Bacterial Chromosome Organization and Segregation

If fully stretched out, a typical bacterial chromosome would be nearly 1 mm long, approximately 1,000 times the length of a cell. Not only must cells massively compact their genetic material, but they must also organize their DNA in a manner that is compatible with a range of cellular processes, including DNA replication, DNA repair, homologous recombination, and horizontal gene transfer. Recent work, driven in part by technological advances, has begun to reveal the general principles of chromosome organization in bacteria. Here, drawing on studies of many different organisms, we review the emerging picture of how bacterial chromosomes are structured at multiple length scales, highlighting the functions of various DNA-binding proteins and the impact of physical forces. Additionally, we discuss the spatial dynamics of chromosomes, particularly during their segregation to daughter cells. Although there has been tremendous progress, we also highlight gaps that remain in understanding chromosome organization and segregation.National Institutes of Health (U.S.) (Grant R01GM082899

Time to death in the presence of E. coli: a mass-scale method for assaying pathogen resistance in Drosophila

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Role of MukBEF in Escherichia coli chromosome organization

In E. coli cells, spatial chromosome organization reflects the genetic map, with the origin located at mid-cell and left and right replichores in either cell half. In contrast to the eukaryotic cell cycle, chromosome segregation occurs progressively as replication proceeds. The highly conserved SMC complex, MukBEF, plays a central role in chromosome organization and segregation, with null mutants having temperature sensitive growth and mispositioned chromosomal regions. However, despite much effort, their mechanism of action in vivo remains elusive.The current work aimed to shed light on MukBEF function by developing tools to investigate its mode of action in live cells. Degron derivatives of MukBEF were developed as a rapid way of depleting cells of MukBEF to observe its effects on chromosome organization. “Real-time” Muk depletion/ repletion shows that MukBEF can establish and maintain position of chromosomal loci independent of the cell cycle. Origin relocation from cell-pole to mid-cell is a slow process, preceded by the formation of MukBEF foci at various locations on the chromosome. Hence, Muk-mediated shaping of the chromosome is a replication-independent phenomenon. In addition, while ATP binding is required for focus formation, ATP hydrolysis is required for MukBEF function in the cell. ATPase mutants are inviable in vivo, highlighting the importance of MukB ATPase activity.In a complementary approach, the composition and dynamics of MukBEF foci were assessed in vivo. The stoichiometries of wild type and ATP-hydrolysis deficient (Walker B) MukBEF complexes in fluorescent foci were estimated using slimfield microscopy. These results suggest that MukBEF complexes are ATP bound in foci with a broad and heterogeneous distribution. Only 19% of total MukBEF molecules are present at a given time in foci. Furthermore, unlike wild type foci, which localize around the origin, Walker B foci colocalize with the terminus. One model proposed is that the terminus may represent the loading site for wild type complexes and that Walker B MukBEF may represent the loading complex, which can associate with the loading site, but requires ATP hydrolysis for movement to the origin.This work provides insight into the role of MukBEF in spatial chromosome organization as well as the in vivo significance of MukB ATPase activity.</p

Role of MukBEF in Escherichia coli chromosome organization

In E. coli cells, spatial chromosome organization reflects the genetic map, with the origin located at mid-cell and left and right replichores in either cell half. In contrast to the eukaryotic cell cycle, chromosome segregation occurs progressively as replication proceeds. The highly conserved SMC complex, MukBEF, plays a central role in chromosome organization and segregation, with null mutants having temperature sensitive growth and mispositioned chromosomal regions. However, despite much effort, their mechanism of action in vivo remains elusive. The current work aimed to shed light on MukBEF function by developing tools to investigate its mode of action in live cells. Degron derivatives of MukBEF were developed as a rapid way of depleting cells of MukBEF to observe its effects on chromosome organization. "Real-time" Muk depletion/ repletion shows that MukBEF can establish and maintain position of chromosomal loci independent of the cell cycle. Origin relocation from cell-pole to mid-cell is a slow process, preceded by the formation of MukBEF foci at various locations on the chromosome. Hence, Muk-mediated shaping of the chromosome is a replication-independent phenomenon. In addition, while ATP binding is required for focus formation, ATP hydrolysis is required for MukBEF function in the cell. ATPase mutants are inviable in vivo, highlighting the importance of MukB ATPase activity. In a complementary approach, the composition and dynamics of MukBEF foci were assessed in vivo. The stoichiometries of wild type and ATP-hydrolysis deficient (Walker B) MukBEF complexes in fluorescent foci were estimated using slimfield microscopy. These results suggest that MukBEF complexes are ATP bound in foci with a broad and heterogeneous distribution. Only 19% of total MukBEF molecules are present at a given time in foci. Furthermore, unlike wild type foci, which localize around the origin, Walker B foci colocalize with the terminus. One model proposed is that the terminus may represent the loading site for wild type complexes and that Walker B MukBEF may represent the loading complex, which can associate with the loading site, but requires ATP hydrolysis for movement to the origin. This work provides insight into the role of MukBEF in spatial chromosome organization as well as the in vivo significance of MukB ATPase activity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Role of MukBEF in Escherichia coli chromosome organization

In E. coli cells, spatial chromosome organization reflects the genetic map, with the origin located at mid-cell and left and right replichores in either cell half. In contrast to the eukaryotic cell cycle, chromosome segregation occurs progressively as replication proceeds. The highly conserved SMC complex, MukBEF, plays a central role in chromosome organization and segregation, with null mutants having temperature sensitive growth and mispositioned chromosomal regions. However, despite much effort, their mechanism of action in vivo remains elusive.The current work aimed to shed light on MukBEF function by developing tools to investigate its mode of action in live cells. Degron derivatives of MukBEF were developed as a rapid way of depleting cells of MukBEF to observe its effects on chromosome organization. “Real-time” Muk depletion/ repletion shows that MukBEF can establish and maintain position of chromosomal loci independent of the cell cycle. Origin relocation from cell-pole to mid-cell is a slow process, preceded by the formation of MukBEF foci at various locations on the chromosome. Hence, Muk-mediated shaping of the chromosome is a replication-independent phenomenon. In addition, while ATP binding is required for focus formation, ATP hydrolysis is required for MukBEF function in the cell. ATPase mutants are inviable in vivo, highlighting the importance of MukB ATPase activity.In a complementary approach, the composition and dynamics of MukBEF foci were assessed in vivo. The stoichiometries of wild type and ATP-hydrolysis deficient (Walker B) MukBEF complexes in fluorescent foci were estimated using slimfield microscopy. These results suggest that MukBEF complexes are ATP bound in foci with a broad and heterogeneous distribution. Only 19% of total MukBEF molecules are present at a given time in foci. Furthermore, unlike wild type foci, which localize around the origin, Walker B foci colocalize with the terminus. One model proposed is that the terminus may represent the loading site for wild type complexes and that Walker B MukBEF may represent the loading complex, which can associate with the loading site, but requires ATP hydrolysis for movement to the origin.This work provides insight into the role of MukBEF in spatial chromosome organization as well as the in vivo significance of MukB ATPase activity.</p

Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex in Live Escherichia coli Bacteria

MukBEF, a structural maintenance of chromosome (SMC) complex, is an important molecular machine for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or "foci") composed of molecular assemblies in the cell, where it is thought to carry out most of its chromosome-associated activities. Here, we outline the technique of fluorescence recovery after photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci. This method can provide important insight into the dynamics of MukB on DNA and be used to study its biochemical properties in vivo

Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria

Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB.National Institutes of Health (U.S.) (Grant R01GM082899)Gordon and Betty Moore Foundation (Postdoctoral Fellow of the Life Sciences Research Foundation)Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship

Solving the variational problems by making use of GPUs

Import 01/09/2009Tématem této diplomové práce je seznámit se s problematikou segmentační metody level set a zvládnout její implementaci v C++. Další náplň práce spočívá v základním pohledu na architekturu akcelerace výpočtů pomocí grafických karet NVIDIA s technologií CUDA. Výsledkem práce tak bude nejen kód v C++, ale rovněž budou segmentační funkce naprogramovány s NVIDIA CUDA, porovnán výkon a vhodnost jednotlivých řešení.The main task of this diploma work is to take up with level set segmentation method and to implement application using this method in programming language C++. Then I should deal with hardware acceleration using GPUs and NVIDIA CUDA technology. The final goal is to solve the C++ and CUDA version of level set method and compare their results in the meaning of achieved speed and usability.Prezenční456 - Katedra informatikyvýborn