Download or read book Molecular Engineering of the Escherichia Coli Global Transcription Factor Fnr to Improve Its Stability to Oxygen written by 单越 and published by . This book was released on 2017-01-26 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Molecular Engineering of the Escherichia Coli Global Transcription Factor FNR to Improve Its Stability to Oxygen" by 单越, Yue, Shan, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: The ability to sense and rapidly respond to oxygen availability is crucial to the survival and physiology of facultative anaerobes. In many gram negative bacteria such as Escherichia coli, this process is primarily controlled by the dimeric, [4Fe〖-4S]〗 DEGREES(2+) containing global transcription factor FNR, which regulates transcription of genes necessary for the anaerobic metabolism. Activity of FNR is directly regulated by the presence of oxygen, which inactivates FNR by oxidizing the [4Fe〖-4S]〗 DEGREES(2+) cluster and causing the dissociation of the FNR dimer. Although the biological function of FNR has been well established, structural and biochemical characterization of the FNR dimer has been limited due to its extreme lability to oxygen. In the current study, I conduct molecular engineering on FNR protein and obtain oxygen stable variants that are suitable for in vitro biochemical studies. By combining several approaches including covalently linking two FNR monomers using a flexible peptide linker, amino acid substitutions to promote dimerization, and removal of protease recognition sites to prevent proteolysis, a series of FNR variants which are potentially active in the presence of oxygen are constructed. Various in vivo and in vitro assays led to the identification of the construct (FNRD154A)2 which covalently links two copies of FNRD154A, an FNR variant that has greater dimerization capability, in tandem displays significantly improved transcription regulation and DNA binding to various FNR regulated promoters in the presence of O2. Circular Dichroism analysis showed that this variant maintains a similar secondary structure as that of native FNRD154A and in vivo transcription assay demonstrated that this protein retains other properties of the native FNR dimer including [4Fe〖-4S]〗 DEGREES(2+) cluster binding, oxygen sensing, and capability to support the anaerobic growth of E. coli. All these together led the conclusion that an FNR variant that retains structural and functional properties of native FNR has been constructed, but with significantly improved O2 stability. Thus, it has the potential to be widely used in various biochemical and structural studies of FNR in the presence of oxygen. In addition to the major project of molecular engineering of FNR protein, in this thesis, I also initiated the study of using metabolomics approaches to identify the cellular substrates of the multidrug efflux pump MdtEF. MdtEF is an important efflux pump in E. coli and its expression has been shown to be induced under a number of stressed conditions. It is thus proposed to have a general detoxification function in E. coli, but the cellular substrates it expels have not been identified. In this study we established and applied metabolite profiling on the wild type and ΔmdtEF E. coli strains and confirmed that indole red, a metabolic by-product formed during anaerobic respiration of nitrate, is one of the cellular substrates of MdtEF under anaerobic conditions. This study provides a general methodology to identify endogenous substrates of efflux pumps and contributes to the understanding of the physiological roles of multidrug efflux pumps in bacteria. DOI: 10.5353/th_b4961737 Subjects: Escherichia coli - Molecular aspects Transcription factors
Download or read book Molecular Engineering of the Escherichia Coli Global Transcription Factor FNR to Improve Its Stability to Oxygen written by Yue Shan and published by . This book was released on 2012 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Molecular Engineering of the Escherichia Coli Global Transcription Factor FNR to Improve Its Stability to Oxygen written by Yue Shan and published by . This book was released on 2012 with total page 268 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Mechanism for Oxygen Regulation Escherichia Coli Transcription Factor FNR written by Beth A. Lazazzera and published by . This book was released on 1995 with total page 380 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Functions and Physiological Significance of the N And C Terminal Regions of the Escherichia Coli Global Transcription Factor Fnr written by Qing Pan and published by Open Dissertation Press. This book was released on 2017-01-26 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Functions and Physiological Significance of the N- and C- Terminal Regions of the Escherichia Coli Global Transcription Factor FNR" by Qing, Pan, 潘庆, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: A facultative anaerobe such as Escherichia coli is able to switch between the aerobic and anaerobic modes of metabolism in response to O2 availability. This adaptation is primarily controlled by a global transcription regulator called FNR (fumarate nitrate reduction). The key property that allows FNR to act as an O2 responsive transcription factor is its capability to dimerize and being activated upon binding of an O2 labile [4Fe-4S]2+ cluster. Previous functional studies have largely focused on the regions of FNR analogous to CRP (cAMP receptor protein), a prototype CRP/FNR family protein which X-ray crystal structure has been resolved. However, E. coli FNR contains extra N- and C-terminal regions that are conserved among various FNR orthologs but are absent in CRP. The functions of these two regions have not been resolved. In this study, their functions and physiological significance to the O2 sensing capacity of FNR were systematically investigated. A three-alanine (3-Ala) scanning library on amino acid 2-19 and 236-250 of FNR was constructed and selective 3-Ala substitution mutants exhibited variable defects. These defects were found to be due to their impairment of intracellular FNR protein levels which was unique only among FNR mutations in these two regions. Introduction of 3-Ala substitution at the residues 239-244, resulting in LAQ239-241A3 and LAG242-244A3 respectively, caused an especially accelerated degradation and decrease of intracellular FNR proteins. These variants were found to be degraded by the ClpXP protease. Sequence alignment of FNR orthologs revealed a highly conserved "L239XXL242XG244" motif, and my experimental data further revealed that L239 and L242 were important residues and were responsible for the defects of LAQ239-241A3 and LAG242-244A3, respectively. Circular dichroism analysis revealed that introduction of LAQ239-241A3 caused conformational changes with a significant loss of secondary structures in FNR. These studies taken together suggest that the N- and C-terminal regions of FNR play an important role in mediating the intracellular protein level of FNR. My studies also specified the ClpXP signals as the N-terminal RR9-10 and C-terminal VA249-250, and indicated that VA249-250 is a more important site than RR9-10 in targeting FNR to proteolysis. The second topic of the thesis involves exploration of the regulatory mechanism of an anaerobically activated multidrug efflux pump MdtEF in E. coli. MdtEF is an important multidrug efflux pump that causes antibiotic resistance upon overexpression. Previous studies revealed that expression of MdtEF was significantly upregulated under anaerobic conditions, but its regulatory mechanism was unknown. In the current study, systematic analyses on the unusually long promoter region of the gadE-mdtEF operon which drives the expression of MdtEF were performed. It was found that unlike FNR, mdtEF was not regulated at post-translational level by proteolysis, but at transcriptional level through the promoter region of gadE. My study showed that anaerobic activation of mdtEF was mediated by the anaerobic regulator ArcA and nitrate responsive regulators NarL and NarP. Important promoter regions P3 and P1 were also identified. This study provides essential molecular basis for the upregulation of MdtEF in a host and clinically relevant conditions. DOI: 10.5353/th
Download or read book Oxygen Sensing in Escherichia Coli and the Role of Cyclic Regulation written by Dean Alexander Tolla and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This work examines the role of a cyclic genetic regulatory circuit acting as an environmental sensor. As a representative of this class of systems, we focus on the FNR regulatory circuit of Escherichia coli for which a large body of experimental evidence describing its molecular components and their individual interactions exists. However, until recently this information had never been integrated into a coherent model of the intact system. The FNR protein is an oxygen sensor that acts as a global transcription factor and when active modifies gene expression to adapt the cell to anaerobic growth. Our study provides strong support for an integrated model of FNR regulation that demonstrates self-consistency of a large body of experimental work, makes predictions of additional mutant behavior that are validated by experimental data, predicts the dynamics of the aerobic-to-anaerobic transition, provides estimates of active FNR in vivo, and makes it possible to predict in situ the dimerization rate of the transcription factor. We provide an original definition of what is meant by phenotype at the molecular level that is based on the concept of a "system design space". This definition allows us to rigorously characterize the regulatory phenotypes exhibited by the master transcription factor FNR. We identify 15 qualitatively-distinct phenotypes, analyze the alternatives, and compare their relative fitness. Our analysis reveals desirable and undesirable phenotypes as well as an optimal band of operation corresponding to the transition between aerobic and anaerobic environments. We analyze specific mutations and predict their associated phenotypic changes. By identifying the phenotypic repertoire our design space analysis provides a means to evaluate the evolutionary pressures that have shaped this system. Our application of the design space approach to the FNR network provides a detailed framework for characterizing other environmental sensing regulatory networks, and this approach is sufficiently general that it can be used to analyze the distinct behaviors presented by essentially any biochemical network. By examining a family of FNR models each with different cycling speeds but mathematically constrained to be otherwise equivalent, we are able to elucidate the relationship between the cycling rate and response time of the FNR circuit. We identify an optimum cycling rate for the FNR system. Our analysis of a currently uncharacterized double mutant argues in favor of these predicted results and provides a means to test our predictions in the laboratory. We examine the phenotype of increasing the number of fnr gene copies and show that the copy-number mutant exaggerates the predicted dynamics of active FNR induction. We provide an experimental method capable of capturing the dynamics of active FNR induction and repression, and predict its results for the wild-type, double mutant, and copy-number mutant.
Download or read book YeiL the Third Member of the CRP FNR Family of Global Transcription Factors in Escherichia Coli written by Sarah Louise Cookson and published by . This book was released on 2006 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Dissertation Abstracts International written by and published by . This book was released on 2001 with total page 834 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Understanding the Genome written by Editors of Scientific American and published by Hachette UK. This book was released on 2002-06-01 with total page 160 pages. Available in PDF, EPUB and Kindle. Book excerpt: Drawn from the pages of Scientific American and collected here for the first time, this work contains updated and condensed information, made accessible to a general popular science audience, on the subject of understanding the genome.
Download or read book Prokaryotic Metabolism and Physiology written by Byung Hong Kim and published by Cambridge University Press. This book was released on 2019-05-16 with total page 509 pages. Available in PDF, EPUB and Kindle. Book excerpt: Extensive and up-to-date review of key metabolic processes in bacteria and archaea and how metabolism is regulated under various conditions.
Download or read book Systems Metabolic Engineering written by Christoph Wittmann and published by Springer Science & Business Media. This book was released on 2012-06-15 with total page 391 pages. Available in PDF, EPUB and Kindle. Book excerpt: Systems Metabolic Engineering is changing the way microbial cell factories are designed and optimized for industrial production. Integrating systems biology and biotechnology with new concepts from synthetic biology enables the global analysis and engineering of microorganisms and bioprocesses at super efficiency and versatility otherwise not accessible. Without doubt, systems metabolic engineering is a major driver towards bio-based production of chemicals, materials and fuels from renewables and thus one of the core technologies of global green growth. In this book, Christoph Wittmann and Sang-Yup Lee have assembled the world leaders on systems metabolic engineering and cover the full story – from genomes and networks via discovery and design to industrial implementation practises. This book is a comprehensive resource for students and researchers from academia and industry interested in systems metabolic engineering. It provides us with the fundaments to targeted engineering of microbial cells for sustainable bio-production and stimulates those who are interested to enter this exiting research field.
Download or read book Escherichia Coli and Salmonella written by Frederick C. Neidhardt and published by . This book was released on 1996 with total page 2822 pages. Available in PDF, EPUB and Kindle. Book excerpt: This is the long–awaited second edition of an invaluable classic! Escherichia coli occupies a central role in contemporary molecular biology. It is the unicellular organism about which most is known – all molecular and cellular biologists will want a copy of this book. In 154 chapters, 250 expert authors and editors present the state of the art. Completely rewritten and restructured, the second edition offers a whole new approach to the subject.
Download or read book Regulation of Photosynthesis written by Eva-Mari Aro and published by Springer Science & Business Media. This book was released on 2006-04-11 with total page 624 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book covers the expression of photosynthesis related genes including regulation both at transcriptional and translational levels. It reviews biogenesis, turnover, and senescence of thylakoid pigment protein complexes and highlights some crucial regulatory steps in carbon metabolism.
Download or read book Microbial Cell Factories Engineering for Production of Biomolecules written by Vijai Singh and published by Academic Press. This book was released on 2021-02-13 with total page 490 pages. Available in PDF, EPUB and Kindle. Book excerpt: Microbial Cell Factories Engineering for Production of Biomolecules presents a compilation of chapters written by eminent scientists worldwide. Sections cover major tools and technologies for DNA synthesis, design of biosynthetic pathways, synthetic biology tools, biosensors, cell-free systems, computer-aided design, OMICS tools, CRISPR/Cas systems, and many more. Although it is not easy to find relevant information collated in a single volume, the book covers the production of a wide range of biomolecules from several MCFs, including Escherichia coli, Bacillus subtilis, Pseudomonas putida, Streptomyces, Corynebacterium, Cyanobacteria, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, and algae, among many others. This will be an excellent platform from which scientific knowledge can grow and widen in MCF engineering research for the production of biomolecules. Needless to say, the book is a valuable source of information not only for researchers designing cell factories, but also for students, metabolic engineers, synthetic biologists, genome engineers, industrialists, stakeholders and policymakers interested in harnessing the potential of MCFs in several fields. - Offers basic understanding and a clear picture of various MCFs - Explains several tools and technologies, including DNA synthesis, synthetic biology tools, genome editing, biosensors, computer-aided design, and OMICS tools, among others - Harnesses the potential of engineered MCFs to produce a wide range of biomolecules for industrial, therapeutic, pharmaceutical, nutraceutical and biotechnological applications - Highlights the advances, challenges, and future opportunities in designing MCFs
Download or read book Sustainable Development of Algal Biofuels in the United States written by National Research Council and published by National Academies Press. This book was released on 2013-01-18 with total page 247 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biofuels made from algae are gaining attention as a domestic source of renewable fuel. However, with current technologies, scaling up production of algal biofuels to meet even 5 percent of U.S. transportation fuel needs could create unsustainable demands for energy, water, and nutrient resources. Continued research and development could yield innovations to address these challenges, but determining if algal biofuel is a viable fuel alternative will involve comparing the environmental, economic and social impacts of algal biofuel production and use to those associated with petroleum-based fuels and other fuel sources. Sustainable Development of Algal Biofuels was produced at the request of the U.S. Department of Energy.
Download or read book The Science of Flavonoids written by Erich Grotewold and published by Springer Science & Business Media. This book was released on 2008 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: This is the only book of its kind to provide an overview of the science of flavonoids in plants.
Download or read book Flavins written by Eduardo Silva and published by Royal Society of Chemistry. This book was released on 2006 with total page 335 pages. Available in PDF, EPUB and Kindle. Book excerpt: Flavins and flavoproteins are a widely investigated and highly versatile group of compounds. Participation of these compounds in photochemistry and photobiology processes are of particular importance in the fields of biology, chemistry and medicine. Written by leading experts in the field each section of the book includes a historical overview of the subject, state of the art developments and future perspectives. Flavins: Photochemistry and Photobiology begins with the properties and applications of flavins, including their photochemistry in aqueous and organic solutions. Subsequent sections discuss riboflavin as a visible light sensitizer in the photo degradation of drugs, antiviral and antibacterial effects, the role of flavins in light induced toxicity and blue light initiated DNA repair by photolyase. Finally there are sections on the flavin based photoreceptors in plants, bacteria and eukaryotic photosynthetic flagelettes. This book brings together leading experts with a unique interdisciplinary emphasis, to provide an authoritative resource on flavins and their role in photochemistry and photobiology.
