Spo0J and SMC are required for normal chromosome segregation in Staphylococcus aureus

Bacterial chromosome segregation is an essential cellular process that is particularly elusive in spherical bacteria such as the opportunistic human pathogen Staphylococcus aureus. In this study, we examined the functional significance of a ParB homologue, Spo0J, in staphylococcal chromosome segregation and investigated the role of the structural maintenance of chromosomes (SMC) bacterial condensin in this process. We show that neither spo0J nor smc is essential in S. aureus; however, their absence causes abnormal chromosome segregation. We demonstrate that formation of complexes containing Spo0J and SMC is required for efficient S. aureus chromosome segregation and that SMC localization is dependent on Spo0J. Furthermore, we found that cell division and cell cycle progression are unaffected by the absence of spo0J or smc. Our results verify the role of Spo0J and SMC in ensuring accurate staphylococcal chromosome segregation and also imply functional redundancy or the involvement of additional mechanisms that might contribute to faithful chromosome inheritance

Compressed Sensing and Adaptive Graph Total Variation for Tomographic Reconstructions

Compressed Sensing (CS) and Total Variation (TV)- based iterative image reconstruction algorithms have received increased attention recently. This is due to the ability of such methods to reconstruct from limited and noisy data. Local TV methods fail to preserve texture details and fine structures, which are tedious for the method to distinguish from noise. In many cases local methods also create additional artifacts due to over smoothing. Non-Local Total Variation (NLTV) has been increasingly used for medical imaging applications. However, it is not updated in every iteration of the algorithm, has a high computational complexity and depends on the scale of pairwise parameters. In this work we propose using Adaptive Graph- based TV in combination with CS (ACSGT). Similar to NLTV our proposed method goes beyond spatial similarity between different regions of an image being reconstructed by establishing a connection between similar regions in the image regardless of spatial distance. However, it is computationally much more efficient and scalable when compared to NLTV due to the use of approximate nearest neighbor search algorithm. Moreover, our method is adaptive, i.e, it involves updating the graph prior every iteration making the connection between similar regions stronger. Since TV is a special case of graph TV the proposed method can be seen as a generalization of CS and TV methods. We test our proposed algorithm by reconstructing a variety of different phantoms from limited and corrupted data and observe that we achieve a better result with ACSGT in every case

Cell shape-independent FtsZ dynamics in synthetically remodeled bacterial cells

FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo

Semi-automatically aligned tilt images in electron tomography

In electron microscope tomography, alignment of tilt series images is a major determinant of resolution in 3D reconstructions. One alignment method uses gold beads deposited on or in the specimen as fiducial markers. We have developed software to semi-automatically align tilt series images. It runs two processes iteratively: (1) Marker picking. In this process, it uses a cross-correlation function to determine the shift between tilt images and predicts marker coordinates. Subsequently it refines them in a local search area, and detects and corrects erroneously picked markers automatically. The coordinates of the picked markers are used to align the images. (2) Image alignment. In this process, it uses a least squares method to estimate image rotation, image shift, and image scale factor

Application of nanotags and nanobodies for live cell single-molecule imaging of the Z-ring in Escherichia coli

Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein, we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA), and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.journal articl

An Extended Field-Based Method for Noise Removal From Electron Tomographic Reconstructions

Molecular structure determination is important for understanding functionalities and dynamics of macromolecules, such as proteins and nucleic acids. Cryo-electron tomography (ET) is a technique that can be used to determine the structures of individual macromolecules, thus providing the snapshots of their native conformations. Such 3-D reconstructions encounter several types of imperfections due to missing, corrupted, and low-contrast data. In this paper, we demonstrate that extending the reconstruction space, which increases the dimensionality of the linear system being solved during reconstruction, facilitates the separation of signal and noise. A considerable amount of the noise associated with collected projection data arises independently from the geometric constraint of image formation, whereas the solution to the reconstruction problem must satisfy such geometric constraints. Increasing the dimensionality thereby allows for a redistribution of such noise within the extended reconstruction space, while the geometrically constrained approximate solution stays in an effectively lower dimensional subspace. Employing various tomographic reconstruction methods with a regularization capability we performed extensive simulation and testing and observed that enhanced dimensionality significantly improves the accuracy of the reconstruction. Our results were validated with reconstructions of colloidal silica nanoparticles as well as P. falciparum erythrocyte membrane protein 1. Although the proposed method is used in the context of Cryo-ET, the method is general and can be extended to a variety of other tomographic modalities

A method for the preparation of heavy-atom derivatives of yeast cytochrome c peroxidase

This Letter describes a strategy for preparation of heavy-atom derivatives which has succeeded with yeast cytochrome c peroxidase and which may be useful for other crystalline proteins. Transfer of the crystals from methylpentanediol solutions, in which they were grown, to solutions of polyethylene glycol (Mr 6000) maintains the crystal structure so that crystals tolerate heavy-atom concentrations at least ten times larger than could be attained in methylpentanediol mother liquors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23653/1/0000619.pd

Network interdiction

Network Interdiction-problem innehåller två mot varandra stående styrkor, en användare och en angripare, somär inbegripna i en krigsliknande konflikt. Användaren använder ett nätverk för att optimera en funktion, t.ex.att förflytta en underhållskonvoj så snabbt som möjligt, eller maximera mängden materiel som transporterasgenom nätverket. Det innebär att användaren vill använda den kortaste eller snabbaste vägen vid transporter,och han vill maximera flödet genom nätverket. Nätverket kan t.ex. vara ett vägnät, kraftförsörjningsnät eller ettdatornätverk. Angriparen försöker begränsa användarens möjlighet att optimera sin funktion. Angriparenssyftet är att maximera den kortaste/snabbaste vägen eller att minimera det maximala flödet genom nätverket.Angriparen uppnår detta genom att angripa bågar eller noder i nätverket och förstöra dem totalt eller reduceraderas kapacitet. Angriparens resurser är begränsade och det finns ett behov av att optimera användandet. Imånga fall är nätverket stort och många parametrar påverkar planeringen. Detta ger en komplexplaneringsförutsättning för angriparen. Genomförs planläggningen av Network Interdiction på traditionellt sätt,tvingas planeraren att använda sin intuition. Resultatet beror till stor del på planläggarens förmåga och tid tillförfogande. Om algoritmer kunde användas för att stödja planläggaren, skulle resursutnyttjandet och effektenav angreppen kunna optimeras. Uppsatsen undersöker om det är möjligt att använda Network Interdictionalgoritmervid planering av Network Interdiction.Network interdiction problems involve two opposing forces, a user and anattacker, who are engaged in a warlike conflict. The user operates a network inorder to optimize a function such as moving a supply convoy through thenetwork as quickly as possible, or maximizing the amount of materieltransported through the network. This means that the user is trying to use theshortest or the fastest route to perform transports, and he is trying to maximizethe flow trough the network. The network could be a road net, an electric powergrid or a computer network system. The attacker attempts to limit the user’spossibility to optimize his function. The purpose is to maximize the shortest andfastest route or to minimize the maximum flow through the network. Theattacker obtains this by interdicting arcs or nodes, e.g. by attacking arcs or nodesin order to destroy them entirely or to reduce their capacity. The attacker’sresources are limited and there is a need to optimize the use of them. In manycases the network is big and numerous parameters influence the planning. Thismakes the conditions for planning complex and difficult for the attacker. If theplanning of network interdiction is performed in the traditional way, the planneris forced to use intuition. The result will depend on the planner’s capacity and thetime at his disposal. If algorithms could be used to support the planner, theresources and the effect of the attack would be optimized. This thesis examines ifit is possible to use network interdiction algorithms to plan network interdiction.Avdelning: ALB - Slutet Mag 3 C-upps.Hylla: Upps. ChP T 01-0