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  • 1
    Electronic Resource
    Electronic Resource
    Polyphosphate Metabolism in Escherichia coli (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 745 (1994), S. 0 
    Details
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1749-6632.1994.tb44365.x
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  • 2
    Electronic Resource
    Electronic Resource
    A Monte Carlo simulation of plasmid replication during the bacterial division cycle (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 633-647 
    Details
    ISSN: 0006-3592
    Keywords: Monte Carlo simulation ; plasmid replication ; bacterial division cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plasmids have cell cycle replication patterns that need to be considered in models of their replication dynamics. To compare current theories for control of plasmid replication with experimental data for timing of plasmid replication with the cell cycle, a Monte Carlo simulation of plasmid replication and partition was developed. High-copy plasmid replication was simulated by incorporating equations previously developed from the known molecular biology of ColE1-type plasmids into the cell-cycle simulation. Two types of molecular mechanisms for low-copy plasmid replication were tested: accumulation of an initiator protein in proportion to cell mass and binding of the plasmid origin to the cell membrane. The low-copy plasmids were partitioned actively, with a specific mechanism to mediate the transfer from mother to daughter cells, whereas the high-copy plasmids were partitioned passively with cell mass.The simulation results and experimental data demonstrate cell-cycle-specific replication for the low-copy F plasmid and cell-cycle-independent replication for the high-copy pBR322, ColBM, and R6K plasmids. The simulation results indicate that synchronous replication at multiple plasmid origins is critical for the cell-cycle-specific pattern observed in rapidly growing cells. Variability in the synchrony of initiation of multiple plasmid origins give rise to a cell-cycle-independent pattern and is offered as a plausible explanation for the controversy surrounding the replication pattern of the low-copy plasmids. A comparison of experimental data and simulation results for the low-copy F plasmid at several growth rates indicates that either initiation mechanism would be sufficient to explain the timing of replication with the cell cycle. The simulation results also demonstrate that, although cell-cycle-specific and cell-cycle independent replication patterns give rise to very different gene-expression patterns during short induction periods in age-selected populations, long-term expression of genes encoded on low-copy and high-copy plasmids in exponentially growing cells have nearly the same patterns. These results may be important for the future use of low-copy plasmids as expression vectors and validate the use of simpler models for high-copy plasmids that do not consider cell-cycle phenomena. © 1996 John Wiley & Sons, Inc.
    Additional Material: 11 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    Kinetics of toluene degradation by a nitrate-reducing bacterium isolated from a groundwater aquifer (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 82-90 
    Details
    ISSN: 0006-3592
    Keywords: toluene degradation ; xylene degradation ; Azoarcus tolulyticus ; nitrate reduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Groundwater from a xylene-contaminated acquifer was enriched in the laboratory in the presence of toluene, xylenes, ethylbenzene, and benzene. A pure culture that degrades toluene and m-xylene under nitrate-reducing conditions was isolated. Fatty acid analysis, 16S rRNA sequencing, and morphological traits indicate that the isolate was a strain of Azoarcus tolulyticus. The kinetics of toluene degradation under nitrate-reducing conditions by this isolate was determined. Nitrate reduction does not proceed beyond nitrite. Nitrate and toluene are substrate limiting at low concentrations, whereas toluene, nitrate, and nitrite are inhibitory at high concentrations. Several inhibition models were compared to experimental data to represent inhibition by these substrates. A kinetic model for toluene and nitrate degradation as well as for cell growth and nitrite production was developed and compared to experimental data. The results of this work may find important application in the remediation of groundwater aquifers contaminated with aromatic hydrocarbons. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 82-90, 1997.
    Additional Material: 4 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    Stoichiometric model of Escherichia coli metabolism: Incorporation of growth-rate dependent biomass composition and mechanistic energy requirements (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 398-421 
    Details
    ISSN: 0006-3592
    Keywords: Escherichia coli ; metabolism ; flux ; linear optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A stoichiometric model of metabolism was developed to describe the balance of metabolic reactions during steady-state growth of Escherichia coli on glucose (or metabolic intermediates) and mineral salts. The model incorporates 153 reversible and 147 irreversible reactions and 289 metabolites from several metabolic data bases for the biosynthesis of the macromolecular precursors, coenzymes, and prosthetic groups necessary for synthesis of all cellular macromolecules. Correlations describing how the cellular composition changes with growth rate were developed from experimental data and were used to calculate the drain of precursors to macromolecules, coenzymes, and prosthetic groups from the metabolic network for the synthesis of those macromolecules at a specific growth rate. Energy requirements for macromolecular polymerization and proofreading, transport of metabolites, and maintenance of transmembrane gradients were included in the model rather than a lumped maintenance energy term. The underdetermined set of equations was solved using the Simplex algorithm, employing realistic objective functions and constraints; the drain of precursors, coenzymes, and prosthetic groups and the energy requirements for the synthesis of macromolecules served as the primary set of constraints. The model accurately predicted experimentally determined metabolic fluxes for aerobic growth on acetate or acetate plus glucose. In addition, the model predicted the genetic and metabolic regulation that must occur for growth under different conditions, such as the opening of the glyoxylate shunt during growth on acetate and the branching of the tricarboxylic acid cycle under anaerobic growth. Sensitivity analyses were performed to determine the flexibility of pathways and the effects of different rates and growth conditions on the distribution of fluxes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 398-421, 1997.
    Additional Material: 5 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    Effect of Escherichia coli biomass composition on central metabolic fluxes predicted by a stoichiometric model (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 230-238 
    Details
    ISSN: 0006-3592
    Keywords: Escherichia coli ; metabolism ; flux ; linear optimization ; biomass composition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The amino acid composition of proteins and the fatty acid composition of the cell membranes were measured in Escherichia coli growing exponentially in batch culture on glucose, succinate, glycerol, pyruvate, and acetate, and growing under continuous culture conditions on glucose at dilutions rates equivalent to the growth rates of the batch cultures. Although the fatty acid composition of the membranes did change significantly with carbon source and dilution rate, the amino acid content of proteins did not change significantly under either condition. A previously developed stoichiometric model of metabolism was used to calculate the fluxes through the metabolic reactions and to determine their sensitivity to changes in fatty acid and amino acid composition. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 230-238, 1998.
    Additional Material: 2 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Modulation of the phosphate-starvation response in Escherichia coli by genetic manipulation of the polyphosphate pathways (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 434-438 
    Details
    ISSN: 0006-3592
    Keywords: polyphosphate ; Escherichia coli ; phosphate starvation ; gene expression ; heterologous ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of intracellular polyphosphate on the phosphate-starvation response in Escherichia coli was studied by genetically manipulating the intracellular polyphosphate levels and by performing phosphate shifts on the genetically engineered strains. Strains that produced large quantities of polyphosphate and were able to degrade it induced the phosphate-starvation response to a lesser extent than wild-type strains, whereas strains that were unable to degrade a large intracellular polyphosphate pool induced the phosphate-starvation response to a greater extent than wild-type strains. These results have important implications for expression of heterologous genes under control of the phoA promoter. © 1996 John Wiley & Sons, Inc.
    Additional Material: 4 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 754-761 
    Details
    ISSN: 0006-3592
    Keywords: polyphosphate ; Escherichia coli ; phosphate starvation ; phosphate secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:754-761, 1998.
    Additional Material: 5 Ill.
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  • 8
    Electronic Resource
    Electronic Resource
    mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 666-672 
    Details
    ISSN: 0006-3592
    Keywords: mRNA stability ; plasmid copy number ; gene expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5′ hairpin structures. Product formation and cell growth were evaluated under a number of inducer concentrations. The introduction of a 5′ hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated. With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate. At inducer concentrations between 1 × 10-4 and 4 × 10-4%, the productivity of low-copy plasmids containing the 5′-hairpin was equal to or greater than that from multicopy plasmids. Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:666-672, 1998.
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  • 9
    Electronic Resource
    Electronic Resource
    Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5′ cassette system (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 577-580 
    Details
    ISSN: 0006-3592
    Keywords: mRNA stability ; hairpins ; gene expression control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An expression system has been developed for the introduction of DNA cassettes into the region between the transcription and translation start sites of a gene of interest. This cassette system was used to engineer mRNA stability through the introduction of hairpins at the 5′ end. A synthetic DNA cassette was designed so that the resulting mRNA hairpin would be positioned one nucleotide from the 5′ mRNA end. The hairpin-containing mRNA exhibited a half-life 3 times that of the mRNA with no hairpin, resulting in increases in both mRNA and protein levels. These results indicate that it is possible to engineer mRNA stability as an additional means of controlling gene expression. © 1997 John Wiley & Sons Inc. Biotechnol Bioeng 55: 557-580, 1997
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  • 10
    Electronic Resource
    Electronic Resource
    Engineering polyphosphate metabolism in Escherichia coli: Implications for bioremediation of inorganic contaminants (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 231-239 
    Details
    ISSN: 0006-3592
    Keywords: polyphosphate metabolism ; metabolic engineering ; Escherichia coli ; bioremediation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municiple wastewater, and may play a key role in heavy metal tolerance and bioremediation. However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity. We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters. Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals. Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals. Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:231-239, 1998.
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