL activity was detected by lactate dehydrogenase (LDH) assay. Results The peptide PYCDETSSV was chosen as an antigen epitope of breast cancer. The T7-EA highly activated in vitro lymphocytes in a dose-dependent manner, which wasn't affected by other relevant peptides. The combination of T7-EA and ROR1 stimulated the secretion of IFN-γ and IL-12 by lymphocytes and TNF-α by bone marrow DCs. The growth of tumor in vivo was significantly inhibited by T7-EA combined with ROR1 compared with T7-EA or ROR1 alone. The specific CTL activity triggered by T7-EA combined with ROR1 was much stronger than that triggered by T7-EA or ROR1 alone. The titer of anti-4T1 tumor protein IgG induced by T7-EA combined with ROR1 was higher than that induced by T7-EA or ROR1. Conclusion The combination of T7-EA and ROR1 has a better killing effect on breast cancer.

  1. Could CoRoT-7b and Kepler-10b be remnants of evaporated gas or ice giants?

    PubMed Central

    Leitzinger, M.; Odert, P.; Kulikov, Yu.N.; Lammer, H.; Wuchterl, G.; Penz, T.; Guarcello, M.G.; Micela, G.; Khodachenko, M.L.; Weingrill, J.; Hanslmeier, A.; Biernat, H.K.; Schneider, J.

    2011-01-01

    We present thermal mass loss calculations over evolutionary time scales for the investigation if the smallest transiting rocky exoplanets CoRoT-7b (∼1.68REarth) and Kepler-10b (∼1.416REarth) could be remnants of an initially more massive hydrogen-rich gas giant or a hot Neptune-class exoplanet. We apply a thermal mass loss formula which yields results that are comparable to hydrodynamic loss models. Our approach considers the effect of the Roche lobe, realistic heating efficiencies and a radius scaling law derived from observations of hot Jupiters. We study the influence of the mean planetary density on the thermal mass loss by placing hypothetical exoplanets with the characteristics of Jupiter, Saturn, Neptune, and Uranus to the orbital location of CoRoT-7b at 0.017 AU and Kepler-10b at 0.01684 AU and assuming that these planets orbit a K- or G-type host star. Our findings indicate that hydrogen-rich gas giants within the mass domain of Saturn or Jupiter cannot thermally lose such an amount of mass that CoRoT-7b and Kepler-10b would result in a rocky residue. Moreover, our calculations show that the present time mass of both rocky exoplanets can be neither a result of evaporation of a hydrogen envelope of a “Hot Neptune” nor a “Hot Uranus”-class object. Depending on the initial density and mass, these planets most likely were always rocky planets which could lose a thin hydrogen envelope, but not cores of thermally evaporated initially much more massive and larger objects. PMID:21969736

  2. Could CoRoT-7b and Kepler-10b be remnants of evaporated gas or ice giants?

    NASA Astrophysics Data System (ADS)

    Leitzinger, M.; Odert, P.; Kulikov, Yu. N.; Lammer, H.; Wuchterl, G.; Penz, T.; Guarcello, M. G.; Micela, G.; Khodachenko, M. L.; Weingrill, J.; Hanslmeier, A.; Biernat, H. K.; Schneider, J.

    2011-10-01

    We present thermal mass loss calculations over evolutionary time scales for the investigation if the smallest transiting rocky exoplanets CoRoT-7b (∼1.68REarth) and Kepler-10b (∼1.416REarth) could be remnants of an initially more massive hydrogen-rich gas giant or a hot Neptune-class exoplanet. We apply a thermal mass loss formula which yields results that are comparable to hydrodynamic loss models. Our approach considers the effect of the Roche lobe, realistic heating efficiencies and a radius scaling law derived from observations of hot Jupiters. We study the influence of the mean planetary density on the thermal mass loss by placing hypothetical exoplanets with the characteristics of Jupiter, Saturn, Neptune, and Uranus to the orbital location of CoRoT-7b at 0.017 AU and Kepler-10b at 0.01684 AU and assuming that these planets orbit a K- or G-type host star. Our findings indicate that hydrogen-rich gas giants within the mass domain of Saturn or Jupiter cannot thermally lose such an amount of mass that CoRoT-7b and Kepler-10b would result in a rocky residue. Moreover, our calculations show that the present time mass of both rocky exoplanets can be neither a result of evaporation of a hydrogen envelope of a “Hot Neptune” nor a “Hot Uranus”-class object. Depending on the initial density and mass, these planets most likely were always rocky planets which could lose a thin hydrogen envelope, but not cores of thermally evaporated initially much more massive and larger objects.

  3. Could CoRoT-7b and Kepler-10b be remnants of evaporated gas or ice giants?

    PubMed

    Leitzinger, M; Odert, P; Kulikov, Yu N; Lammer, H; Wuchterl, G; Penz, T; Guarcello, M G; Micela, G; Khodachenko, M L; Weingrill, J; Hanslmeier, A; Biernat, H K; Schneider, J

    2011-10-01

    We present thermal mass loss calculations over evolutionary time scales for the investigation if the smallest transiting rocky exoplanets CoRoT-7b (∼1.68REarth) and Kepler-10b (∼1.416REarth) could be remnants of an initially more massive hydrogen-rich gas giant or a hot Neptune-class exoplanet. We apply a thermal mass loss formula which yields results that are comparable to hydrodynamic loss models. Our approach considers the effect of the Roche lobe, realistic heating efficiencies and a radius scaling law derived from observations of hot Jupiters. We study the influence of the mean planetary density on the thermal mass loss by placing hypothetical exoplanets with the characteristics of Jupiter, Saturn, Neptune, and Uranus to the orbital location of CoRoT-7b at 0.017 AU and Kepler-10b at 0.01684 AU and assuming that these planets orbit a K- or G-type host star. Our findings indicate that hydrogen-rich gas giants within the mass domain of Saturn or Jupiter cannot thermally lose such an amount of mass that CoRoT-7b and Kepler-10b would result in a rocky residue. Moreover, our calculations show that the present time mass of both rocky exoplanets can be neither a result of evaporation of a hydrogen envelope of a "Hot Neptune" nor a "Hot Uranus"-class object. Depending on the initial density and mass, these planets most likely were always rocky planets which could lose a thin hydrogen envelope, but not cores of thermally evaporated initially much more massive and larger objects.

  4. WATER PRODUCTION IN COMETS 2001 Q4 (NEAT) AND 2002 T7 (LINEAR) DETERMINED FROM SOHO/SWAN OBSERVATIONS

    SciTech Connect

    Combi, M. R.; Lee, Y.; Maekinen, J. T. T.; Bertaux, J.-L.; Quemerais, E.

    2009-06-15

    The SWAN all-sky camera on the Solar and Heliospheric Observatory (SOHO) spacecraft detected the hydrogen Lyman-alpha (Ly{alpha}) comae of comets 2001 Q4 NEAT and 2002 T7 LINEAR for large portions of their perihelion apparitions in 2003 and 2004. C/2001 Q4 NEAT was observed from 2003 September 14 through 2004 November 2, covering heliocentric distances from 3.23 AU before perihelion to 2.75 AU after, and C/2002 T7 LINEAR was observed from 2003 December 4 through 2004 August 6, covering heliocentric distances from 2.52 AU before perihelion to 2.09 AU after. We combined the full set of comet specific and full-sky observations and used our time-resolved model (TRM), which enables us to extract continuous values of the daily-average value of the water production rate throughout most of this entire period. The average power-law fit to the production rate variation of C/2001 Q4 NEAT with heliocentric distance, r, gives 3.5 x 10{sup 29} r {sup -1.7} and that for C/2002 T7 LINEAR gives 4.6 x 10{sup 29} r {sup -2.0}. Both comets show roughly a factor of 2 asymmetry in activity about perihelion, being more active before perihelion. C/2001 Q4 NEAT showed a production rate outburst about 30 days before perihelion (2004 April 15) and then a large extended increase above the nominal trend from 50 to 70 days after perihelion (2004 July 5-July 25)

  5. Crystallization and preliminary X-ray analysis of ocr, the product of gene 0.3 of bacteriophage T7.

    PubMed

    Sturrock, S S; Dryden, D T; Atanasiu, C; Dornan, J; Bruce, S; Cronshaw, A; Taylor, P; Walkinshaw, M D

    2001-11-01

    Ocr, the product of gene 0.3 of bacteriophage T7, prevents the action of restriction endonucleases of the host bacteria. The amino-acid sequence of ocr has less than 20% similarity to any protein of known three-dimensional structure. Ocr has been crystallized in a number of different crystal forms and X-ray data for the seleno-L-methionine-substituted form has been collected to a resolution of 1.8 A. The presence of caesium was found to be required for good crystal growth. Anomalous X-ray data was used to identify possible positions for Se and Cs atoms in the unit cell.

  6. Abnormal rapid non-linear RNA production induced by T7 RNA polymerase in the absence of an exogenous DNA template

    NASA Astrophysics Data System (ADS)

    Kakimoto, Y.; Fujinuma, A.; Fujita, S.; Kikuchi, Y.; Umekage, S.

    2015-02-01

    Although recombinant T7 RNA polymerase is commonly used for in vitro RNA synthesis, several reports have pointed out that T7 RNA polymerase can also induce RNA-directed RNA polymerization or replication. In addition, here we show a new aberrant transcription when using T7 RNA polymerase. This polymerization was observed in the presence of both ribonucleotides and a purchasable T7 RNA polymerase, Thermo T7 RNA polymerase, as well as in the absence of an exogenous DNA template. This cryptic RNA production was detectable after several hours of incubation and was inhibited by adding DNase I. These findings suggested that some contaminated DNA along with the Thermo stable T7 RNA polymerase could be used as template DNA. However, to our surprise, RNA production showed a rapid non-linear increase. This finding strongly indicated that a self-replication cycle emerged from the RNA-directed polymerization or replication by T7 RNA polymerase, triggering the abnormal explosive increase.

  7. A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA.

    PubMed

    Bloom, Kristie; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-01

    Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.

  8. Segregation of the AML t(7;11)(p15;p15) translocation chromosomes in somatic cell hybrids

    SciTech Connect

    Borrow, J.; Munroe, D.; Housman, D.E.

    1994-09-01

    The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated predominately with acute myeloid leukemia (AML) FAB M2 and occasionally with other types of AML or CML blast crisis. High resolution banding techniques have previously localized the breakpoints to 7q15.1 and 11p15.5. We have fused t(7;11)(p15;p15) blast cells from an AML patient to CHTG (hamster) cells in order to segregate the translocated chromosomes from their normal counterparts in somatic cell hybrids. Fusion events containing the derivative chromosomes or the normal chromosome 11 were enriched by panning with the antibodies M1C1 and MER2. These antibodies recognize cell surface markers which are expressed from genes which map to opposite sides of the breakpoint on chromosome 11 (11p13 and 11p15.5, respectively). Individual hybrids were expanded and typed with a series of ordered STSs from chromosomes 7 and 11, and hybrids containing the der(7) and der(11) chromosomes were identified. The segregation of the STSs between the two derivatives is in full agreement with the consensus breakpoint positions as determined cytogenetically. These hybrids may prove useful in further delineation of the breakpoint regions on chromosomes 7 and 11.

  9. Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability.

    PubMed

    Donmez, Ilker; Rajagopal, Vaishnavi; Jeong, Yong-Joo; Patel, Smita S

    2007-07-20

    Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.

  10. A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis.

    PubMed

    Huang, Mo Chao; Cheong, Wai Chye; Lim, Li Shi; Li, Mo-Huang

    2012-03-01

    Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.

  11. Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template*

    PubMed Central

    Lee, Seung-Joo; Zhu, Bin; Akabayov, Barak; Richardson, Charles C.

    2012-01-01

    The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis. PMID:23024359

  12. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    NASA Astrophysics Data System (ADS)

    Shokri, Leila; Rouzina, Ioulia; Williams, Mark C.

    2009-06-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork.

  13. Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

    PubMed

    Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

    2012-01-01

    We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.

  14. SPITZER INFRARED OBSERVATIONS AND INDEPENDENT VALIDATION OF THE TRANSITING SUPER-EARTH CoRoT-7 b

    SciTech Connect

    Fressin, Francois; Torres, Guillermo; Charbonneau, David; Pont, Frederic; Knutson, Heather A.; Mazeh, Tsevi; Aigrain, Suzanne; Fridlund, Malcolm; Guillot, Tristan; Rauer, Heike

    2012-01-20

    The detection and characterization of the first transiting super-Earth, CoRoT-7 b, has required an unprecedented effort in terms of telescope time and analysis. Although the star does display a radial-velocity signal at the period of the planet, this has been difficult to disentangle from the intrinsic stellar variability and pinning down the velocity amplitude has been very challenging. As a result, the precise value of the mass of the planet-and even the extent to which it can be considered to be confirmed-has been debated in the recent literature, with six mass measurements published so far based on the same spectroscopic observations, ranging from about 2 to 8 Earth masses. Here we report on an independent validation of the planet discovery using one of the fundamental properties of a transit signal: its achromaticity. We observed four transits of CoRoT-7 b at 4.5 {mu}m and 8.0 {mu}m with the Infrared Array Camera (IRAC) on board the Spitzer Space Telescope in order to determine whether the depth of the transit signal in the near-infrared is consistent with that observed in the CoRoT bandpass, as expected for a planet. We detected the transit and found an average depth of 0.426 {+-} 0.115 mmag at 4.5 {mu}m, which is in good agreement with the depth of 0.350 {+-} 0.011 mmag (ignoring limb darkening) found by CoRoT. The observations at 8.0 {mu}m did not yield a significant detection. The 4.5 {mu}m observations place important constraints on the kinds of astrophysical false positives that could mimic the signal. Combining this with additional constraints reported earlier, we performed an exhaustive exploration of possible blend scenarios for CoRoT-7 b using the BLENDER technique. We are able to rule out the vast majority of false positives, and the remaining ones are found to be much less likely than a true transiting planet. We thus validate CoRoT-7 b as a bona fide planet with a very high degree of confidence, independently of any radial-velocity information

  15. Shape of Ocr, the gene 0.3 protein of bacteriophage T7: modeling based on light scattering experiments.

    PubMed

    Blackstock, J J; Egelhaaf, S U; Atanasiu, C; Dryden, D T; Poon, W C

    2001-08-21

    Ocr, the first protein expressed by bacteriophage T7, inhibits type Iota DNA restriction enzymes by preventing them from binding to DNA. This inhibition allows the phage to successfully infect the host. The shape of ocr is modeled on the basis of static and dynamic light scattering measurements. The static light scattering data confirm previous observations that ocr exists in solution as a dimer. The diffusion constant determined by dynamic light scattering indicates a nonspherical shape of the ocr dimer. Hydrodynamic models of ellipsoids are presented, and it is argued that ocr is best described by a prolate ellipsoid with dimensions of 10.4 nm by 2.6 nm. The size and shape predicted by this model are consistent with ocr acting as a mimic of the DNA structure bound by type Iota restriction enzymes.

  16. Spitzer Infrared Observations and Independent Validation of the Transiting Super-Earth CoRoT-7 b

    NASA Astrophysics Data System (ADS)

    Fressin, Francois; Torres, Guillermo; Pont, Frederic; Knutson, Heather A.; Charbonneau, David; Mazeh, Tsevi; Aigrain, Suzanne; Fridlund, Malcolm; Henze, Christopher E.; Guillot, Tristan; Rauer, Heike

    2012-01-01

    The detection and characterization of the first transiting super-Earth, CoRoT-7 b, has required an unprecedented effort in terms of telescope time and analysis. Although the star does display a radial-velocity signal at the period of the planet, this has been difficult to disentangle from the intrinsic stellar variability and pinning down the velocity amplitude has been very challenging. As a result, the precise value of the mass of the planet—and even the extent to which it can be considered to be confirmed—has been debated in the recent literature, with six mass measurements published so far based on the same spectroscopic observations, ranging from about 2 to 8 Earth masses. Here we report on an independent validation of the planet discovery using one of the fundamental properties of a transit signal: its achromaticity. We observed four transits of CoRoT-7 b at 4.5 μm and 8.0 μm with the Infrared Array Camera (IRAC) on board the Spitzer Space Telescope in order to determine whether the depth of the transit signal in the near-infrared is consistent with that observed in the CoRoT bandpass, as expected for a planet. We detected the transit and found an average depth of 0.426 ± 0.115 mmag at 4.5 μm, which is in good agreement with the depth of 0.350 ± 0.011 mmag (ignoring limb darkening) found by CoRoT. The observations at 8.0 μm did not yield a significant detection. The 4.5 μm observations place important constraints on the kinds of astrophysical false positives that could mimic the signal. Combining this with additional constraints reported earlier, we performed an exhaustive exploration of possible blend scenarios for CoRoT-7 b using the BLENDER technique. We are able to rule out the vast majority of false positives, and the remaining ones are found to be much less likely than a true transiting planet. We thus validate CoRoT-7 b as a bona fide planet with a very high degree of confidence, independently of any radial-velocity information. Our Spitzer

  17. A de nevo complex t(7;13;8) translocation with a deletion in the TRPS gene region.

    PubMed

    Brandt, C A; Lüdecke, H J; Hindkjaer, J; Strømkjaer, H; Pinkel, D; Herlin, T; Bolund, L; Friedrich, U

    1997-09-01

    Molecular cytogenetic analyses have resolved the pathogenetic aberration of an 8-year-old girl with tricho-rhino-phalangeal syndrome type I (TRPS I), normal intelligence, and a karyotype originally described as 46,XX,t(8;13)(q24;q21). R- and Q-banding and high resolution R-banding analyses have also disclosed a seemingly mosaic abnormality of the distal short arm of chromosome 7 but have not fully characterized this abnormality. Combined primed in situ labelling and chromosome painting, and three-colour chromosome painting have revealed a complex, apparently balanced translocation t(7;13;8). Fluorescence in situ hybridization with yeast artificial chromosome and cosmid clones from 8q24.1 has shown an interstitial deletion of at least 3 Mb covering most of the TRPS I critical region.

  18. Effects of deposited nuclear and electronic energy on the hardness of R7T7-type containment glass

    NASA Astrophysics Data System (ADS)

    Peuget, S.; Noël, P.-Y.; Loubet, J.-L.; Pavan, S.; Nivet, P.; Chenet, A.

    2006-05-01

    The effects of elastic and inelastic interactions induced by cumulative alpha decay on the hardness of R7T7-type nuclear containment glass were investigated on actinide-doped glass specimens and by external irradiation of inactive glass by light and heavy ions. Vickers microindentation and nanoindentation hardness measurements showed that in the deposited energy range investigated (below 3 × 10 22 keV/cm 3) inelastic effects have no influence on the plastic response of the glass. Conversely, identical hardness variations versus the nuclear energy deposited in the material were observed on curium-doped glass and on glass irradiated by ion bombardment. The observed hardness variation stabilized after the deposited energy reached about 3 × 10 20 keV nucl/cm 3. These findings indicate that the change in the plastic response of the glass is a consequence of ballistic effects.

  19. New trends in the cyber and street market of recreational drugs? The case of 2C-T-7 ('Blue Mystic').

    PubMed

    Schifano, Fabrizio; Deluca, Paolo; Agosti, Lisa; Martinotti, Giovanni; Corkery, John M; Alex, Baldacchino; Caterina, Bonan; Heikki, Bothas; Raffaella, Brigada; Anna, Comacchio; Lucia, Di Furia; Dorte, Duarte Rui Eastwood; Magi, Farré; Susana, Ferreira; Irene, Flores; Claude, Guionnet; Lisbet, Harder; Lene, Stokholm Jensen; Mauro, Leoni; Christopher, Littlejohn; Aino, Majava; Teuvo, Peltoniemi; Milena, Pizza; Salman, Rawaf; Damien, Robert; Angela, Rossi Maria; Francesco, Rovetto; Norbert, Scherbaum; Holger, Siemann; Josep, Tarrago; Marta, Torrens; Francesco, Zambello

    2005-11-01

    2C-T-7 ('Blue Mystic'), an illicit compound which shows similarities with MDMA and other designer drugs, has been only occasionally identified in the EU, but discussion on the Internet between experimenters has recently grown significantly. We aimed at collecting together in a review the available information on 2C-T-7, both at the cyber and at the street market level. 2C-T-7 was first synthesized in 1986; its desired effects include both a sense of empathy and of well-being. Hallucinations, nausea, anxiety, panic attacks and paranoid ideation are anecdotally reported. According to the different European sources here approached, the availability of 2C-T-7 at street level seems to be currently very low, although one death related to a mono-intoxication with 2C-T-7 has been documented in the USA. With respect to information on 2C-T-7 available online, due to both redundancy and relevance issues the initial identified sample of 360 was reduced to 118 websites. In 14 (11.9%) websites, the detailed description of the 2C-T-7 synthesis was given. Harm Reduction websites appeared significantly earlier in the search engines results' list than Anti drugs (p 0.006) websites. Five (4.2%) websites apparently offered 2C-T-7 for sale. The large body of knowledge available online seems to contrast with small numbers of seizures at street level; an exhaustive web mapping of drug-related issues may be of interest for the clinician. Projects aimed at designing more 'attractive' prevention websites should be planned and future studies should better assess the characteristics of those consumers who take advantage of the online information of hallucinogenic compounds.

  20. Two new reciprocal translocations in the German cockroach. Cytology and genetics of T(3;12) and T(7;12).

    PubMed

    Ross, M H; Cochran, D G

    1975-01-01

    Two new reciprocal translocations in the German cockroach have been analyzed. They were identified cytologically to be T(3;12) and T(7;12). Linkage studies showed that groups XI, IX, and IV are on chromosomes 12, 3, and 7, respectively, and clearly demonstrated sex differences in recombination. Each of these chromosomes have distinctive morphological features that facilitate their identification, and permit breakpoint and centromere localization. A sex difference in fecundity is associated with T(7;12), but not T(3;12). About 40 percent mortality occurred when T(3;12) males or females and T(7;12) females were outcrossed. Outcrossing T(7;12) males produced the expected 50 percent mortality. Cell counts at metaphase I revealed that T(3;12) males exhibit directed segregation, while T(7;12) males do not. Tests for homozygosity indicated that the T(7;12) homozygote is viable. A map of chromosome 12 is presented showing the tentative placement of linkage group XI with respect to interchange breakpoints and chromosome morphology. The results are discussed in relation to possible sex differences in chiasma localization.

  1. Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification.

    PubMed

    van Bakel, Harm; van Werven, Folkert J; Radonjic, Marijana; Brok, Mariel O; van Leenen, Dik; Holstege, Frank C P; Timmers, H T Marc

    2008-03-01

    Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is a powerful technique to detect in vivo protein-DNA interactions. Due to low yields, ChIP assays of transcription factors generally require amplification of immunoprecipitated genomic DNA. Here, we present an adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (double-T7). Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis. In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p. The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR. Both LM-PCR and double-T7 identified strongly bound genomic regions, but the double-T7 method increased sensitivity and specificity to allow detection of weaker binding sites.

  2. Mannose-inhibitable adhesins and T3-T7 receptors of Klebsiella pneumoniae inhibit phagocytosis and intracellular killing by human polymorphonuclear leukocytes.

    PubMed Central

    Pruzzo, C; Debbia, E; Satta, G

    1982-01-01

    It has recently been shown that Klebsiella pneumoniae strains adhere to human epithelial cells and that adherence is mediated by mannose-inhibitable adhesins which are also receptors for coliphages T3 and T7. We have now found that Klebsiella strain K59, which adheres to human epithelial cells and carries the receptors for coliphages T3 and T7, adheres to human polymorphonuclear leukocytes (PMN) at 4 degrees C. Strains KRTT1 and KRTT2, which are spontaneous mutants unable to adsorb coliphages T3 and T7 and adhere to human epithelial cells, at this temperature did not adhere to PMN. Adherence of K59 cells to PMN at 4 degrees C was inhibited by D-mannose, by UV-inactivated T7 phages, and by pepsin-digested anti-K59 antibodies absorbed with KRTT1 cells. At 37 degrees C the number of PMN with KRTT bacteria associated was fourfold higher than at 4 degrees C. On the contrary, the number of PMN with K59 bacteria associated at this temperature was fourfold lower than at 4 degrees C. Phagocytosis and intracellular killing experiments performed at 37 degrees C showed that KRTT1 and KRTT2 were phagocytized and killed at a higher rate than K59. After blocking of the mannose-inhibitable adhesins and T3-T7 receptors (MIAT) by D-mannose, UV-inactivated bacteriophage T7, or specific antibodies, K59 cells became more sensitive to phagocytosis and intracellular killing at 37 degrees C. K59 cells lysogenic for prophage AP3 were approximately as sensitive to phagocytosis and intracellular killing by human PMN as strains KRTT1 and KRTT2. Unencapsulated Klebsiella strains isolated from clinical specimens were found to carry MIAT most often. Four such strains were found much more resistant to phagocytosis and intracellular killing than their spontaneous mutants resistant to bacteriophages T3 and T7. PMID:7047402

  3. Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in bacteriophage T7 early mRNA.

    PubMed Central

    Nicholson, A W; Niebling, K R; McOsker, P L; Robertson, H D

    1988-01-01

    To test the ability of an RNA processing enzyme to cleave chemically-modified RNA substrates, RNA transcripts containing RNase III cleavage sites were enzymatically synthesized in vitro to contain specific phosphorothioate diester internucleotide linkages. One transcript (R1.1 RNA) was generated using phage T7 RNA polymerase and a cloned segment of phage T7 DNA containing the R1.1 RNase III processing site. The second transcript was the phage T7 polycistronic early mRNA precursor, which was synthesized using E. coli RNA polymerase and T7 genomic DNA. The RNA transcripts contained phosphorothioate diester groups at positions including the scissile bonds. The modified RNAs were stable to incubation in Mg2+-containing buffer, and were specifically cleaved by RNase III. RNA oligonucleotide sequence analysis showed that the modified R1.1 RNA processing site was the same as the canonical site and contained a phosphorothioate bond. Furthermore, RNase III cleaved the phosphorothioate internucleotide bond with 5' polarity. RNase III cleavage of phosphorothioate substituted T7 polycistronic early mRNA precursor produced the same gel electrophoretic pattern as that obtained with the control transcript. Thus, RNase III cleavage specificity is not altered by phosphorothioate internucleotide linkages. Images PMID:3279395

  4. Overexpression of the tcp Gene Cluster Using the T7 RNA Polymerase/Promoter System and Natural Transformation-Mediated Genetic Engineering of Vibrio cholerae

    PubMed Central

    Borgeaud, Sandrine; Blokesch, Melanie

    2013-01-01

    The human pathogen and aquatic bacterium Vibrio cholerae belongs to the group of naturally competent bacteria. This developmental program allows the bacterium to take up free DNA from its surrounding followed by a homologous recombination event, which allows integration of the transforming DNA into the chromosome. Taking advantage of this phenomenon we genetically engineered V. cholerae using natural transformation and FLP recombination. More precisely, we adapted the T7 RNA polymerase/promoter system in this organism allowing expression of genes in a T7 RNA polymerase-dependent manner. We naturally transformed V. cholerae by adding a T7-specific promoter sequence upstream the toxin-coregulated pilus (tcp) gene cluster. In a V. cholerae strain, which concomitantly produced the T7 RNA polymerase, this genetic manipulation resulted in the overexpression of downstream genes. The phenotypes of the strain were also in line with the successful production of TCP pili. This provides a proof-of-principle that the T7 RNA polymerase/promoter system is functional in V. cholerae and that genetic engineering of this organism by natural transformation is a straightforward and efficient approach. PMID:23308292

  5. groE genes affect SOS repair in Escherichia coli

    SciTech Connect

    Liu, S.K.; Tessman, I. )

    1990-10-01

    Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes. When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13. Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copy-number umuD+C+ plasmid and a UV dose that increases groE expression. Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes. By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host.

  6. The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

    PubMed

    Melkina, Olga E; Goryanin, Ignatiy I; Zavilgelsky, Gennadii B

    2016-11-01

    The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria.

  7. Sequence and Structural Characterization of Great Salt Lake Bacteriophage CW02, a Member of the T7-Like Supergroup

    PubMed Central

    Shen, Peter S.; Sanz-García, Eduardo; Makaju, Aman; Taylor, Ryan M.; Hoggan, Ryan; Culumber, Michele D.; Oberg, Craig J.; Breakwell, Donald P.; Prince, John T.

    2012-01-01

    Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1. PMID:22593163

  8. Interaction of a trehalose lipid biosurfactant produced by Rhodococcus erythropolis 51T7 with a secretory phospholipase A2.

    PubMed

    Zaragoza, Ana; Teruel, José A; Aranda, Francisco J; Ortiz, Antonio

    2013-10-15

    Trehalose-containing glycolipid biosurfactants form an emerging group of interesting compounds, which alter the structure and properties of phospholipid membranes, and interact with enzymatic and non-enzymatic proteins. Phospholipases A2 constitute a class of enzymes that hydrolyze the sn-2 ester of glycerophospholipids, and are classified into secreted phospholipases A2 (sPLA2) and intracellular phospholipases A2. In this work, pancreatic sPLA2 was chosen as a model enzyme to study the effect of the trehalose lipid biosurfactant on enzymes acting on interfaces. By using this enzyme, it is possible to study the modulation of enzyme activity, either by direct interaction of the biosurfactant with the protein, or as a result of the incorporation of the glycolipid on the phospholipid target membrane. It is shown that the succinoyl trehalose lipid isolated from Rhodococcus erythropolis 51T7 interacts with porcine pancreatic sPLA2 and inhibits its catalytic activity. Two modes of inhibition are observed, which are clearly differentiated by its timescale. First, a slow inhibition of sPLA2 activity upon preincubation of the enzyme with trehalose lipid in the absence of substrate is described. Second, incorporation of trehalose lipid into the phospholipid target membrane gives rise to a fast enzyme inhibition. These results are discussed in the light of previous data on sPLA2 inhibitors and extend the list of interesting biological activities reported for this R. erythropolis trehalose lipid biosurfactant.

  9. Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7

    PubMed Central

    Li, Ping; Wang, Lei

    2013-01-01

    The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. PMID:23417490

  10. Bima Array Detections of HCN in Comets Linear (C/2002 T7) and Neat (C/2001 Q4)

    NASA Technical Reports Server (NTRS)

    Friedel, D. N.; Remijan, A.; Snyder, L. E.; AHearn, M. F.; Blake, Geoffrey A.; dePater, Imke; Dickel, H. R.; Forster, J. R.; Hogerheijde, M. R.

    2004-01-01

    We present interferometric detections of HCN in comets LINEAR (C/2002 T7) and NEAT (C/2001 Q4) with the Berkeley-Illinois-Maryland Association (BIMA) Array in its D-configuration cross-correlation mode. We detected the HCN J = 1 - 0 emission line in both comets. With a 25".4 x 20".3 synthesized beam around Comet LINEAR, we found a total beam averaged HCN column density (assuming a rotation temperature of 146 K) of < N(sub T) > = 2.1(11)x 10(sup 13) cm(exp -2), and a HCN production rate of Q(HCN)=2.8(15)x 10(sup 27) s(exp -1). With a 21".3 x 17".5 synthesized beam around Comet NEAT, we found a total beam averaged HCN column density (assuming a rotation temperature of 107 K) of < N(sub T) > = 5.7(30) x 10(sup l2) cm(exp -2), and a HCN production rate of Q(HCN)=8.3(44) x 10(sup 26) s(exp -l) giving a production rate of HCN relative to H2O of approximately 0.09(5)%. The production rates relative to H2O and spatial extent of HCN are similar to previous comet observations.

  11. Immunologic relatedness of extracellular ligninases from the actinomycetes Streptomyces viridosporus T7A and Streptomyces badius 252

    SciTech Connect

    Magnuson, T.S.; Roberts, M.A.; Crawford, D.L.; Hertel, G.

    1991-12-31

    Four isoforms of the extracellular lignin peroxidase of the ligninolytic actinomycete Streptomyces viridosporus T7A (ALip-P1, P2, P3, and P4) were individually purified by ultrafiltration and ammonium sulfate precipitation, followed by electro-elution using polyacrylamide gel electrophoresis. Three of the purified peroxidases were compared for their immunologic relatedness by Western blot analysis using a polyclonal antibody preparation produced in rabbits against pure isoform P3. The anti-P3 antibody was also tested for its reactivity towards a lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and another ligninolytic actinomycete Streptomyces badius 252. Results showed that peroxidases ALip-P1 through ALip-P3 are immunologically related to one another. The peroxidases of S. badius, but not the peroxidase of P. chrysosporium, also reacted with the antibody, thus indicating that the lignin peroxidases of S. viridosporus and S. badius are immunologically related. Based upon its specific affinity, fignin peroxidase isoform ALip-P3 of S. viridosporus was readily purified using an anti-P3 antibody affinity column.

  12. Pyrovanadolysis: a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate MN2plus and DNA Polymerase of Bacteriophage T7

    SciTech Connect

    B Akabayov; A Kulczyk; S Akabayov; C Thiele; L McLaughlin; B Beauchamp; C Richardson

    2011-12-31

    DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP{sub i}). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP{sub i}, a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP{sub i} complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn{sup 2+}, larger than Mg{sup 2+}, fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

  13. DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure Function Study of the Zinc-Binding Domain

    SciTech Connect

    Akabayov, B.; Lee, S; Akabayov, S; Rekhi, S; Zhu, B; Richardson, C

    2009-01-01

    Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.

  14. Genome, Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

    PubMed

    Frampton, Rebekah A; Acedo, Elena Lopez; Young, Vivienne L; Chen, Danni; Tong, Brian; Taylor, Corinda; Easingwood, Richard A; Pitman, Andrew R; Kleffmann, Torsten; Bostina, Mihnea; Fineran, Peter C

    2015-06-24

    Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.

  15. Nonequilibrium Relaxation of Conformational Dynamics Facilitates Catalytic Reaction in an Elastic Network Model of T7 DNA Polymerase.

    PubMed

    Zhao, Ziqing W; Xie, X Sunney; Ge, Hao

    2016-03-24

    Nucleotide-induced conformational closing of the finger domain of DNA polymerase is crucial for its catalytic action during DNA replication. Such large-amplitude molecular motion is often not fully accessible to either direct experimental monitoring or molecular dynamics simulations. However, a coarse-grained model can offer an informative alternative, especially for probing the relationship between conformational dynamics and catalysis. Here we investigate the dynamics of T7 DNA polymerase catalysis using a Langevin-type elastic network model incorporating detailed structural information on the open conformation without the substrate bound. Such a single-parameter model remarkably captures the induced conformational dynamics of DNA polymerase upon dNTP binding, and reveals its close coupling to the advancement toward transition state along the coordinate of the target reaction, which contributes to significant lowering of the activation energy barrier. Furthermore, analysis of stochastic catalytic rates suggests that when the activation energy barrier has already been significantly lowered and nonequilibrium relaxation toward the closed form dominates the catalytic rate, one must appeal to a picture of two-dimensional free energy surface in order to account for the full spectrum of catalytic modes. Our semiquantitative study illustrates the general role of conformational dynamics in achieving transition-state stabilization, and suggests that such an elastic network model, albeit simplified, possesses the potential to furnish significant mechanistic insights into the functioning of a variety of enzymatic systems.

  16. Transcriptional bypass of regioisomeric ethylated thymidine lesions by T7 RNA polymerase and human RNA polymerase II

    PubMed Central

    You, Changjun; Wang, Pengcheng; Dai, Xiaoxia; Wang, Yinsheng

    2014-01-01

    Alkylative damage to DNA can be induced by environmental chemicals, endogenous metabolites and some commonly prescribed chemotherapeutic agents. The regioisomeric N3-, O2- and O4-ethylthymidine (N3-, O2- and O4-EtdT, respectively) represent an important class of ethylated DNA lesions. Using nonreplicative double-stranded vectors containing an N3-EtdT, O2-EtdT or O4-EtdT at a defined site in the template strand, herein we examined the effects of these lesions on DNA transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. We found that O4-EtdT is highly mutagenic and exclusively induces the misincorporation of guanine opposite the lesion, whereas N3-EtdT and O2-EtdT display promiscuous miscoding properties during transcription. In addition, N3-EtdT and O2-EtdT were found to inhibit strongly DNA transcription in vitro and in certain human cells. Moreover, N3-EtdT, but not O2-EtdT or O4-EtdT, is an efficient substrate for transcription-coupled nucleotide excision repair. These findings provide new important insights into how these alkylated DNA lesions compromise the flow of genetic information, which may help to understand the risk of these lesions in living cells. PMID:25404131

  17. Analysis Of The Surface Roughness Obtained During The Dry Turning Of UNS A97050-T7 Aluminium Alloys

    NASA Astrophysics Data System (ADS)

    de Agustina, B.; Rubio, E. M.; Villeta, M.; Sebastián, M. A.

    2009-11-01

    Currently, in the aeronautical, aerospace and automotive industries there is high demand of materials such as the aluminium alloys that have high resistance even at high temperatures as well as a low density. For this reason, these alloys are widely used for the production of different elements that compose aircraft and aerospace vehicles. Nevertheless, in spite of the important role these materials have from the competitive point of view, they can commonly show problems of machinability associated with the tool wear. That has made that traditionally cutting fluids had been used in machining processes. However, they can contain environmentally harmful constituents and increase considerably the total cost of the process. Therefore, researches have been focused on the development of cleaner production technologies applications as dry machining. This leads to the search for combinations of cutting parameters and type of tools (types of coatings and different geometries) that could improve the machining under such conditions. The aim of this study is to analyse the relationship between the surface roughness obtained during the dry turning of aluminium UNS A97050-T7 bars and the cutting parameters (cutting speed and feed) using three different tools. As a first conclusion it could be affirmed that the feed was the cutting parameter more influential on the surface roughness and to a lesser extend the cutting speed, the type of tool and the interaction between the type of tool and the feed.

  18. 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...

  19. 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...

  20. Emerging Escherichia Pathogen

    PubMed Central

    Permpalung, Nitipong; Sentochnik, Deborah E.

    2013-01-01

    Escherichia hermannii was first identified as a new species in 1982. It has rarely been reported as a human pathogen. We report the first case of E. hermannii as the sole pathogen in a catheter-related bloodstream infection. PMID:23740732

  1. C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

    PubMed

    Karnati, Srikanth; Palaniswamy, Saranya; Alam, Mohammad Rashedul; Oruqaj, Gani; Stamme, Cordula; Baumgart-Vogt, Eveline

    2016-03-01

    In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

  2. K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

    PubMed

    Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

    2017-03-11

    Amino-acid mutations of Gly(12) (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

  3. Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

    PubMed Central

    Guo, Fei; Liu, Zheng; Fang, Ping-An; Zhang, Qinfen; Wright, Elena T.; Wu, Weimin; Zhang, Ci; Vago, Frank; Ren, Yue; Jakana, Joanita; Chiu, Wah; Serwer, Philip; Jiang, Wen

    2014-01-01

    Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses. PMID:25313071

  4. Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions.

    PubMed

    Guo, Fei; Liu, Zheng; Fang, Ping-An; Zhang, Qinfen; Wright, Elena T; Wu, Weimin; Zhang, Ci; Vago, Frank; Ren, Yue; Jakana, Joanita; Chiu, Wah; Serwer, Philip; Jiang, Wen

    2014-10-28

    Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.

  5. A novel molecular beacon-based method for isothermal detection of sequence-specific DNA via T7 RNA polymerase-aided target regeneration.

    PubMed

    Yin, Bin-Cheng; Wu, Shan; Ma, Jin-Liang; Ye, Bang-Ce

    2015-06-15

    Developing molecular beacon (MB)-based method for DNA detection has been of great interest to many researchers because of its intrinsic advantages of simplicity, rapidity, and specificity. In this work, we have developed a novel MB-based method for isothermal detection of sequence-specific DNA via T7 RNA polymerase-aided target regeneration strategy. The proposed method involves three primary processes of target-mediated ligation by T4 DNA ligase, transcription reaction by T7 RNA polymerase, and MB switch for signal output. Upon the hybridization with DNA target, a rationally designed MB and a pair of primers encoded with T7 promoter sequence were ligated via the formation of a phosphodiester bond by T4 DNA ligase. The resultant joint fragment acted as template to initiate T7 RNA polymerase-mediated transcription reaction. Correspondingly, a great amount of RNA strands complementary to MB and partial primers were transcribed to initiate new cyclic reactions of MB switch, ligation, and transcription. With such signal amplification strategy of the regeneration of target-like RNA fragments, our proposed assay achieved a detection limit as low as ∼10 pM, which was ∼3 orders of magnitude lower than the traditional MB-based method with a recognition mechanism in 1:1 stoichiometric ratio between MB and target molecule.

  6. Switching from single-stranded to double-stranded DNA limits the unwinding processivity of ring-shaped T7 DNA helicase.

    PubMed

    Jeong, Yong-Joo; Rajagopal, Vaishnavi; Patel, Smita S

    2013-04-01

    Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3'-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted.

  7. An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

  8. 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

  9. Hydrotalcite formed by alteration of R7T7 nuclear waste glass and basaltic glass in salt brine at 190{degrees}C

    SciTech Connect

    Abdelouas, A.; Crovisier, J.L.; Lutze, W.; Mueller, R.; Bernotat, W.

    1994-12-31

    The R7T7 and synthetic basaltic glasses were submitted to corrosion in a saline MgCl{sub 2} dominated solution at 190{degrees}C. For both glasses, the early alteration product is a hydrotalcite-like compound in which HPO{sub 4}{sup 2{minus}}, SO{sub 4}{sup 2{minus}} and Cl{sup {minus}} substitutes to CO{sub 3}{sup 2{minus}}. The measured d{sub 003} spacing is 7.68 {angstrom} for the hydrotalcite formed from R7T7 glass and 7.62 {angstrom} for the hydrotalcite formed from basaltic glass which reflect the high aluminium content. Chemical microanalyses show that the hydrotalcite is subsequently covered by a silica-rich gel which evolves into saponite after few months.

  10. Coupled orbital and spin evolution of the CoRoT-7 two-planet system using a Maxwell viscoelastic rheology

    NASA Astrophysics Data System (ADS)

    Rodríguez, A.; Callegari, N.; Correia, A. C. M.

    2016-12-01

    We investigate the orbital and rotational evolution of the CoRoT-7 two-planet system, assuming that the innermost planet behaves like a Maxwell body. We numerically resolve the coupled differential equations governing the instantaneous deformation of the inner planet together with the orbital motion of the system. We show that, depending on the relaxation time for the deformation of the planet, the orbital evolution has two distinct behaviours: for relaxation times shorter than the orbital period, we reproduce the results from classic tidal theories, for which the eccentricity is always damped. However, for longer relaxation times, the eccentricity of the inner orbit is secularly excited and can grow to high values. This mechanism provides an explanation for the present high eccentricity observed for CoRoT-7 b, as well as for other close-in super-Earths in multiple planetary systems.

  11. A New Case of a Complex Small Supernumerary Marker Chromosome: A Der(9)t(7;9)(p22;q22) due to a Maternal Balanced Rearrangement

    PubMed Central

    Manvelyan, Marine; Simonyan, Izabella; Hovhannisyan, Galina; Aroutiounian, Rouben; Hamid, Ahmed B.; Liehr, Thomas

    2015-01-01

    Complex small supernumerary marker chromosomes (sSMCs) constitute one of the smallest subsets within the patients with an sSMC. Complex sSMCs consist of chromosomal material derived from more than one chromosome, for example, the derivative der(22)t(11;22)(q23;q11.2) in Emanuel syndrome. Here, a yet unreported case of a complex sSMC formed due to a t(7;9)(p22;q22)mat is presented. PMID:27617132

  12. A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase.

    PubMed

    Piccirilli, J A; Moroney, S E; Benner, S A

    1991-10-22

    With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

  13. 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

  14. 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.

  15. A system for dual protein expression in Pichia pastoris and Escherichia coli.

    PubMed

    Lueking, A; Holz, C; Gotthold, C; Lehrach, H; Cahill, D

    2000-12-01

    We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.

  16. Re-analysis of the ╣ of NH3 in C/2002 LINEAR T7 Using CSHELL at the NASA IRTF

    NASA Astrophysics Data System (ADS)

    Magee-Sauer, Karen P.; Villanueva, G. L.; Mumma, M. J.; Bonev, B.; DiSanti, M. A.; Dello Russo, N.; Lippi, M.; Gibb, E. L.

    2008-09-01

    NH3 is the dominant (observable) nitrogen-containing volatile in comets, present at 1% relative to water. At infrared wavelengths, NH3 (ν1 band) is difficult to detect since it requires a significant geocentric velocity to shift the strongest NH3 emissions (Q-branch) out of the terrestrial atmospheric water absorption. The P- and R- branches are less dependent on geocentric velocity, but since they are generally weaker lines, they are challenging to detect. The 3-micron region is rich in molecular emissions (HCN, OH, H2O, C2H2, NH2, and other minor species), so that for many lines of the P- and R- branches, the ability to resolve these faint emissions from nearby brighter emission lines requires careful spectral registration of images. Previously, we applied and tested our model of fluorescence efficiency factors for NH3 in C/2002 T7 (LINEAR). The high Doppler shift (- 66 km/s) of C/T7 during early May 2004 made it an excellent target for searching for NH3 through its strongest IR emission lines. While the Q-branch lines were detected with high signal to noise, the sQ33 line of NH3 was on the wing of a deep atmospheric absorption. Other lines were fainter and near brighter emissions or atmospheric absorptions. The ability to accurately use the sQ33 emission and P- and R-branch lines, we discovered that our atmospheric model and algorithms needed improvement. Upgrades to our algorithms include an improved spectral transmittance model and spectral registration routines (Villanueva et al. 2006). This work will present a re-analysis of the 3-micron region from the high quality data set of C/2002 LINEAR T7 using the improved atmospheric transmittance models and data analysis algorithms from Villanueva et al. KM-S would like to acknowledge support from the National Science Foundation RUI Program No. 0407052.

  17. 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.

  18. 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

  19. The silicate and carbon-rich models of CoRoT-7b, Kepler-9d and Kepler-10b

    NASA Astrophysics Data System (ADS)

    Gong, Yan-Xiang; Zhou, Ji-Lin

    2012-06-01

    Possible bulk compositions of the super-Earth exoplanets CoRoT-7b, Kepler-9d, and Kepler-10b are investigated by applying a commonly used silicate model and a non-standard carbon model. Their internal structures are deduced using a suitable equation of state for the materials. The degeneracy problems of their compositions can be partly overcome, based on the fact that all three planets are extremely close to their host stars. By analyzing the numerical results, we conclude: 1) the iron core of CoRoT-7b is not more than 27% of its total mass within 1σ mass-radius error bars, so an Earth-like composition is less likely, but its carbon rich model can be compatible with an Earth-like core/mantle mass fraction; 2) Kepler-10b is more likely to have a Mercury-like composition, with its old age implying that its high iron content may be a result of strong solar wind or giant impact; 3) the transiting-only super-Earth Kepler-9d is also discussed. Combining its possible composition with the formation theory, we can place some constraints on its mass and bulk composition.

  20. A T7exonuclease-assisted target recycling amplification with graphene oxide acting as the signal amplifier for fluorescence polarization detection of human immunodeficiency virus (HIV) DNA.

    PubMed

    Wang, Lijun; Tian, Jianniao; Yang, Wen; Zhao, Yanchun; Zhao, Shulin

    2016-03-01

    We report a fluorescence polarization (FP) platform for human immunodeficiency virus (HIV) DNA detection based on T7exonuclease-assisted target recycling amplification with graphene oxide (GO) acting as a FP signal amplifier. In the sensing method, the presence of the target DNA leads to target recycling with the assistance of T7exonuclease, furthermore, the amplification products are absorbed onto the surface of GO, so the all FP values are enhanced by GO. More importantly, this FP sensor exhibits high detection sensitivity; under optimal conditions, the change in FP is linear with the concentration of the target DNA within a concentration range of 50-2000 pmol/L, and the detection limit of this method is as low as 38.6 pmol/L. This FP sensor also exhibits high selectivity, even single-base mismatched DNA can be effectively discriminated from complementary target DNA. Above all, the proposed FP sensor may serve as a general platform for the sensitive assay of disease-related genes.

  1. A survey of volatile species in Oort cloud comets C/2001 Q4 (NEAT) and C/2002 T7 (LINEAR) at millimeter wavelengths

    NASA Astrophysics Data System (ADS)

    de Val-Borro, M.; Küppers, M.; Hartogh, P.; Rezac, L.; Biver, N.; Bockelée-Morvan, D.; Crovisier, J.; Jarchow, C.; Villanueva, G. L.

    2013-11-01

    Context. The chemical composition of comets can be inferred using spectroscopic observations in submillimeter and radio wavelengths. Aims: We aim to compare the production rates ratio of several volatiles in two comets, C/2001 Q4 (NEAT) and C/2002 T7 (LINEAR), which are generally regarded as dynamically new and likely to originate in the Oort cloud. This type of comets is considered to be composed of primitive material that has not undergone considerable thermal processing. Methods: The line emission in the coma was measured in the comets, C/2001 Q4 (NEAT) and C/2002 T7 (LINEAR), that were observed on five consecutive nights, 7-11 May 2004, at heliocentric distances of 1.0 and 0.7 AU, respectively, by means of high-resolution spectroscopy using the 10-m Submillimeter Telescope at the Arizona Radio Observatory. Both objects became very bright and reached naked-eye visibility during their perihelion passage in the spring of 2004. Results: We present a search for six parent- and product-volatile species (HCN, H2CO, CO, CS, CH3OH, and HNC) in both comets. Multiline observations of the CH3OH J = 5-4 series allow us to estimate the rotational temperature using the rotation diagram technique. We derive rotational temperatures of 54(9) K for C/2001 Q4 (NEAT) and 119(34) K for C/2002 T7 (LINEAR). The gas production rates are computed using the level distribution obtained with a spherically symmetric molecular excitation code that includes collisions between neutrals and electrons. The effects of radiative pumping of the fundamental vibrational levels by infrared photons from the Sun are considered for the case of HCN. We find an HCN production rate of 2.96(5) × 1026 molec.s-1 for comet C/2001 Q4 (NEAT), corresponding to a mixing ratio with respect to H2O of 1.12(2) × 10-3. The mean HCN production rate during the observing period is 4.54(10) × 1026 molec.s-1 for comet C/2002 T7 (LINEAR), which gives a mixing ratio of 1.51(3) × 10-3. Relative abundances of CO, CH3OH, H2CO

  2. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor.

    PubMed

    Poon, S K; Peacock, L; Gibson, W; Gull, K; Kelly, S

    2012-02-01

    Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

  3. Physical mapping of the chromosome 7 breakpoint region in an SLOS patient with t(7;20)X(q32.1;q13.2)

    SciTech Connect

    Alley, T.L.; Wallace, M.R.; Scherer, S.W.

    1997-01-31

    Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by multiple congenital anomalies and mental retardation. SLOS has an associated defect in cholesterol biosynthesis, but the molecular genetic basis of this condition has not yet been elucidated. Previously our group reported a patient with a de novo balanced translocation [t(7;20)(q32.1;q13.2)] fitting the clinical and biochemical profile of SLOS. Employing fluorescence in situ hybridization (FISH), a 1.8 Mb chromosome 7-specific yeast artificial chromosome (YAC) was identified which spanned the translocation breakpoint in the reported patient. The following is an update of the on-going pursuit to physically and genetically map the region further, as well as the establishment of candidate genes in the 7q32.1 breakpoint region. 11 refs., 1 fig.

  4. Screening of paclitaxel-binding molecules from a library of random peptides displayed on T7 phage particles using paclitaxel-photoimmobilized resin.

    PubMed

    Aoki, Sota; Morohashi, Kengo; Sunoki, Takashi; Kuramochi, Kouji; Kobayashi, Susumu; Sugawara, Fumio

    2007-01-01

    Paclitaxel (Taxol), an effective anticancer agent, is known to bind to tubulin and induce tubulin polymerization. Several other binding proteins of paclitaxel, such as Bcl-2, heat shock proteins, and NSC-1, have also been reported. Here, we describe a T7 phage-based display to screen for paclitaxel-binding molecules from a random peptide library using paclitaxel-photoimmobilized TentaGel resin. Specific phage particles that bind the paclitaxel-immobilized resin were obtained. Among them, two phage clones included the same consensus amino acid sequence (KACGRTRVTS). Analysis of the protein database using BLAST revealed that a portion of this sequence is conserved in the zinc finger domain of human NFX1. Binding affinity of paclitaxel against the partial recombinant protein of NFX1 (424aa-876aa) was confirmed by pull-down assays and surface plasmon resonance analyses.

  5. Hepatosplenic gamma/delta T-cell lymphoma with isochromosome 7q, translocation t(7;21), and tetrasomy 8 in a 9-year-old girl.

    PubMed

    Rossbach, Hans-Christoph; Chamizo, Wilfredo; Dumont, Doris P; Barbosa, Jerry L; Sutcliffe, Maxine J

    2002-02-01

    The authors report a child younger than age 15 years with a rare hepatosplenic gamma/delta T-cell lymphoma, which is highly aggressive and primarily seen in young men. A 9-year-old girl presented with thrombocytopenia and hepatosplenomegaly. Bone marrow analysis revealed a metastatic pleomorphic lymphoma of peripheral T-cell phenotype, with rearrangement of the T-cell receptor gamma/delta and expression of CD3 and CD16/56. Instead of the previously reported primary, nonrandom, chromosomal abnormalities, isochromosome 7q and trisomy 8, this patient had four copies each of chromosome 7q, including isochromosome 7[i(7)(q10)] and der(21)t(7;21), as well as chromosome 8. This entity needs to be considered in women and children with lymphoma. Conventional therapy appears to be inadequate for cure.

  6. Molecular analysis of TCRB and ABL in a t(7; 9)-containing cell line (SUP-T3) from a human T-cell leukemia

    SciTech Connect

    Westbrook, C.A.; Rubin, C.M.; Le Beau, M.M.; Kaminer, L.S.; Smith, S.D.; Rowley, J.D.; Diaz, M.O.

    1987-01-01

    A translocation between chromosomes 7 and 9, t(7;9), has been described in cell lines derived from the malignant cells of children with acute T-cell lymphoblastic leukemia or lymphoma. The cytogenetic analysis of one such cell line, SUP-T3, demonstrates that the breakpoints on chromosomes 7 and 9 lie within bands q36 and q34, respectively, corresponding to the location of the gene encoding the ..beta.. chain of the T-cell receptor, TCRB, and the gene homologous to the transforming gene of the Abelson murine leukemia virus, ABL. The authors investigated the role of these genes in the t(7;9). In situ chromosomal hybridization of TCRB and ABL probes to metaphase cells from SUP-T3 demonstrated that ABL is translocated from chromosome 9 to 7 and that all or part of TCRB is translocated from chromosome 7 to 9. Southern blot analysis revealed that both TCRB alleles were rearranged; however, it could not be determined whether the translocation breakpoint lies within this gene. Pulsed-field gel electrophoresis and Southern blot analysis were used to examine more than 500 kilobases on the ABL locus; they concluded that there are no rearrangements within 250 kb in either direction of the sequences homologous to v-abl. These results indicate that, in SUP-T3, the breakpoint on chromosome 9 lies proximal to ABL and the break results in no apparent alteration of the ABL protein. They therefore hypothesize that another gene of chromosome 9, at band q34, plays a role in this translocation. This study also demonstrates that pulsed-field gel electrophoresis is a powerful new tool for the analysis of human chromosomal translocations.

  7. Spinach ferredoxin i: overproduction in Escherichia coli and purification.

    PubMed

    Piubelli, L; Aliverti, A; Bellintani, F; Zanetti, G

    1995-06-01

    Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.

  8. Mosquito larvicidal activity of Escherichia coli with combinations of genes from Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    Ben-Dov, E; Boussiba, S; Zaritsky, A

    1995-01-01

    The genes cryIVA and cryIVD, encoding 134- and 72-kDa proteins, respectively, and the gene for a regulatory 20-kDa polypeptide of Bacillus thuringiensis subsp. israelensis (serovar H14) were cloned in all seven possible combinations by the Escherichia coli expression vectors pT7 and pUHE. The four combinations containing cryIVA (cryIVA alone, with cryIVD, with the 20-kDa-protein gene, and with both) displayed high levels of mosquito larvicidal activity in pUHE. The toxicity of the combination of cryIVA and cryIVD, with or without the 20-kDa-protein gene, was higher than has ever been achieved with delta-endotoxin genes in recombinant E. coli. Fifty percent lethal concentrations against third-instar Aedes aegypti larvae for these clones decreased (i.e., toxicity increased) continuously to about 3 x 10(5) cells ml-1 after 4 h of induction. Larvicidal activities, obtained after 30 min of induction, were lower for clones in pT7 and decreased for an additional 3.5 h. Induction of either cryIVD or the 20-kDa-protein gene alone resulted in no larvicidal activity in either pT7 or pUHE20. Cloned together, these genes were slightly toxic in pT7 but not in pUHE20. Five minutes of induction of this combination (cryIVD with the 20-kDa-protein gene) in pT7 yielded a maximal mortality of about 40%, which decreased rapidly and disappeared completely after 50 min. CryIVD is thus apparently degraded in E. coli and partially stabilized by the 20-kDa regulatory protein. Larvicidal activity of the combination of cryIVA and cryIVD was sevenfold higher than that of cryIVA alone, probably because of the cross-stabilization of the polypeptides or the synergism between their activities. PMID:7751296

  9. Transiting exoplanets from the CoRoT space mission. VIII. CoRoT-7b: the first super-Earth with measured radius

    NASA Astrophysics Data System (ADS)

    Léger, A.; Rouan, D.; Schneider, J.; Barge, P.; Fridlund, M.; Samuel, B.; Ollivier, M.; Guenther, E.; Deleuil, M.; Deeg, H. J.; Auvergne, M.; Alonso, R.; Aigrain, S.; Alapini, A.; Almenara, J. M.; Baglin, A.; Barbieri, M.; Bruntt, H.; Bordé, P.; Bouchy, F.; Cabrera, J.; Catala, C.; Carone, L.; Carpano, S.; Csizmadia, Sz.; Dvorak, R.; Erikson, A.; Ferraz-Mello, S.; Foing, B.; Fressin, F.; Gandolfi, D.; Gillon, M.; Gondoin, Ph.; Grasset, O.; Guillot, T.; Hatzes, A.; Hébrard, G.; Jorda, L.; Lammer, H.; Llebaria, A.; Loeillet, B.; Mayor, M.; Mazeh, T.; Moutou, C.; Pätzold, M.; Pont, F.; Queloz, D.; Rauer, H.; Renner, S.; Samadi, R.; Shporer, A.; Sotin, Ch.; Tingley, B.; Wuchterl, G.; Adda, M.; Agogu, P.; Appourchaux, T.; Ballans, H.; Baron, P.; Beaufort, T.; Bellenger, R.; Berlin, R.; Bernardi, P.; Blouin, D.; Baudin, F.; Bodin, P.; Boisnard, L.; Boit, L.; Bonneau, F.; Borzeix, S.; Briet, R.; Buey, J.-T.; Butler, B.; Cailleau, D.; Cautain, R.; Chabaud, P.-Y.; Chaintreuil, S.; Chiavassa, F.; Costes, V.; Cuna Parrho, V.; de Oliveira Fialho, F.; Decaudin, M.; Defise, J.-M.; Djalal, S.; Epstein, G.; Exil, G.-E.; Fauré, C.; Fenouillet, T.; Gaboriaud, A.; Gallic, A.; Gamet, P.; Gavalda, P.; Grolleau, E.; Gruneisen, R.; Gueguen, L.; Guis, V.; Guivarc'h, V.; Guterman, P.; Hallouard, D.; Hasiba, J.; Heuripeau, F.; Huntzinger, G.; Hustaix, H.; Imad, C.; Imbert, C.; Johlander, B.; Jouret, M.; Journoud, P.; Karioty, F.; Kerjean, L.; Lafaille, V.; Lafond, L.; Lam-Trong, T.; Landiech, P.; Lapeyrere, V.; Larqué, T.; Laudet, P.; Lautier, N.; Lecann, H.; Lefevre, L.; Leruyet, B.; Levacher, P.; Magnan, A.; Mazy, E.; Mertens, F.; Mesnager, J.-M.; Meunier, J.-C.; Michel, J.-P.; Monjoin, W.; Naudet, D.; Nguyen-Kim, K.; Orcesi, J.-L.; Ottacher, H.; Perez, R.; Peter, G.; Plasson, P.; Plesseria, J.-Y.; Pontet, B.; Pradines, A.; Quentin, C.; Reynaud, J.-L.; Rolland, G.; Rollenhagen, F.; Romagnan, R.; Russ, N.; Schmidt, R.; Schwartz, N.; Sebbag, I.; Sedes, G.; Smit, H.; Steller, M. B.; Sunter, W.; Surace, C.; Tello, M.; Tiphène, D.; Toulouse, P.; Ulmer, B.; Vandermarcq, O.; Vergnault, E.; Vuillemin, A.; Zanatta, P.

    2009-10-01

    Aims: We report the discovery of very shallow (Δ F/F ≈ 3.4× 10-4), periodic dips in the light curve of an active V = 11.7 G9V star observed by the CoRoT satellite, which we interpret as caused by a transiting companion. We describe the 3-colour CoRoT data and complementary ground-based observations that support the planetary nature of the companion. Methods: We used CoRoT colours information, good angular resolution ground-based photometric observations in- and out- of transit, adaptive optics imaging, near-infrared spectroscopy, and preliminary results from radial velocity measurements, to test the diluted eclipsing binary scenarios. The parameters of the host star were derived from optical spectra, which were then combined with the CoRoT light curve to derive parameters of the companion. Results: We examined all conceivable cases of false positives carefully, and all the tests support the planetary hypothesis. Blends with separation >0.40´´or triple systems are almost excluded with a 8 × 10-4 risk left. We conclude that, inasmuch we have been exhaustive, we have discovered a planetary companion, named CoRoT-7b, for which we derive a period of 0.853 59 ± 3 × 10-5 day and a radius of Rp = 1.68 ± 0.09 R_Earth. Analysis of preliminary radial velocity data yields an upper limit of 21 M_Earth for the companion mass, supporting the finding. Conclusions: CoRoT-7b is very likely the first Super-Earth with a measured radius. This object illustrates what will probably become a common situation with missions such as Kepler, namely the need to establish the planetary origin of transits in the absence of a firm radial velocity detection and mass measurement. The composition of CoRoT-7b remains loosely constrained without a precise mass. A very high surface temperature on its irradiated face, ≈1800-2600 K at the substellar point, and a very low one, ≈50 K, on its dark face assuming no atmosphere, have been derived. The CoRoT space mission, launched on 27

  10. Cloning of the lambda resistant genes from Brevibacterium albidum and Proteus vulgaris into Escherichia coli.

    PubMed

    Chae, K S; Yoo, O J

    1986-11-14

    Genes from Proteus vulgaris ATCC13315 and Brevibacterium albidum ATCC15831 were introduced into Escherichia coli, which rendered the host resistant to coliphage lambda. The clones transformed by any one of the two recombinant plasmids, pRMG101 or pRMG216, were totally resistant against the infection of virulent lambda and N4, but sensitive to ø80, T4 and T7. However, when maltose transport systems of the clones were induced by maltose, the clones were no more resistant to the phage: thus, this phenotype was thought to be due to the inhibition of phage adsorption onto the cell surface. The gene product was shown by SDS-PAGE of membrane protein-enriched extract of the clone. Molecular weight as measured was about 40,000 dalton, which coincide with that inferred from the nucleotide sequences.

  11. An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.

    PubMed

    Seo, Jeong-Woo; Hong, Won-kyung; Rairakhwada, Dina; Seo, Pil-Soo; Choi, Min Ho; Song, Ki-Bang; Rhee, Sang-Ki; Kim, Chul Ho

    2009-06-01

    The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli.

  12. Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme.

    PubMed

    Atanasiu, C; Su, T-J; Sturrock, S S; Dryden, D T F

    2002-09-15

    The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over approximately 24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme. Ocr can also bind to the component subunits of EcoKI. Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change. This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme. This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.

  13. Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases.

    PubMed Central

    Hanna, M M; Zhang, Y; Reidling, J C; Thomas, M J; Jou, J

    1993-01-01

    A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin. Images PMID:7684833

  14. Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding

    PubMed Central

    2016-01-01

    The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7–10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2–12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography. PMID:27387136

  15. Characterization, Genome Sequence, and Analysis of Escherichia Phage CICC 80001, a Bacteriophage Infecting an Efficient L-Aspartic Acid Producing Escherichia coli.

    PubMed

    Xu, Youqiang; Ma, Yuyue; Yao, Su; Jiang, Zengyan; Pei, Jiangsen; Cheng, Chi

    2016-03-01

    Escherichia phage CICC 80001 was isolated from the bacteriophage contaminated medium of an Escherichia coli strain HY-05C (CICC 11022S) which could produce L-aspartic acid. The phage had a head diameter of 45-50 nm and a tail of about 10 nm. The one-step growth curve showed a latent period of 10 min and a rise period of about 20 min. The average burst size was about 198 phage particles per infected cell. Tests were conducted on the plaques, multiplicity of infection, and host range. The genome of CICC 80001 was sequenced with a length of 38,810 bp, and annotated. The key proteins leading to host-cell lysis were phylogenetically analyzed. One protein belonged to class II holin, and the other two belonged to the endopeptidase family and N-acetylmuramoyl-L-alanine amidase family, respectively. The genome showed the sequence identity of 82.7% with that of Enterobacteria phage T7, and carried ten unique open reading frames. The bacteriophage resistant E. coli strain designated CICC 11021S was breeding and its L-aspartase activity was 84.4% of that of CICC 11022S.

  16. Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli.

    PubMed

    Narayanan, Niju; Hsieh, Ming-Yi; Xu, Yali; Chou, C Perry

    2006-01-01

    Arabinose was shown to serve as an effective inducer for induction of the lac-derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-beta-d-thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by the trc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac-derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LL pac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression.

  17. Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli.

    PubMed

    Boghigian, Brett A; Salas, Daniel; Ajikumar, Parayil Kumaran; Stephanopoulos, Gregory; Pfeifer, Blaine A

    2012-02-01

    Taxa-4(5),11(12)-diene is the first dedicated intermediate in the metabolic pathway responsible for synthesizing the anticancer compound Taxol. In this study, the heterologous production of taxadiene was established in and analyzed between K- and B-derived Escherichia coli strains. First, recombinant parameters associated with precursor metabolism (the upstream methylerythritol phosphate (MEP) pathway) and taxadiene biosynthesis (the downstream pathway) were varied to probe the effect different promoters and cellular backgrounds have on taxadiene production. Specifically, upstream MEP pathway genes responsible for the taxadiene precursors, dimethylallyl diphosphate and isopentenyl diphosphate, were tested with an inducible T7 promoter system within K and B E. coli strains. Whereas, inducible T7, Trc, and T5 promoters were tested with the plasmid-borne geranylgeranyl diphosphate synthase and taxadiene synthase genes responsible for the downstream pathway. The K-derivative produced taxadiene roughly 2.5-fold higher than the B-derivative. A transcriptomics study revealed significant differences in pyruvate metabolism between the K and B strains, providing insight into the differences observed in taxadiene biosynthesis and targets for future metabolic engineering efforts. Next, the effect of temperature on cell growth and taxadiene production was analyzed in these two strains, revealing similar phenotypes between the two with 22°C as the optimal production temperature. Lastly, the effect of indole on cell growth was investigated between the two strains, showing that the K-derivative demonstrated greater growth inhibition compared to the B-derivative.

  18. Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.

    SciTech Connect

    Scholle, M. D.; Banach, B. S.; Hamdan, S. M.; Richardson, C. C.; Kay, B. K.; Biosciences Division; Amunix, Inc.; Univ. of Illinois at Chicago; Harvard Medical School

    2008-11-01

    Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, under oxidizing conditions, with an IC50 of {approx} 10 {micro}M.

  19. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.

    PubMed

    Sun, Zachary Z; Hayes, Clarmyra A; Shin, Jonghyeon; Caschera, Filippo; Murray, Richard M; Noireaux, Vincent

    2013-09-16

    Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems. The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented. Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted. The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters. Accompanying mathematical models are available. The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

  20. Development of a hamster kidney cell line expressing stably T7 RNA polymerase using retroviral gene transfer technology for efficient rescue of infectious foot-and-mouth disease virus.

    PubMed

    Zheng, Haixue; Tian, Hong; Jin, Ye; Wu, Jinyan; Shang, Youjun; Yin, Shuanghui; Liu, Xiangtao; Xie, Qingge

    2009-03-01

    Reverse genetics systems, with the ability to manipulate viral genomes at the DNA molecular level, are an important platform for study of the assembly and function of viruses. Genome manipulation, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for increasing the efficiency of rescuing recombinant viruses. To develop an efficient, helper virus-free viral recovery system (reverse genetics), a retroviral gene transfer technology was used to establish a stable BHK-21 cell line (designated as BHKT7) which expressed constitutively bacteriophage T7 RNA polymerase (T7 RNAP). An improved method for rescue of infectious foot-and-mouth disease virus (FMDV) was then developed. FMDV full-length cDNA under control of a T7 promotor, was transfected into BHKT7 of differing passages. FMDV virus was rescued efficiently from the BHKT7 cells, the passage number not having an effect on the efficiency of recovery. As a result, the cell line was stable even after multiple passages, expressing sufficient T7 RNAP to support ex vivo transcription and efficient rescue. The reverse genetics system described below is efficient, stable, and convenient. The system could provide not only the basis of gene function research into FMDV, but could also be used for reverse genetics research into other positive-strand RNA viruses, without the need for helper viruses.

  1. A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

    PubMed

    Serwer, P; Hayes, S J; Moreno, E T; Park, C Y

    1992-09-15

    Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Mutation rate is reduced by increased dosage of mutL gene in Escherichia coli K-12.

    PubMed

    Galán, Juan-Carlos; Turrientes, María-Carmen; Baquero, María-Rosario; Rodríguez-Alcayna, Manuel; Martínez-Amado, Jorge; Martínez, José-Luis; Baquero, Fernando

    2007-10-01

    A variable but substantial proportion of wild Escherichia coli isolates present consistently lower mutation frequencies than that found in the ensemble of strains. The genetic mechanisms responsible for the hypo-mutation phenotype are much less known than those involved in hyper-mutation. Changes in E. coli mutation frequencies derived from the gene-copy effect of mutS, mutL, mutH, uvrD, mutT, mutY, mutM, mutA, dnaE, dnaQ, and rpoS are explored. When present in a very high copy number ( approximately 300 copies cell(-1)), mutL, mutH, and mutA gene copies yielded >/=twofold decrease in mutation rates determined by Luria-Delbrück fluctuation tests. Nevertheless, when the copy number was not such high ( approximately 15 copies cell(-1)), only mutL results in a consistent twofold decrease in the mutation rate. This reduction seems to be independent from the RecA background, phase of growth, or from the presence of proficient MutS. An increase in mutL gene copies was also able to partially compensate the hypermutator phenotype of a mutS-defective E. coli derivative.

  3. Genomics of Escherichia and Shigella

    NASA Astrophysics Data System (ADS)

    Perna, Nicole T.

    The laboratory workhorse Escherichia coli K-12 is among the most intensively studied living organisms on earth, and this single strain serves as the model system behind much of our understanding of prokaryotic molecular biology. Dense genome sequencing and recent insightful comparative analyses are making the species E. coli, as a whole, an emerging system for studying prokaryotic population genetics and the relationship between system-scale, or genome-scale, molecular evolution and complex traits like host range and pathogenic potential. Genomic perspective has revealed a coherent but dynamic species united by intraspecific gene flow via homologous lateral or horizontal transfer and differentiated by content flux mediated by acquisition of DNA segments from interspecies transfers.

  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. An Escherichia coli system for assay of F1p site-specific recombination on substrate plasmids.

    PubMed

    Snaith, M R; Kilby, N J; Murray, J A

    1996-11-21

    We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the F1p site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40.pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying F1p-recognition targets (FRT) are transformed into BL-FLP, and the consequences of F1p-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect F1p-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used F1p substrate plasmid, pNEO beta GAL (O'Gorman et al. (1991) Science 251, 1351-1355).

  6. 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

  7. 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

  8. Genetic mapping of a mutation that causes ribonucleases III deficiency in Escherichia coli.

    PubMed Central

    Studier, F W

    1975-01-01

    the mutation that causes ribonuclease III (RNase III) deficiency in strain AB301-105 of Kindler et al. (1973) has been mapped by use of F' merodiploids, Hfr matings, and P1 transduction. This mutation, rnc-105, lies close to nadB, near 49 min on the genetic map of Escherichia coli. The rnc-105 mutation has been transferred from its original genetic background by transduction and conjugation, and these new strains have the same defects in ribonucleic acid processing reported previously for AB301-105. Strains that carry rnc-105 grow more slowly than parental rnc+ strains, but the difference in growth rate seems to depend on the genetic background of each strain. Bacteriophage T7 grows about equally well in RNase III+ and III- female strains of E. coli, even though the specific cuts that RNase III makes in T7 ribonucleic acid are not made in the RNase III- strains. A low-phosphate defined medium in which most E. coli strains seem to grow well was developed. This medium is equally useful for labeling ribonucleic acids with 32PO4 and as a selective medium for genetic manipulations. It was used to determine the growth requirements of strain AB301-105, which are biotin and succinate in addition to the methionine and histidine requirements of the parental strain. The biotin mutation lies near the position expected from known mutations of E. coli, but the succinate mutation apparently does not. The possibility that the succinate requirement could be due to the RNase III deficiency is discussed. A uraP mutation was isolated for use in transferring rnc-105 between strains by conjugation. It lies near 47 min, somewhat removed from the commonly accepted position for uraP. PMID:1100605

  9. 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

  10. 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.

  11. 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.

  12. 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...

  13. 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,...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

    PubMed

    Nagahari, K; Sakaguchi, K

    1978-01-17

    The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu+ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B.leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu+. However, B. subtilis ilvB and ilvc auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. beta-Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.

  19. 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.…

  20. 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…

  1. 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...

  2. Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production.

    PubMed

    Li, Zheng-Jun; Cai, Lei; Wu, Qiong; Chen, Guo-Qiang

    2009-07-01

    NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16-35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y (PHB/glucose) showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y (PHB/glucose). These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.

  3. Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli.

    PubMed

    Pai, Jen C; Entzminger, Kevin C; Maynard, Jennifer A

    2012-02-15

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3' primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.

  4. Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli

    SciTech Connect

    Plotch, S.J.; Palant, O.; Gluzman, Y.

    1989-01-01

    A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter. This poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it. The enzyme exhibits poly(A)-dependent oligo(U)-primed ply(U) polymerase activity as well as RNA polymerase activity. In the presence of an oligo(U) primer, the enzyme catalyzes the synthesis of a full-length copy of either poliovirus or globin RNA templates. In the absence of added primer, RNA products up to twice the length of the template are synthesized. When incubated in the presence of a single nucleoside triphosphate, (..cap alpha..-/sup 32/P)UTP, the enzyme catalyzes the incorporation of radioactive label into template RNA. These results are discussed in light of previously proposed models of poliovirus RNA synthesis in vitro.

  5. Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli.

    PubMed

    Harrison, S J; McManus, A M; Marcus, J P; Goulter, K C; Green, J L; Nielsen, K J; Craik, D J; Maclean, D J; Manners, J M

    1999-03-01

    MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 with an additional ATG start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coli extract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coli. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens.

  6. 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

  7. The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

    PubMed Central

    Blasche, Sonja; Wuchty, Stefan; Rajagopala, Seesandra V.

    2013-01-01

    Although most of the 73 open reading frames (ORFs) in bacteriophage λ have been investigated intensively, the function of many genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs for interactions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of 62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions between the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda infection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns of human viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous to other phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually large number of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens. PMID:24049175

  8. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.

  9. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    PubMed

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  10. Stable, high-level expression of a type I antifreeze protein in Escherichia coli.

    PubMed

    Solomon, R G; Appels, R

    1999-06-01

    The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.

  11. RNA structure-dependent uncoupling of substrate recognition and cleavage by Escherichia coli ribonuclease III

    PubMed Central

    Calin-Jageman, Irina; Nicholson, Allen W.

    2003-01-01

    Members of the ribonuclease III superfamily of double-strand-specific endoribonucleases participate in diverse RNA maturation and decay pathways. Ribonuclease III of the gram-negative bacterium Escherichia coli processes rRNA and mRNA precursors, and its catalytic action can regulate gene expression by controlling mRNA translation and stability. It has been proposed that E.coli RNase III can function in a non-catalytic manner, by binding RNA without cleaving phosphodiesters. However, there has been no direct evidence for this mode of action. We describe here an RNA, derived from the T7 phage R1.1 RNase III substrate, that is resistant to cleavage in vitro by E.coli RNase III but retains comparable binding affinity. R1.1[CL3B] RNA is recognized by RNase III in the same manner as R1.1 RNA, as revealed by the similar inhibitory effects of a specific mutation in both substrates. Structure-probing assays and Mfold analysis indicate that R1.1[CL3B] RNA possesses a bulge– helix–bulge motif in place of the R1.1 asymmetric internal loop. The presence of both bulges is required for uncoupling. The bulge–helix–bulge motif acts as a ‘catalytic’ antideterminant, which is distinct from recognition antideterminants, which inhibit RNase III binding. PMID:12711683

  12. Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System

    PubMed Central

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

    2016-01-01

    Purpose: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. Methods: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. Results: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. Conclusion: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space. PMID:27478780

  13. Bayesian analysis of interiors of HD 219134b, Kepler-10b, Kepler-93b, CoRoT-7b, 55 Cnc e, and HD 97658b using stellar abundance proxies

    NASA Astrophysics Data System (ADS)

    Dorn, Caroline; Hinkel, Natalie R.; Venturini, Julia

    2017-01-01

    Aims: Using a generalized Bayesian inference method, we aim to explore the possible interior structures of six selected exoplanets for which planetary mass and radius measurements are available in addition to stellar host abundances: HD 219134b, Kepler-10b, Kepler-93b, CoRoT-7b, 55 Cnc e, and HD 97658b. We aim to investigate the importance of stellar abundance proxies for the planetary bulk composition (namely Fe/Si and Mg/Si) on prediction of planetary interiors. Methods: We performed a full probabilistic Bayesian inference analysis to formally account for observational and model uncertainties while obtaining confidence regions of structural and compositional parameters of core, mantle, ice layer, ocean, and atmosphere. We determined how sensitive our parameter predictions depend on (1) different estimates of bulk abundance constraints and (2) different correlations of bulk abundances between planet and host star. Results: The possible interior structures and correlations between structural parameters differ depending on data and data uncertainty. The strongest correlation is generally found between size of rocky interior and water mass fraction. Given the data, possible water mass fractions are high, even for most potentially rocky planets (HD 219134b, Kepler-93b, CoRoT-7b, and 55 Cnc e with estimates up to 35%, depending on the planet). Also, the interior of Kepler-10b is best constrained with possible interiors similar to Earth. Among all tested planets, only the data of Kepler-10b and Kepler-93b allow to put a higher probability on the planetary bulk Fe/Si to be stellar compared to extremely sub-stellar. Conclusions: Although the possible ranges of interior structures are large, structural parameters and their correlations are constrained by the sparse data. The probability for the tested exoplanets to be Earth-like is generally very low. Furthermore, we conclude that different estimates of planet bulk abundance constraints mainly affect mantle composition and

  14. Escherichia albertii in Wild and Domestic Birds

    PubMed Central

    Besser, Thomas E.; Walk, Seth T.; Gordon, David M.; Beckmen, Kimberlee B.; Burek, Kathy A.; Haldorson, Gary J.; Bradway, Dan S.; Ouellette, Lindsey; Rurangirwa, Fred R.; Davis, Margaret A.; Dobbin, Greg; Whittam, Thomas S.

    2010-01-01

    Escherichia albertii has been associated with diarrhea in humans but not with disease or infection in animals. However, in December 2004, E. albertii was found, by biochemical and genetic methods, to be the probable cause of death for redpoll finches (Carduelis flammea) in Alaska. Subsequent investigation found this organism in dead and subclinically infected birds of other species from North America and Australia. Isolates from dead finches in Scotland, previously identified as Escherichia coli O86:K61, also were shown to be E. albertii. Similar to the isolates from humans, E. albertii isolates from birds possessed intimin (eae) and cytolethal distending toxin (cdtB) genes but lacked Shiga toxin (stx) genes. Genetic analysis of eae and cdtB sequences, multilocus sequence typing, and pulsed-field gel electrophoresis patterns showed that the E. albertii strains from birds are heterogeneous but similar to isolates that cause disease in humans. PMID:20350378

  15. 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.

  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. Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes.

    PubMed

    Hino, Mami; Kataoka, Masatoshi; Kajimoto, Kazuaki; Yamamoto, Takenori; Kido, Jun-Ichi; Shinohara, Yasuo; Baba, Yoshinobu

    2008-01-20

    The efficiency of protein synthesis for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined with several in vitro coupled transcription/translation protein synthesis systems based on Escherichia coli lysate, wheat germ, or reticulocyte lysate, and an in vitro translation system based on wheat germ extract. A significant amount of protein synthesis was observed only in systems based on E. coli using pET/G3PDH as the expression vector. A remarkable increase of protein synthesis was obtained in wheat germ using a pT(N)T expression vector which contains a 5'-globin leader sequence and a synthetic poly(A)(30) tail instead of pET. A significant difference of T7 RNA polymerase presence by Western blot analysis was not observed in the first four systems, and the difference of total RNA presence in each reaction mixture by Northern blot analysis seemed unrelated to protein synthesis. Although a small amount of protein was synthesized using RNA-encoding G3PDH transcribed in vitro with pET/G3PDH by an in vitro translation system, an extreme increase was observed using transcribed RNA with pEU/G3PDH, which contains T7 RNA promoter and a translation enhancer, Omega sequence. These results suggest that the presence of an enhancer sequence for translation is one of the critical steps for protein synthesis by a eukaryotic cell-free protein synthesis system.

  18. 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.

  19. 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

  20. 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...

  1. Escherichia marmotae sp. nov., isolated from faeces of Marmota himalayana.

    PubMed

    Liu, Sha; Jin, Dong; Lan, Ruiting; Wang, Yiting; Meng, Qiong; Dai, Hang; Lu, Shan; Hu, Shoukui; Xu, Jianguo

    2015-07-01

    The taxonomic position of a group of seven closely related lactose-negative enterobacterial strains, which were isolated from fresh faecal samples of Marmota himalayana collected from the Qinghai-Tibetan plateau, China, was determined by using a polyphasic approach. Cells were Gram-reaction-negative, non-sporulating, non-motile, short rods (0.5-1 × 1-2.5 μm). By 16S rRNA gene sequences, the representative strain, HT073016(T), showed highest similarity values with Escherichia fergusonii ATCC 35469(T) at 99.3%, Escherichia coli ATCC 11775(T) at 99.2%, Escherichia albertii LMG 20976(T) at 98.9%, Escherichia hermannii CIP 103176(T) at 98.4%, and Escherichia vulneris ATCC 33821(T) at 97.7%. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the seven strains formed a monophyletic group with five other species of the genus Escherichia. Digital DNA-DNA hybridization studies between strain HT073016(T) and five other species of the genus Escherichia showed that it shared less than 70% DNA-DNA relatedness with all known species of the genus Escherichia, supporting the novel species status of the strain. The DNA G+C content of strain HT073016(T) was 53.8 mol%. On the basis of phenotypic and phylogenetic characteristics, strain HT073016(T) and the six other HT073016(T)-like strains were clearly distinct from the type strains of other recognized species of the genus Escherichia and represent a novel species of the genus Escherichia, for which the name Escherichia marmotae sp. nov. is proposed, with HT073016(T) ( = CGMCC 1.12862(T) = DSM 28771(T)) as the type strain.

  2. 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

  3. 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.

  4. Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences.

    PubMed

    Magliano, Ana C M; Teixeira, Marta M G; Alfieri, Silvia C

    2012-01-01

    Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.

  5. Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.

    PubMed

    Hu, Zhonglan; Chen, Zhenyu; Huang, Nongyu; Teng, Xiu; Zhang, Jun; Wang, Zhen; Wei, Xiaoqiong; Qin, Ke; Liu, Xiao; Wu, Xueping; Tang, Huan; Zhu, Xiaofeng; Cui, Kaijun; Li, Jiong

    2015-03-01

    Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined.

  6. An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli.

    PubMed

    Chhetri, Gaurav; Kalita, Parismita; Tripathi, Timir

    2015-01-01

    Bacterial cells can be engineered to express non-native genes, resulting in the production of, recombinant proteins, which have various biotechnological and pharmaceutical applications. In eukaryotes, such as yeast or mammalian cells, which have large genomes, a higher recombinant protein expression can be troublesome. Comparatively, in the Escherichia coli (E. coli) expression system, although the expression is induced with isopropyl β-d-1-thiogalactopyranoside (IPTG), studies have shown low expression levels of proteins. Irrespective of the purpose of protein production, the production process requires the accomplishment of three individual factors: expression, solubilization and purification. Although several efforts, including changing the host, vector, culture parameters of the recombinant host strain, co-expression of other genes and changing of the gene sequences, have been directed towards enhancing recombinant protein expression, the protein expression is still considered as a significant limiting step. Our protocol explains a simple method to enhance the recombinant protein expression that we have optimized using several unrelated proteins. It works with both T5 and T7 promoters. This protocol can be used to enhance the expressions of most of the proteins. The advantages of this technique are presented below:•It produces several fold increase in the expression of poorly expressed, less expressed or non-expressed recombinant proteins.•It does not employ any additional component such as chaperones, heat shock proteins or co-expression of other genes.•In addition to being inexpensive, easy to manage, universal, and quick to perform, the proposed method does not require any commercial kits and, can be used for various recombinant proteins expressed in the E. coli expression system.

  7. Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

    PubMed

    Park, Chan-Hwan; Ju, Hye-Kyoung; Han, Jae-Yeong; Park, Jong-Seo; Kim, Ik-Hyun; Seo, Eun-Young; Kim, Jung-Kyu; Hammond, John; Lim, Hyoun-Sub

    2017-04-01

    Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.

  8. 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

  9. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    PubMed

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

  10. Efficient production of mutant phytase (phyA-7) derived from Selenomonas ruminantium using recombinant Escherichia coli in pilot scale.

    PubMed

    Chi-Wei Lan, John; Chang, Chih-Kai; Wu, Ho-Shing

    2014-09-01

    A mutant gene of rumen phytase (phyA-7) was cloned into pET23b(+) vector and expressed in the Escherichia coli BL21 under the control of the T7 promoter. The study of fermentation conditions includes the temperature impacts of mutant phytase expression, the effect of carbon supplements over induction stage, the inferences of acetic acid accumulation upon enzyme expression and the comparison of one-stage and two-stage operations in batch mode. The maximum value of phytase activity was reached 107.0 U mL(-1) at induction temperature of 30°C. Yeast extract supplement demonstrated a significant increase on both protein concentration and phytase activity. The acetic acid (2 g L(-1)) presented in the modified synthetic medium demonstrated a significant decrease on expressed phytase activity. A two-stage batch operation enhanced the level of phytase activity from 306 to 1204 U mL(-1) in the 20 L of fermentation scale. An overall 3.7-fold improvement in phytase yield (35,375.72-1,31,617.50 U g(-1) DCW) was achieved in the two-stage operation.

  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. 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. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  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. 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

  7. 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...

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. [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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  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. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant.

    PubMed

    Lee, J H; Kim, M S; Cho, J H; Kim, S C

    2002-05-01

    The tandem multimeric expression of various peptides has been explored by many researchers. However, expression levels have usually not been proportional to the degree of multimerization. To increase the expression level in Escherichia coli of tandem multimers of a cationic antimicrobial peptide, buforin II, fused to an anionic peptide, we studied the effect of the DEAD-box protein and the trxB mutant on the expression of tandem multimers. An expression vector with a tac promoter was more effective in directing multimeric expression than one with a T7 promoter. The expression level of large multimers was substantially increased with the tac promoter, possibly through stabilization of long transcripts by synchronization of transcription and translation. Coexpression of the DEAD-box protein, an RNA-binding protein, with the T7 expression system increased the expression level of multimers, especially large multimers, due to protection of the long RNA transcripts. In addition, the use of the trxB mutant also enhanced the expression level of tandem multimers, which contain two cysteine residues at both ends of the monomeric unit. It seems that disulfide bonds formed in the multimers in the trxB mutant might help efficient charge neutralization for inclusion body formation of the multimers, resulting in enhancement of expression. Our results show that the expression of multimers can be improved through the stabilization of the long transcripts by the DEAD-box protein or the expression, under an oxidizing environment, of the trxB mutant in which covalent cross-links through disulfide bonds facilitate inclusion body formation of the multimeric fusion peptide.

  3. Role of wild birds as carriers of multi-drug resistant Escherichia coli and Escherichia vulneris.

    PubMed

    Shobrak, Mohammed Y; Abo-Amer, Aly E

    2014-01-01

    Emergence and distribution of multi-drug resistant (MDR) bacteria in environments pose a risk to human and animal health. A total of 82 isolates of Escherichia spp. were recovered from cloacal swabs of migrating and non-migrating wild birds. All bacterial isolates were identified and characterized morphologically and biochemically. 72% and 50% of isolates recovered from non-migrating and migrating birds, respectively, showed positive congo red dye binding (a virulence factor). Also, hemolysin production (a virulence factor) was showed in 8% of isolates recovered from non-migrating birds and 75% of isolates recovered from migrating birds. All isolates recovered from non-migrating birds were found resistant to Oxacillin while all isolates recovered from migrating birds demonstrated resistance to Oxacillin, Chloramphenicol, Oxytetracycline and Lincomycin. Some bacterial isolates recovered from non-migrating birds and migrating birds exhibited MDR phenotype. The MDR isolates were further characterized by API 20E and 16S rRNA as E. coli and E. vulneris. MDR Escherichia isolates contain ~1-5 plasmids of high-molecular weights. Accordingly, wild birds could create a potential threat to human and animal health by transmitting MDR bacteria to water streams and other environmental sources through their faecal residues, and to remote regions by migration.

  4. The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.

    PubMed

    Suziedeliené, E; Suziedélis, K; Garbenciūté, V; Normark, S

    1999-04-01

    Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

    PubMed

    Przystałowska, Hanna; Zeyland, Joanna; Szymanowska-Powałowska, Daria; Szalata, Marlena; Słomski, Ryszard; Lipiński, Daniel

    2015-02-01

    1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol₁,₃-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming

  17. Production of aromatic compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway.

    PubMed

    Koma, Daisuke; Yamanaka, Hayato; Moriyoshi, Kunihiko; Ohmoto, Takashi; Sakai, Kiyofumi

    2012-09-01

    Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by

  18. Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

    PubMed Central

    Yamanaka, Hayato; Moriyoshi, Kunihiko; Ohmoto, Takashi; Sakai, Kiyofumi

    2012-01-01

    Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by

  19. 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

  20. 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

  1. 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].

  2. 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

  3. [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.

  4. Chimera-free, high copy number YAC libraries and efficient methods of analysis

    SciTech Connect

    Not Available

    1992-01-01

    Evidence is accumulating to indicate that the mouse genome YAC library constructed in a rad52 host strain is quite low in chimeric YACs and in unstable YACs. We have optimized the transformation efficiency of two recombination-deficient strains which are isogenic with CGY2570. They are the rad52 strain CGY2872 and the rad52 radl strain CGY2897. The genotype of these gene-disrupted strains was verified by testing the uv and MMS sensitivity of the strains. These recombination-deficient strains grow about 10% slower than the isogenic wild-type strain and transform somewhat less efficiently. However, by optimizing the parameters described above, we have obtained reproducible transformation efficiencies which are only about 4-fold and 8-fold poorer than that of the isogenic wild-type strain. Using this procedure, we have obtained over 5,000 YACs of about 200 kb average size in the rad52 host and several thousand YACs of about 400--500 kb average size in the rad52 and rad52 radl hosts. Therefore, all of the procedures are now in place to build a total human genome library in a recombination deficient host.

  5. Chimera-free, high copy number YAC libraries and efficient methods of analysis. Technical progress report

    SciTech Connect

    Not Available

    1992-12-31

    Evidence is accumulating to indicate that the mouse genome YAC library constructed in a rad52 host strain is quite low in chimeric YACs and in unstable YACs. We have optimized the transformation efficiency of two recombination-deficient strains which are isogenic with CGY2570. They are the rad52 strain CGY2872 and the rad52 radl strain CGY2897. The genotype of these gene-disrupted strains was verified by testing the uv and MMS sensitivity of the strains. These recombination-deficient strains grow about 10% slower than the isogenic wild-type strain and transform somewhat less efficiently. However, by optimizing the parameters described above, we have obtained reproducible transformation efficiencies which are only about 4-fold and 8-fold poorer than that of the isogenic wild-type strain. Using this procedure, we have obtained over 5,000 YACs of about 200 kb average size in the rad52 host and several thousand YACs of about 400--500 kb average size in the rad52 and rad52 radl hosts. Therefore, all of the procedures are now in place to build a total human genome library in a recombination deficient host.

  6. 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...

  7. 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.

  8. 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

  9. A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.

    PubMed

    Wang, Liying; Zhang, Lihong; Liu, Zhanzhi; Liu, Zhangzhi; Zhao, Dehua; Liu, Xiaomeng; Zhang, Bo; Xie, Jianbo; Hong, Yuanyuan; Li, Pengfei; Chen, Sanfeng; Dixon, Ray; Li, Jilun

    2013-01-01

    Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ(70) (σ(A))-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.

  10. A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.

    PubMed

    Cai, Qi; Hanson, Jeffrey A; Steiner, Alexander R; Tran, Cuong; Masikat, Mary Rose; Chen, Rishard; Zawada, James F; Sato, Aaron K; Hallam, Trevor J; Yin, Gang

    2015-01-01

    Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics.

  11. Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures

    SciTech Connect

    Wiberg, J.S.; Mowrey-Mckee, M.F.; Stevens, E.J.

    1988-01-01

    Production of bacteriophages T2, T4, and T6 at 42.8 to 44/sup 0/C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30/sup 0/C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44/sup 0/C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70 and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44/sup 0/C exhibited a partial tryptophan deficiency. Production of bacteriophage T7 at 44/sup 0/C was increased two- to fourfold by adapting the host to 44/sup 0/C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage delta appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature.

  12. Production of 3-Hydroxypropionic Acid via the Propionyl-CoA Pathway Using Recombinant Escherichia coli Strains

    PubMed Central

    Luo, Hui; Zhou, Dafeng; Liu, Xiaohui; Nie, Zhihua; Quiroga-Sánchez, Diego Leandro; Chang, Yanhong

    2016-01-01

    Our study aimed to produce the commercially promising platform chemical 3-hydroxypropionic acid (3-HP) via the propionyl-CoA pathway in genetically engineered Escherichia coli. Recombinant E. coli Ec-P overexpressing propionyl-CoA dehydrogenase (PACD, encoded by the pacd gene from Candida rugosa) under the T7 promoter produced 1.33 mM of 3-HP in a shake flask culture supplemented with 0.5% propionate. When propionate CoA-transferase (PCT, encoded by the pct gene from Megasphaera elsdenii) and 3-hydroxypropionyl-CoA dehydratase (HPCD, encoded by the hpcd gene from Chloroflexus aurantiacus) were expressed along with PACD, the 3-HP titer of the resulting E. coli Ec-PPH strain was improved by 6-fold. The effect of the cultivation conditions on the 3-HP yield from propionate in the Ec-PPH strain was also investigated. When cultured at 30°C with 1% glucose in addition to propionate, 3-HP production by Ec-PPH increased 2-fold and 12-fold compared to the cultivation at 37°C (4.23 mM) or without glucose (0.68 mM). Deletion of the ygfH gene encoding propionyl-CoA: succinate CoA-transferase from Ec-PPH (resulting in the strain Ec-△Y-PPH) led to increase of 3-HP production in shake flask experiments (15.04 mM), whereas the strain Ec-△Y-PPH with deletion of the prpC gene (encoding methylcitrate synthase in the methylcitrate cycle) produced 17.76 mM of 3-HP. The strain Ec-△Y-△P-PPH with both ygfH and prpC genes deleted produced 24.14 mM of 3-HP, thus showing an 18-fold increase in the 3-HP titer in compare to the strain Ec-P. PMID:27227837

  13. 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.

  14. 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...

  15. 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.

  16. 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

  17. 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...

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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....

  5. 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...

  6. 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...

  7. 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

  8. Sensitivity of Escherichia albertii to food preservation treatments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia albertii is a potential foodborne pathogen because of its documented ability to cause diarrheal disease by producing attachment and effacement lesions. Its tolerance to food preservation treatments has not been investigated. Heat, acid, and pressure tolerance were determined for stationa...

  9. 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.

  10. 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

  11. Unusual “Flesh-Eating” Strain of Escherichia coli▿

    PubMed Central

    Grimaldi, David; Bonacorsi, Stéphane; Roussel, Hélène; Zuber, Benjamin; Poupet, Hélène; Chiche, Jean-Daniel; Poyart, Claire; Mira, Jean-Paul

    2010-01-01

    We report an exceptional case of life-threatening Escherichia coli-induced necrotizing fasciitis. A combined host-pathogen genetic analysis explained the phenotype: the host displayed a susceptibility to intravascular coagulation, and the strain was capable of producing a necrotic toxin (cytotoxic necrotizing factor 1), showing how E. coli can be a dermonecrotic pathogen. PMID:20686096

  12. 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...

  13. 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

  14. 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

  15. Cold-inducible cloning vectors for low-temperature protein expression in Escherichia coli: application to the production of a toxic and proteolytically sensitive fusion protein.

    PubMed

    Mujacic, M; Cooper, K W; Baneyx, F

    1999-10-01

    TolAI-beta-lactamase a fusion protein consisting of the inner membrane anchoring domain of the Escherichia coli transenvelope protein TolA followed by TEM-beta-lactamase was found to be toxic and highly unstable when transcribed from the bacteriophage T7 promoter at 37 degrees C. Expression at 15 or 23 degrees C alleviated toxicity, but led to only partial stabilization of the fusion protein. To evaluate the usefulness of cold-shock promoters for the production of proteolytically sensitive proteins at low temperatures, we constructed a set of cloning vectors suitable for rapidly positioning PCR products under cspA transcriptional control. TolAI-beta-lactamase degradation was completely abolished when cspA-driven transcription was induced by temperature downshift to 15 or 23 degrees C. Our results suggest that the cspA promoter system may be a valuable tool for the production of proteins containing membrane-spanning domains or otherwise unstable gene products in E. coli.

  16. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

    PubMed

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Ali, Syed A

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  17. Targeted Capture and Heterologous Expression of the Pseudoalteromonas Alterochromide Gene Cluster in Escherichia coli Represents a Promising Natural Product Exploratory Platform

    PubMed Central

    2015-01-01

    Marine pseudoalteromonads represent a very promising source of biologically important natural product molecules. To access and exploit the full chemical capacity of these cosmopolitan Gram-(−) bacteria, we sought to apply universal synthetic biology tools to capture, refactor, and express biosynthetic gene clusters for the production of complex organic compounds in reliable host organisms. Here, we report a platform for the capture of proteobacterial gene clusters using a transformation-associated recombination (TAR) strategy coupled with direct pathway manipulation and expression in Escherichia coli. The ∼34 kb pathway for production of alterochromide lipopeptides by Pseudoalteromonas piscicida JCM 20779 was captured and heterologously expressed in E. coli utilizing native and E. coli-based T7 promoter sequences. Our approach enabled both facile production of the alterochromides and in vivo interrogation of gene function associated with alterochromide’s unusual brominated lipid side chain. This platform represents a simple but effective strategy for the discovery and biosynthetic characterization of natural products from marine proteobacteria. PMID:25140825

  18. 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)....

  19. In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

    PubMed

    Burian, J; Tu, N; Kl'ucár, L; Guller, L; Lloyd-Jones, G; Stuchlík, S; Fejdi, P; Siekel, P; Turna, J

    1998-01-01

    A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested

  20. Transaldolase B of Escherichia coli K-12: cloning of its gene, talB, and characterization of the enzyme from recombinant strains.

    PubMed Central

    Sprenger, G A; Schörken, U; Sprenger, G; Sahm, H

    1995-01-01

    A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai, T. Nagata, A. Ishihama, N. Fujita, K. Isono, K. Mizobuchi, and A. Nakata, Nucleic Acids Res. 20:3305-3308) from the 0.2-min region of the Escherichia coli K-12 chromosome is shown to encode a functional transaldolase activity. After cloning of the gene onto high-copy-number vectors, transaldolase B (D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate dihydroxyacetone transferase; EC 2.2.1.2) was overexpressed up to 12.7 U mg of protein-1 compared with less than 0.1 U mg of protein-1 in wild-type homogenates. The enzyme was purified from recombinant E. coli K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit size of 35,000 +/- 1,000 Da. As the enzyme had a molecular mass of 70,000 Da by gel filtration, transaldolase B is likely to form a homodimer. N-terminal amino acid sequencing of the protein verified its identity with the product of the cloned gene talB. The specific activity of the purified enzyme determined at 30 degrees C with the substrates fructose-6-phosphate (donor of C3 compound) and erythrose-4-phosphate (acceptor) at an optimal pH (50 mM glycylglycine [pH 8.5]) was 60 U mg-1.Km values for the substrates fructose-6-phosphate and erythrose-4-phosphate were determined at 1,200 and 90 microM, respectively. Kinetic constants for the other two physiological reactants, D,L-glyceraldehyde 3-phosphate (Km, 38 microM; relative activity [V(rel)], 8%) and sedoheptulose-7-phosphate (K(m), 285 microM; V(rel), 5%) were also determined. Fructose acted as a C(3) donor at a high apparent K(m) (>/=M) and with a V(rel) of 12%. The enzyme was inhibited by Tris-HCl, phosphate, or

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Alkylation Induced DNA Repair and Mutagenesis in Escherichia coli.

    DTIC Science & Technology

    1987-11-23

    III (Gates and inn, 1977), Micrococcus luteus UV endo- nuclease (Grossman et al, 1978) and bacteriophage T UV endonuclease (Warner et al, 1980) have DNA...34, Garland Publishing, Inc. New York & London USA. Ather, A., Z. Ahmed and S. Riazxxddin, 1984. Adaptive response of Micrococcus luteus to alkylating...Laval, J., 3. Pierre and F. Laval. 1981. Release of 7-nmthylguanine residues frain alkylated ENA by extracts of Micrococcus luteus and Escherichia

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. [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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  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. 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

  16. Overexpression of Recombinant Human Teriparatide, rhPTH (1–34) in Escherichia coli : An Innovative Gene Fusion Approach

    PubMed Central

    Bakhtiari, Nahid; Amini Bayat, Zahra; Sagharidouz, Sepideh; Vaez, Mohsen

    2017-01-01

    Background: Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1–34 amino acids. The recombinant form of hormone (1–34) has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1–34 amino acids of hPTH. Methods: Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to Escherichia coli (E. coli) β-galactosidase (LacZ) gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH (1–34) gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in E. coli (DH5α) cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 mM IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase. Results: By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting. Conclusion: In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial β-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point (pI). This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for

  17. Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures.

    PubMed Central

    Wiberg, J S; Mowrey-McKee, M F; Stevens, E J

    1988-01-01

    Production of bacteriophages T2, T4, and T6 at 42.8 to 44 degrees C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30 degrees C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44 degrees C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70, and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44 degrees C exhibited a partial tryptophan deficiency; this correlated with reduced uptake of uracil that is probably caused by partial induction of stringency. Production of bacteriophage T7 at 44 degrees C was increased two- to fourfold by adapting the host to 44 degrees C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage lambda (C. Waghorne and C.R. Fuerst, Virology 141:51-64, 1985) appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature. Images PMID:2446014

  18. 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 ...

  19. Growth of Escherichia albertii strains in ground turkey at three temperatures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia albertii is the newest species designated in the genus Escherichia and has been associated with diarrheal disease in developing nations. The impacts of food preservation treatments against E. albertii have been studied, but data on the behavior of the organism in food are lacking. The ob...

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. Feces of feedlot cattle contain a diversity of bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli.

    PubMed

    Wang, Jiaying; Niu, Yan D; Chen, Jinding; Anany, Hany; Ackermann, Hans-W; Johnson, Roger P; Ateba, Collins N; Stanford, Kim; McAllister, Tim A

    2015-07-01

    This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

  13. Isolation of a Gene Responsible for the Oxidation of trans-Anethole to para-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

    PubMed Central

    Han, Dongfei; Ryu, Ji-Young; Kanaly, Robert A.

    2012-01-01

    A plasmid, pTA163, in Escherichia coli contained an approximately 34-kb gene fragment from Pseudomonas putida JYR-1 that included the genes responsible for the metabolism of trans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyze trans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter in E. coli catalyzed oxidative cleavage of a propenyl group of trans-anethole to an aldehyde group, resulting in the production of para-anisaldehyde, and this gene was designated tao (trans-anethole oxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein of Agrobacterium vitis S4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water into p-anisaldehyde using 18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase from Pseudomonas putida IE27 and Pseudomonas nitroreducens Jin1, TAO from P. putida JYR-1 catalyzed isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts of E. coli (pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future. PMID:22610435

  14. Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs.

    PubMed Central

    Xu, S; Xiao, J; Posfai, J; Maunus, R; Benner, J

    1997-01-01

    BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector. PMID:9321648

  15. Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

    PubMed

    Chung, Wen-Jen; Huang, Chi-Lung; Gong, Hong-Yi; Ou, Tsung-Yin; Hsu, Jue-Liang; Hu, Shao-Yang

    2015-06-01

    Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.

  16. An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

    PubMed

    Ta, Duy Tien; Redeker, Erik Steen; Billen, Brecht; Reekmans, Gunter; Sikulu, Josephine; Noben, Jean-Paul; Guedens, Wanda; Adriaensens, Peter

    2015-10-01

    In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.

  17. Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system.

    PubMed

    Khasa, Yogender Pal; Khushoo, Amardeep; Mukherjee, Krishna Jyoti

    2013-03-01

    The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.5 mg g(-1) DCW h(-1) at a dilution rate (D) of 0.3 h(-1). The maximum volumetric product concentration achieved at this dilution rate was 474 mg l(-1), which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h(-1) and 0.4 h(-1) respectively. The specific product yield (Y(P/x)) peaked at 138 mg g(-1) DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q(p) was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l(-1) with a specific hGM-CSF yield (Y(P/x)) of 107 mg g(-1) DCW.

  18. Non-Invasive Analysis of Recombinant mRNA Stability in Escherichia coli by a Combination of Transcriptional Inducer Wash-Out and qRT-PCR

    PubMed Central

    Kucharova, Veronika; Strand, Trine Aakvik; Almaas, Eivind; Naas, Adrian E.; Brautaset, Trygve; Valla, Svein

    2013-01-01

    mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research. PMID:23840466

  19. 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.

  20. Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli.

    PubMed Central

    Visick, J E; Whiteley, H R

    1991-01-01

    CytA, a 27-kDa cytolytic crystal protein of Bacillus thuringiensis subsp. israelensis, is produced only at very low levels by recombinant Escherichia coli cells unless a 20-kDa B. thuringiensis subsp. israelensis protein is also present (K. M. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987; L. F. Adams, J. E. Visick, and H. R. Whiteley, J. Bacteriol. 171:521-530, 1989). However, the data reported here demonstrate that the 20-kDa protein is not required for high-level CytA production in E. coli strains carrying mutations in rpoH, groEL, or dnaK, all of which affect the proteolytic ability of the cells. The 20-kDa protein also increases the amount of CryIVD (another B. thuringiensis subsp. israelensis crystal protein) and LacZX90 (a mutant of beta-galactosidase) made by E. coli. The latter phenomenon is attributable to an increase in the half-life of LacZX90, suggesting that the 20-kDa protein may stabilize this protein. The effect of the 20-kDa protein was also examined in vitro and in a T7 RNA polymerase expression system, and the possible significance of these results for the timing of proteolysis and of 20-kDa protein activity is discussed. Finally, the ability of a single antibody to coimmunoprecipitate CytA and the 20-kDa protein from E. coli extracts provides evidence for a protein-protein interaction that may be related to the mechanism of action of the 20-kDa protein. Images PMID:1900280

  1. Biofilm Formation by and Thermal Niche and Virulence Characteristics of Escherichia spp.▿

    PubMed Central

    Ingle, Danielle J.; Clermont, Olivier; Skurnik, David; Denamur, Erick; Walk, Seth T.; Gordon, David M.

    2011-01-01

    In order to better understand the ecological and virulence characteristics of the various clades of Escherichia, in vitro and in vivo experiments were undertaken. Members of the recently described cryptic clades of Escherichia (clades III, IV, and V) were found to have an enhanced ability to form biofilms compared to strains of Escherichia coli, E. fergusonii, or E. albertii. Members of the cryptic clades were also able to replicate at a lower temperature (5°C versus 11°C) than strains of the named species of Escherichia. Neither a strain's maximal growth rate nor its optimal temperature for growth varied with respect to the strain's phylogenetic affiliation. Escherichia strains not belonging to the species E. coli were positive for a mix of traits thought to enhance a strain's ability to cause either intestinal or extraintestinal disease. However, no non-E. coli Escherichia strain was virulent in a mouse model of extraintestinal infection. The frequency of resistance to antibiotics was low, and none of the strains tested harbored class 1, 2, or 3 integrons. The results of these experiments support the hypothesis that members of the cryptic Escherichia clades may be better able to persist in the external environment compared to E. coli, E. fergusonii, or E. albertii, isolates. PMID:21335385

  2. 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

  3. [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.

  4. 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/

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  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. 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. 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.

  17. 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

  18. 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

  19. 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

  20. 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