Download or read book Spatial Biology of Transcription and Translation in Live E Coli Using Super resolution Fluorescence Microscopy written by and published by . This book was released on 2014 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Due to the lack of membrane bound organelles in bacteria, both transcription and translation take place inside the same compartment. The objective of this thesis is to understand the fundamental, as yet unanswered question of where transcription and translation take place inside bacteria. I have focused on the bacterium E. coli, as it is the best characterized organism, both molecularly and physiologically. I have used superresolution microscopy techniques in live E. coli cells to study the spatio-temporal organization of transcription and translation machinery. By localizing individual molecules with 30 nm scale accuracy, I was able to resolve the spatial distribution and dynamics of RNA polymerase (RNAP) and of the ribosomes. The spatial distribution of RNAP mimicked the spatial distribution of the chromosomal DNA, imaged by DNA-stains and superresolution imaging of DNA-binding protein HU. Ribosomes on the other hand were found to be segregated from the chromosomal DNA. By relating the position, dynamics and copy numbers of ribosomes and RNAPs, I was able to conclude that most translation in E. coli is taking place away from the nucleoid in the ribosome-rich regions without transcriptional coupling. This finding is consistent with the timescale of transcription and life time of mRNA. Based on the comparison of spatio-temporal dynamics of chromosomal loci and RNA polymerase, I was able to infer that transcription of protein genes is scattered more or less uniformly throughout the nucleoid. Here the mRNA is translated co-transcriptionally. Once the transcription is terminated, the mRNA diffuses out into the ribosomes-rich regions of the end cap of cytoplasm and is translated until it is degraded by the degradosome machinery. In sharp contrast, transcription of the ribosomal RNA seems to be localized to the periphery of the chromosomal DNA. Such placement of rRNA transcription at the interface between the dense nucleoids and the ribosome-rich regions may be functionally important for efficient ribosome assembly. I also found significant numbers of transcribing RNAPs and translating ribosomes near the cytoplasmic membrane. This provides circumstantial evidence for "transertion hypothesis", which posits the co-transcriptional translation of membrane proteins, which are evidently inserted into the membrane by the Sec-machinery.
Download or read book Functional Mapping of the Components of the E Coli Translational Machinery written by Sonisilpa Mohapatra and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial cells generally lack membrane-bound organelles. Therefore, traditionally they were viewed as bags of freely diffusing macromolecules devoid of any internal organization. The development in imaging techniques in the last two decades has enabled better visualization of bacterial cells and led to careful investigation of their internal organization. Consequently, they are now shown to have an intricate internal organization of various proteins in their cytoplasmic space despite the absence of membrane boundaries. The spatiotemporal control of various cellular processes in bacteria is possibly regulated by this internal organization. Most cellular processes are regulated by complex networks of intermolecular interactions. A systematic study quantifying the correlation in spatial organization of various biomolecules might help to unravel their underlying interactions. A positive correlation might suggest binding to a common site or common sites of production, degradation or action. A negative correlation might suggest a mechanism that sequesters the molecules from each other. A commonly used method to quantify linear correlation between images of two different species is the Pearson Correlation Coefficient (PCC). PCC however fails both qualitatively and quantitatively in spherocylindrical cells such as E. coli. A modified PCC (MPCC) is proposed that corrects this issue by using a proper reference matrix for spherocylindrical cells (Chapter 2). The application of MPCC to experimental spatial distributions of RNA Polymerase and HU is demonstrated. A significantly positive correlation coefficient was obtained for the spatial distribution of the two DNA binding proteins. The MPCC concept could be generalizable to other cell shapes and can be used for both widefield and superresolution images. Translation is one of the most important cellular process. Ribosomes are the central component of translation engaged in protein synthesis from mRNA. Optimal usage of ribosomes is dependent on their spatial organization. In this dissertation, the spatial and temporal organization of translation and a translation elongation protein, Elongation Factor P (EF-P) was investigated in live E. coli cells using superresolution fluorescence microscopy. In rapidly growing E. coli and B. subtilis, strong nucleoid - ribosome segregation is observed. The picture of spatial organization in slowly growing C. crescentus is however completely different. The effect of growth rate on the segregation of ribosomes and nucleoids in E. coli is therefore investigated using two color superresolution fluorescence imaging in Chapter 3. MPCC was used to quantify the degree of segregation between spatial distribution of ribosomes and DNA in E. coli in the two different growth conditions. Slowly growing E. coli showed significant segregation of ribosomes from DNA, unlike slowly growing C. crescentus. Imaged ribosomes were classified as translating and non-translating based on their diffusive trajectories. The fraction of ribosomes engaged in translation in the two growth conditions was quantified. A small but significant reduction of translating ribosomes was observed in slowly growing cells. Simulated spatial distributions in spherocylinders of dimensions same as E. coli that best fit experimental spatial distributions of translating and non-translating ribosomes were used to obtain a cellular map of ribosomes based on their biological functions. Based on the functional map of translating and non-translating ribosomes, it was concluded that a "circulation model" of ribosomes evidently applies to E. coli in all growth conditions. In cells, the translation elongation is discontinuous, often suffering from periods of pauses. The cells have mechanisms to alleviate these pauses, irrespective of the reason for pausing. EF-P is a translation factor that alleviates pausing during translation of polyproline motifs. The spatial distribution and ribosome binding dynamics of EF-P were investigated in Chapter 4 to understand details of mechanism of interaction of EF-P with ribosomes during the translation elongation process. By localizing individual EF-P molecules, the spatial distribution of EF-P was shown to mimic the "three peaked" ribosome distribution in the cells. Photoactivated Localization Microscopy combined with single particle tracking (sptPALM) was employed to distinguish and quantify the different diffusive states of EF-P. The "slow" diffusing EF-P molecules are associated with translating ribosomes whereas the "fast" diffusing ones are EF-P copies searching for a potential binding site on ribosomes. Nearly 30% of EF-Ps are associated with translating ribosomes, implying 1500-6000 EF-P/ribosome complexes in a cell at any given time. This is significantly larger than the estimated 280 pausing events due to polyproline motifs being translated in the cell, suggesting interrogation of ribosomes by EF-P more frequently than the number of pausing events. In addition, faster imaging rates enabled measurement of binding/unbinding timescales of EF-P interactions with ribosomes (Chapter 4). Overall, the reported works explore the synergism of interactions between different components of translation and their spatiotemporal organization. The famous biologist Jacques Monod noted "What was true for the E. coli would be true for the elephant". A better understanding of the intracellular spatial organization of bacterial cells could elucidate the mechanisms that underlie spatial order in higher organisms
Download or read book Single cell Detection of Antimicrobial Peptide s Attack on Live E Coli by Super resolution Fluorescence Microscopy written by Yanyu Zhu and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The era of multidrug-resistant bacterial infections necessitates the development of new antimicrobial agents. Evolutionarily conserved antimicrobial peptides (AMPs) are promising prototypes for designing new antimicrobial agents in this context. We combined single-cell, real-time fluorescence microscopy and super-resolution, single-particle tracking to study the novel effects of the cationic human AMP LL-37 on the E. coli cytoplasm. After membrane permeabilization, approximately 108 cationic LL-37 copies flood the cell and the chromosomal DNA becomes rigidified on a length scale of ~30 nm. The diffusive motion of ribosomes, DNA binding protein HU and the non-endogenous protein Kaede also decreased. Much of the LL-37 remains bound within the cell after extensive rinsing with fresh growth medium, and growth never recovers. Our results suggest that the high concentration of absorbed polycationic peptides form a dense network of noncovalent, electrostatic "pseudo-crosslinks" within the chromosomal DNA and among 70S-polysomes, thus rigidifying bacterial cytoplasm. The results indicate that the damage of AMPs to bacterial cells goes far beyond the membrane permeabilization and might help explain why bacteria develop resistance very slowly to AMPs. We further extended the method to study other AMPs and synthetic polymers. We also applied super-resolution fluorescence microscopy to study the spatial distributions and diffusive properties of key components of the transcription-translation machinery in stationary phase E. coli cells. Compared to exponential phase, the chromosomal DNA in stationary phase is locally more rigid but remains sufficiently permeable to RNAP and to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. This description contrasts with the usual picture of a rigid stationary phase cytoplasm with DNA highly condensed.
Download or read book The Bacterial Cell Coupling between Growth Nucleoid Replication Cell Division and Shape written by Arieh Zaritsky and published by Frontiers Media SA. This book was released on 2016-05-02 with total page 326 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial Physiology was inaugurated as a discipline by the seminal research of Maaløe, Schaechter and Kjeldgaard published in 1958. Their work clarified the relationship between cell composition and growth rate and led to unravel the temporal coupling between chromosome replication and the subsequent cell division by Helmstetter et al. a decade later. Now, after half a century this field has become a major research direction that attracts interest of many scientists from different disciplines. The outstanding question how the most basic cellular processes - mass growth, chromosome replication and cell division - are inter-coordinated in both space and time is still unresolved at the molecular level. Several particularly pertinent questions that are intensively studied follow: (a) what is the primary signal to place the Z-ring precisely between the two replicating and segregating nucleoids? (b) Is this coupling related to the structure and position of the nucleoid itself? (c) How does a bacterium determine and maintain its shape and dimensions? Possible answers include gene expression-based mechanisms, self-organization of protein assemblies and physical principles such as micro-phase separations by excluded volume interactions, diffusion ratchets and membrane stress or curvature. The relationships between biochemical reactions and physical forces are yet to be conceived and discovered. This e-book discusses the above mentioned and related questions. The book also serves as an important depository for state-of-the-art technologies, methods, theoretical simulations and innovative ideas and hypotheses for future testing. Integrating the information gained from various angles will likely help decipher how a relatively simple cell such as a bacterium incorporates its multitude of pathways and processes into a highly efficient self-organized system. The knowledge may be helpful in the ambition to artificially reconstruct a simple living system and to develop new antibacterial drugs.
Download or read book Single molecule Techniques written by Paul R. Selvin and published by CSHL Press. This book was released on 2008 with total page 511 pages. Available in PDF, EPUB and Kindle. Book excerpt: Geared towards research scientists in structural and molecular biology, biochemistry, and biophysics, this manual will be useful to all who are interested in observing, manipulating and elucidating the molecular mechanisms and discrete properties of macromolecules.
Download or read book Molecular Biology of the Cell written by and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book The BAM Complex written by Susan Buchanan and published by Humana. This book was released on 2015-10-02 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume is comprised of a collection of experimental protocols for common techniques and strategies used to study the biogenesis of b-barrel outer membrane proteins in Gram-negative bacteria. The BAM Complex: Methods and Protocols guides readers through methods on the function of the BAM complex, the roles played by each of the individual components, the expression and purification of the components, crystallization and structure determination of the components, and how the individual Bam components may assemble into a functional complex. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, The BAM Complex: Methods and Protocols will serve as an invaluable reference for those interested in studying the BAM complex.
Download or read book Organelles Advances in Research and Application 2013 Edition written by and published by ScholarlyEditions. This book was released on 2013-06-21 with total page 279 pages. Available in PDF, EPUB and Kindle. Book excerpt: Organelles—Advances in Research and Application: 2013 Edition is a ScholarlyEditions™ book that delivers timely, authoritative, and comprehensive information about Ribosomes. The editors have built Organelles—Advances in Research and Application: 2013 Edition on the vast information databases of ScholarlyNews.™ You can expect the information about Ribosomes in this book to be deeper than what you can access anywhere else, as well as consistently reliable, authoritative, informed, and relevant. The content of Organelles—Advances in Research and Application: 2013 Edition has been produced by the world’s leading scientists, engineers, analysts, research institutions, and companies. All of the content is from peer-reviewed sources, and all of it is written, assembled, and edited by the editors at ScholarlyEditions™ and available exclusively from us. You now have a source you can cite with authority, confidence, and credibility. More information is available at http://www.ScholarlyEditions.com/.
Download or read book Super Resolution Microscopy written by Holger Erfle and published by Humana Press. This book was released on 2017-09-19 with total page 262 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume presents current advanced technologies and methods used in super-resolution microscopy. The chapters in this book cover a wide range of topics such as introducing super-resolution microscopy into a core facility; two-photon STED microscopy for nanoscale imaging of neural morphology in vivo; correlative SIM-STORM microscopy; two-color single-molecule tracking in live cells; and correlative single molecule localization microscopy and confocal microscopy. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Super-Resolution Microscopy: Methods and Protocols is a valuable resource for both established and novel researchers and users in this field.
Download or read book Handbook of Single Molecule Biophysics written by Peter Hinterdorfer and published by Springer Science & Business Media. This book was released on 2009-12-24 with total page 634 pages. Available in PDF, EPUB and Kindle. Book excerpt: This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.
Download or read book Oxidative Stress and Chronic Degenerative Diseases written by Jose Antonio Morales-Gonzalez and published by BoD – Books on Demand. This book was released on 2013-05-22 with total page 516 pages. Available in PDF, EPUB and Kindle. Book excerpt: This work responds to the need to find, in a sole document, the affect of oxidative stress at different levels, as well as treatment with antioxidants to revert and diminish the damage. Oxidative Stress and Chronic Degenerative Diseases - a Role for Antioxidants is written for health professionals by researchers at diverse educative institutions (Mexico, Brazil, USA, Spain, Australia, and Slovenia). I would like to underscore that of the 19 chapters, 14 are by Mexican researchers, which demonstrates the commitment of Mexican institutions to academic life and to the prevention and treatment of chronic degenerative diseases.
Download or read book Quantitative Imaging in Cell Biology written by and published by Academic Press. This book was released on 2014-06-25 with total page 609 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new volume, number 123, of Methods in Cell Biology looks at methods for quantitative imaging in cell biology. It covers both theoretical and practical aspects of using optical fluorescence microscopy and image analysis techniques for quantitative applications. The introductory chapters cover fundamental concepts and techniques important for obtaining accurate and precise quantitative data from imaging systems. These chapters address how choice of microscope, fluorophores, and digital detector impact the quality of quantitative data, and include step-by-step protocols for capturing and analyzing quantitative images. Common quantitative applications, including co-localization, ratiometric imaging, and counting molecules, are covered in detail. Practical chapters cover topics critical to getting the most out of your imaging system, from microscope maintenance to creating standardized samples for measuring resolution. Later chapters cover recent advances in quantitative imaging techniques, including super-resolution and light sheet microscopy. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come. Covers sections on model systems and functional studies, imaging-based approaches and emerging studies Chapters are written by experts in the field Cutting-edge material
Download or read book Bacterial Regulatory RNA written by Kenneth C. Keiler and published by Humana Press. This book was released on 2012-06-28 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The discovery of wide-spread RNA-based regulation in bacteria has led to new evaluations of the importance of bacterial regulatory RNA in every aspect of bacterial physiology. In Bacteria Regulatory RNA: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study bacterial regulatory RNA. These include methods and techniques to identify regulatory RNAs, characterizing the function and expression of regulatory RNAs in bacterial cells, RNA structure prediction, and interactions between regulatory RNAs and proteins. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Bacteria Regulatory RNA: Methods and Protocols seeks to aid scientists in the further study of bacterial regulatory RNA.
Download or read book Quantitative Bioimaging written by Raimund J. Ober and published by CRC Press. This book was released on 2020-12-15 with total page 693 pages. Available in PDF, EPUB and Kindle. Book excerpt: Quantitative bioimaging is a broad interdisciplinary field that exploits tools from biology, chemistry, optics, and statistical data analysis for the design and implementation of investigations of biological processes. Instead of adopting the traditional approach of focusing on just one of the component disciplines, this textbook provides a unique introduction to quantitative bioimaging that presents all of the disciplines in an integrated manner. The wide range of topics covered include basic concepts in molecular and cellular biology, relevant aspects of antibody technology, instrumentation and experimental design in fluorescence microscopy, introductory geometrical optics and diffraction theory, and parameter estimation and information theory for the analysis of stochastic data. Key Features: Comprises four parts, the first of which provides an overview of the topics that are developed from fundamental principles to more advanced levels in the other parts. Presents in the second part an in-depth introduction to the relevant background in molecular and cellular biology and in physical chemistry, which should be particularly useful for students without a formal background in these subjects. Provides in the third part a detailed treatment of microscopy techniques and optics, again starting from basic principles. Introduces in the fourth part modern statistical approaches to the determination of parameters of interest from microscopy data, in particular data generated by single molecule microscopy experiments. Uses two topics related to protein trafficking (transferrin trafficking and FcRn-mediated antibody trafficking) throughout the text to motivate and illustrate microscopy techniques. An online appendix providing the background and derivations for various mathematical results presented or used in the text is available at http://www.routledge.com/9781138598980.
Download or read book Protein Export and Secretion Among Bacterial Pathogens written by Sophie Bleves and published by Frontiers Media SA. This book was released on 2020-02-27 with total page 198 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Antimicrobial Peptides written by Katsumi Matsuzaki and published by Springer. This book was released on 2019-04-12 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents an overview of antimicrobial peptides (AMPs), their mechanisms of antimicrobial action, other activities, and various problems that must still be overcome regarding their clinical application. Divided into four major parts, the book begins with a general overview of AMPs (Part I), and subsequently discusses the various mechanisms of antimicrobial action and methods for researching them (Part 2). It then addresses a range of activities other than antimicrobial action, such as cell penetration, antisepsis, anticancer, and immunomodulatory activities (Part 3), and explores the prospects of clinical application from various standpoints such as the selective toxicity, design, and discovery of AMPs (Part 4). A huge number of AMPs have been discovered in plants, insects, and vertebrates including humans, and constitute host defense systems against invading pathogenic microorganisms. Consequently, many attempts have been made to utilize AMPs as antibiotics. AMPs could help to solve the urgent problem of drug-resistant bacteria, and are also promising with regard to sepsis and cancer therapy. Gathering a wealth of information, this book will be a bible for all those seeking to develop antibiotics, anti-sepsis, or anticancer agents based on AMPs.
Download or read book Cell Free Gene Expression written by Ashty S. Karim and published by Humana. This book was released on 2022-01-06 with total page 435 pages. Available in PDF, EPUB and Kindle. Book excerpt: This detailed volume explores perspectives and methods using cell-free expression (CFE) to enable next-generation synthetic biology applications. The first section focuses on tools for CFE systems, including a primer on DNA handling and reproducibility, as well as methods for cell extract preparation from diverse organisms and enabling high-throughput cell-free experimentation. The second section provides an array of applications for CFE systems, such as metabolic engineering, membrane-based and encapsulated CFE, cell-free sensing and detection, and educational kits. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Cell‐Free Gene Expression: Methods and Protocols serves as an ideal guide for researchers seeking technical methods to current aspects of CFE and related applications.
