Molecular Mechanisms Determining Bacterial Cell Shape

The insights gained from these studies will enable a wide range of applications spanning the identification of novel antibiotic targets and the optimization of biomaterials production by bacterial cells.

Author: Ti-Yu Lin

Publisher:

ISBN: OCLC:1237283520

Category:

Page: 238

View: 872

DOWNLOAD →

Bacteria exhibit a variety of shapes, including: cocci, bacilli, and spirochetes. Cell shape influences the spatial and temporal dynamics of processes that are essential for growth and replication in bacteria and is connected to pathogenicity and evasion of the mammalian immune system. Several bacterial cell-shape determinants have been proposed, including: 1) the cytoskeleton as an internal scaffolding, and 2) the peptidoglycan layer of the cell wall that resists osmotic pressure and maintains cell shape. However, some bacteria lack these subcellular components and yet retain distinct cellular shapes. This observation raises the question of whether bacteria use other shape-determining strategies and provides an opportunity to explore the biochemical evolution of cell shape across bacteria. The phospholipid membrane is another cellular structure that may regulate the shape of bacteria, and yet the impact of this cellular structure on bacterial morphology has been largely overlooked. Bacterial cell membranes consist of the three major families of phospholipids: phosphatidylethanolamine is zwitterionic, and phosphatidylglycerol and cardiolipin are anionic. The composition of cell membranes plays a fundamental role in bacterial cell biology. This dissertation describes how cardiolipin regulates cell morphology and influences bacterial adaptation to environmental stress. We observed that a cardiolipin deficiency in Rhodobacter sphaeroides changes the shape of cells and impairs biofilm formation. We demonstrated that cardiolipin participates in bacterial cell shape determination by regulating peptidoglycan precursor biosynthesis. In this dissertation, we also developed a new method for optimizing the production of recombinant proteins in Escherichia coli by engineering its cell shape. The insights gained from these studies will enable a wide range of applications spanning the identification of novel antibiotic targets and the optimization of biomaterials production by bacterial cells.

Exploring Mechanisms of Cell Shape Control in Helicobacter Pylori

Helicobacter pylori chronically infects over half of the global population and is associated with increased risk for gastric cancer, the third leading cause of cancer deaths worldwide.

Author: Kris Blair

Publisher:

ISBN: OCLC:1044763196

Category:

Page: 133

View: 331

DOWNLOAD →

Helicobacter pylori chronically infects over half of the global population and is associated with increased risk for gastric cancer, the third leading cause of cancer deaths worldwide. H. pylori resides in the gastric mucosa of the human stomach where it utilizes helical cell shape to efficiently colonize the epithelium. However, the underlying molecular mechanisms that H. pylori bacteria use to generate and control their helical cell shape are not known. In this dissertation, I took a multidisciplinary approach using genetic, biochemical, and proteomic approaches to discover and characterize protein interactions among known cell shape determining (Csd) proteins to gain mechanistic insight into helical cell shape determination. We discovered that the non-enzymatic protein Csd5 comprises a multi-protein “Shapeosome” complex that resides in the cytoplasmic membrane where it interacts with ATP synthase, a cytoplasmic peptidoglycan synthase (MurF), a bacterial cytoskeleton protein (CcmA), and the bacterial cell wall. We also performed structure-function studies of Csd5 and the peptidoglycan hydrolase Csd4 to identify functional domains required for protein function. Finally, we show that H. pylori isolates obtained from a chronically infected host exhibit significant heterogeneity in cell shape and find that mutations in cell shape determining genes contribute to this cell shape diversity.

The Bacterial Cell Coupling between Growth Nucleoid Replication Cell Division and Shape Volume 2

The 1st volume of our Research Topic "The Bacterial Cell: Coupling between Growth, Nucleoid Replication, Cell Division and Shape” was published as an eBook in May 2016 (see: http://journal.frontiersin.org/researchtopic/2905/the-bacterial ...

Author: Ariel Amir

Publisher: Frontiers Media SA

ISBN: 9782889631568

Category:

Page: 246

View: 279

DOWNLOAD →

The 1st volume of our Research Topic "The Bacterial Cell: Coupling between Growth, Nucleoid Replication, Cell Division and Shape” was published as an eBook in May 2016 (see: http://journal.frontiersin.org/researchtopic/2905/the-bacterial-cell-coupling-between-growth-nucleoid-replication-cell-division-and-shape). As a sign of growing interest to the topic, two workshops followed the same year: "Stochasticity in the Cell Cycle" in Jerusalem (Israel) by the Hebrew University’s Institute of Advanced Studies and EMBO's "Cell Size Regulation" in Joachimsthal (Germany). From the time of launching the first edition, several new groups have entered the field, and many established groups have made significant advances using state-of-the-art microscopy and microfluidics. Combining these approaches with the techniques pioneered by quantitative microbiologists decades ago, these approaches have provided remarkable amounts of numerical data. Most of these data needed yet to be put into a broader theoretical perspective. Moreover, the molecular mechanisms governing coordination and progression of the main bacterial cell cycle processes have remained largely unknown. These outstanding fundamental questions and the growing interest to the field motivated us to launch the next volume titled “The Bacterial Cell: Coupling between Growth, Nucleoid Replication, Cell Division, and Shape, Volume 2” shortly after completion of the first edition in October 2016. The issue contains 17 contributions from a diverse array of scientists whose field of study spans microbiology, biochemistry, genetics, experimental and theoretical biophysics. The specific questions addressed in the issue include: What triggers initiation of chromosome replication? How is cell division coordinated with replication both spatially and temporally? How is cell size controlled and linked to the rate of mass growth? What role plays physical organization of the chromosomes in their segregation and in regulation of cell division? The publications covering these questions are divided into three topical areas: 1) Cell Cycle Regulation, 2) Growth and Division, and 3) Nucleoid Structure and Replication. New ideas and techniques put forward in these articles bring us closer to understand these fundamental cellular processes, but the quest to resolve them is far from being complete. Plans for the next edition are under way along with further meetings and workshops, e.g., an EMBO Workshop on Bacterial cell biophysics: DNA replication, growth, division, size and shape in Ein Gedi (Israel), May 2020. We hope that via such interdisciplinary exchange of ideas we will come closer to answering the above-mentioned complex and multifaceted questions.

The Bacterial Cell Coupling between Growth Nucleoid Replication Cell Division and Shape

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.

Author: Arieh Zaritsky

Publisher: Frontiers Media SA

ISBN: 9782889198177

Category:

Page:

View: 721

DOWNLOAD →

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.

The Molecular Architecture of Peptidoglycan and the Rate of Peptidoglycan Biosynthesis Relative to Cytoplasmic Expansion Play Essential Roles in Determining Bacterial Size and Shape

Cell size and shape are cell-scale properties many orders of magnitude larger than individual proteins, and it is largely unknown how cells coordinate the growth of such reproducible, uniform, large-scale structures.

Author: Leigh Kathleen Harris

Publisher:

ISBN: OCLC:951370937

Category:

Page:

View: 784

DOWNLOAD →

Cell size and shape are cell-scale properties many orders of magnitude larger than individual proteins, and it is largely unknown how cells coordinate the growth of such reproducible, uniform, large-scale structures. In this thesis I present several lines of inquiry to address what determines cell size and shape in several bacterial species. First, I investigate a point mutation in the morphogenetic protein MreB and find that the resulting variable-width phenotype reveals a role for surface area to volume ratio (SA/V) maintenance in determining cell shape and size. Next, I present a general mechanism for SA/V homeostasis in bacteria and demonstrate that this mechanism underlies size determination in bacteria, resolving many long-standing questions about the link between growth rate and cell size. Finally, I develop a novel method for detecting the underlying architecture of peptidoglycan that can be widely implemented to understand how genetic and chemical perturbations acting locally give rise to cell-scale morphological defects.

Bacterial Cell Wall Structure and Dynamics

This collection of articles aims to contribute to new understandings of bacterial cell wall structure and dynamics.

Author: Tobias Dörr

Publisher: Frontiers Media SA

ISBN: 9782889631520

Category:

Page:

View: 795

DOWNLOAD →

Bacterial cells are encased in a cell wall, which is required to maintain cell shape and to confer physical strength to the cell. The cell wall allows bacteria to cope with osmotic and environmental challenges and to secure cell integrity during all stages of bacterial growth and propagation, and thus has to be sufficiently rigid. Moreover, to accommodate growth processes, the cell wall at the same time has to be a highly dynamic structure: During cell enlargement, division, and differentiation, bacteria continuously remodel, degrade, and resynthesize their cell wall, but pivotally need to assure cell integrity during these processes. Finally, the cell wall is also adjusted according to both environmental constraints and metabolic requirements. However, how exactly this is achieved is not fully understood. The major structural component of the bacterial cell wall is peptidoglycan (PG), a mesh-like polymer of glycan chains interlinked by short-chain peptides, constituting a net-like macromolecular structure that has historically also termed murein or murein sacculus. Although the basic structure of PG is conserved among bacteria, considerable variations occur regarding cross-bridging, modifications, and attachments. Moreover, different structural arrangements of the cell envelope exist within bacteria: a thin PG layer sandwiched between an inner and outer membrane is present in Gram-negative bacteria, and a thick PG layer decorated with secondary glycopolymers including teichoic acids, is present in Gram-positive bacteria. Furthermore, even more complex envelope structures exist, such as those found in mycobacteria. Crucially, all bacteria possess a multitude of often redundant lytic enzymes, termed “autolysins”, and other cell wall modifying and synthesizing enzymes, allowing to degrade and rebuild the various structures covering the cells. However, how cell wall turnover and cell wall biosynthesis are coordinated during different stages of bacterial growth is currently unclear. The mechanisms that prevent cell lysis during these processes are also unclear. This Research Topic focuses on the dynamics of the bacterial cell wall, its modifications, and structural rearrangements during cell growth and differentiation. It pays particular attention to the turnover of PG, its breakdown and recycling, as well as the regulation of these processes. Other structures, for example, secondary polymers such as teichoic acids, which are dynamically changed during bacterial growth and differentiation, are also covered. In recent years, our view on the bacterial cell envelope has undergone a dramatic change that challenged old models of cell wall structure, biosynthesis, and turnover. This collection of articles aims to contribute to new understandings of bacterial cell wall structure and dynamics.

From Atoms to Cells

In this dissertation work, I will further explore core molecular players that regulate bacterial physiology in different growth conditions, with the focus on the essential cell-shape determining protein, MreB.

Author: Handuo Shi

Publisher:

ISBN: OCLC:1095948802

Category:

Page:

View: 672

DOWNLOAD →

Bacterial cells in nature constantly encounter stresses from complex and fluctuating external environments that disrupt growth. In stressed conditions, cells gradually enter a growth-arrested state (stationary phase) with altered cell size and heterogeneous transcriptional reprogramming. My PhD research has focused on important connections among cell shape, growth, and fitness during those transitional processes. While most studies on bacterial cells have focused on fast-growing, exponential-phase cells, stationary phase presents unique challenges for cell growth due to accumulated waste and limited nutrients. Cells slow down growth in stationary phase, and at the same time actively alter their sizes. In the model bacterium Escherichia coli, cells reduce their sizes by approximately 70% when entering stationary phase. Such transition is precisely regulated by a consortium of proteins determining cell shape, with the main player being an actin homolog, MreB. MreB localizes to cell periphery and actively tunes its localization based on geometric cues. The localization of MreB directly guides the synthesis of new cell wall, which eventually dictates cell morphology. In the first chapter of this dissertation work, I will provide a thorough overview of the interplay between protein localization, cell morphology, and growth. Throughout my PhD study, I sought to link the dynamics of bacterial growth and shape by perturbing cells away from steady-state growth and observing the molecular- and cellular-level physiological changes during those processes. In Chapters 2 and 3, I present two concrete examples of how unbalanced growth impact cell viability. Chapter 2 focuses on a mutant protein involved in a lipid transporting pathway in E. coli. While fast-growing cells are virtually unaffected by this mutation, half of the mutant cells die upon entering stationary phase, due to disrupted lipid homeostasis. Similarly, in Chapter 3, I present a set of Bacillus subtilis strains with tunable knockdowns in each essential genes. Mild knockdowns do not substantially alter growth in exponential phase, but inhibit a subpopulation of stationary-phase cells from resuming growth once they are presented with fresh nutrient. In this dissertation work, I will further explore core molecular players that regulate bacterial physiology in different growth conditions, with the focus on the essential cell-shape determining protein, MreB. In Chapter 4, I present the process of isolating and screening a library of E. coli MreB mutants that exhibit a wide range of shapes, and further demonstrate how the changes in cell shape can be related to a number of physiological phenotypes in a cell-size-dependent manner. As an example, MreB localization pattern is growth-dependent, which drives changes of cell shape at different growth phases. Given the growth-dependent MreB localization, I then interrogate the atomic-level molecular changes that drive changes in such localization patterns, using molecular dynamics simulations. In Chapter 5, I combine experimental measurements of cell shape and simulations of MreB to show that cells regulate MreB filament conformation and eventually cell shape through the relative concentration of an MreB-binding protein, RodZ. In Chapter 6, I further extend the molecular dynamics simulations of MreB to a larger scale. Applying the simulations to the MreB mutants I identified previously, I show that those cell-shape mutants have altered MreB filament conformations, highlighting the molecular basis of cell shape regulation and determination. Finally, to tackle the question of why cells actively tune shape at different growth stages, I systematically quantify changes in cellular dimensions across fluctuating environments in Chapter 7, and develop an ODE model explaining the trade-offs of cellular surface area and volume across nutrient conditions, revealing a global regulation of cellular physiology in fluctuating environments. All the projects above involve precise and rapid quantification of single-cell morphology across many strains and conditions. This has been made possible, partially by my implementation of an open-source high-throughput imaging platform that automates screening of thousands of strains in several hours. In Chapter 8, I will introduce the basic setup of this imaging platform and showcase its applications. Taken together, my PhD work has centered on the question of how cells dynamically regulate their morphology and physiology when environments fluctuate. I have combined single-cell imaging, genetic engineering, modeling, and simulations to tackle this question, and have highlighted how cells handle such challenges at multiple length scales.

Molecules in Time and Space

Molecules in Time and Space reviews the data on the formation of subcellular patterns or structures in bacteria, presents observations and hypotheses on the establishment and the maintenance of cell shape, and on the organization of genetic ...

Author: Miguel Vicente

Publisher: Springer Science & Business Media

ISBN: 9780306485787

Category: Science

Page: 275

View: 252

DOWNLOAD →

During the last decade a wealth of new data has arisen from the use of new fluorescent labelling techniques and the sequencing of whole microbial genomes. One important conclusion from these data is that bacterial cells are much more structured than previously thought. The wall and the outer membrane contain topological domains, some proteins localize or move in specific patterns inside the cells, and some genes appear clustered in the chromosome and form conserved evolutionary units. Many of these structures are related to the cell cycle and to the process of cell morphogenesis, two processes that are themselves related to each other. From these observations the dcw gene cluster appears as a phylogenetic trait that is mainly conserved in bacilli. Molecules in Time and Space reviews the data on the formation of subcellular patterns or structures in bacteria, presents observations and hypotheses on the establishment and the maintenance of cell shape, and on the organization of genetic information in the chromosome.

The Relationship Between Bacterial Stress Responses and Cell Shape

How do we respond to our environment? How much does our environment drive our very form, our physical shape? These are fundamental questions that biology must grapple with on all scales of life.

Author: Amanda Miguel

Publisher:

ISBN: OCLC:1050346863

Category:

Page:

View: 211

DOWNLOAD →

How do we respond to our environment? How much does our environment drive our very form, our physical shape? These are fundamental questions that biology must grapple with on all scales of life. Understanding how the shape of an organism is formed, how it is maintained, and how it interacts with external stresses is key to understanding its strengths, limitations, and what it could become. My thesis examines a portion of the complex relationship between organism and environment by studying the physical changes bacteria undergo in both friendly and hostile environments, using the well-studied model organism Escherichia coli. In Chapter one, I will introduce the relevant bacterial physiology necessary for understanding cellular growth and division. I will also define the structural and molecular determinants of cell shape in bacteria, and then examine what is known about how environmental conditions impact cell shape. In chapter two, I will start at the protein level, examining a key protein in the cell division machinery, FtsZ, and its relationship to inhibitors that induce cell death. In chapter three, four and five, I will move to larger cellular structures and discuss how genetic or external perturbations effect the integrity of the bacterial cell wall, the key macromolecule in cell shape determination. In chapter six, I will move from single molecules to molecular networks: I will examine a stress response pathway, the Rcs pathway, and its connection with perturbations to cell width and its influence on cell length. I will show how these observations again relate to the interaction and regulation of the cell wall synthesis machinery governed by cytoskeletal proteins MreB and FtsZ, respectively. In chapter seven, I move finally from single cell behavior to the behavior of cell populations under stress. Specifically, I will utilize previous work examining the relationship between increased cell size and fitness as the basis for generating a cell shape mutant library. I then used this library in a high-throughput chemical genomics screen to further characterize the types of stressful environments in which size plays a key role in fitness, revealing evolutionary pressures on cell shape. I will conclude in chapter eight by re-examining the fundamental questions posed at the beginning of this chapter and reflect on how this work moves us a step forward in illuminating the complex and varied ways in which organisms interact and are changed by their environment.

The Assembly and Interactions of MreB in the Maintenance of Cell Shape in Caulobacter Crescentus

Given that MreC and Pbp2 do not normally colocalize considerably with MreB in wild type cells (Chapter 2), ... the precise molecular mechanism by which MreB contributes to cell wall synthesis and the determination of proper cell shape ...

Author:

Publisher: Stanford University

ISBN: STANFORD:bg008yn0701

Category:

Page:

View: 690

DOWNLOAD →

This work focuses on the mechanism by which MreB contributes to the maintenance of cell shape in the gram-negative alpha-proteobacterium Caulobacter crescentus. The gene mreB encodes a protein that resembles actin, a eukaryotic cytoskeletal protein. Previously, it was shown that mreB is required to maintain a rod-like shape and localizes to a helical pattern near the cytoplasmic membrane. Here, we show that MreB is associated with regions of active growth in Caulobacter, as mutant strains that mislocalize MreB to the cell poles direct new growth at or near the poles. We present evidence to suggest that MreB contributes to the determination of proper length, width, and curvature through partially distinct mechanisms. The determination of proper width involves the essential proteins MreC and Pbp2, which are encoded in the mreB operon. While MreB and MreC are both required to position the cell wall transpeptidase Pbp2 along the lateral sidewalls and away from midcell, the two do not colocalize and each can maintain its localization in the absence of the other. When MreB is mislocalized to the poles, MreC and Pbp2 do not follow. These data argue against the idea that MreB provides a scaffold-like structure to localize enzymes that directly modify the cell wall. The determination of proper curvature, involves the intermediate filament-like protein, Crescentin. We identify a putative binding site on MreB for Crescentin or other curvature-mediating factors. We also show that the extent to which the subcellular localization of MreB changes over the cell cycle is correlated with cell size, indicating that MreB is involved in the coordination between elongation and division. In addition, we show that in vitro purified MreB spontaneously forms very stable polymers in the presence or absence of nucleotide. These polymers are globular or amorphous and only filamentous when placed on a highly positively charged surface of Poly-L-lysine. These in vitro data suggest that MreB is likely to be regulated at the disassembly step in the cell and that the cellular environment may influence the structure of MreB polymers. Lastly, we present biochemical evidence to support the existence of a disassembly factor in cytoplasmic Caulobacter extract. Together our data suggest that the maintenance of the crescent-rod cell shape in Caulobacter is the result of a complicated balance between MreB's dynamic subcellular localization, polymeric structure, and communication with cellular components.