Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Investigation of DNA Repair Pathway Activity
DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage. Here we describe a range of assays used to study DNA ...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Isolation of Dictyostelium Nuclei for Light and Electron Microscopy
The significance of amoebae for studies of nuclear architecture has considerably increased in the recent years. The availability of a protocol for isolation of nuclei in a quality sufficient for high-resolution light and electron microscopy is a prerequisite for such studies. Here we present a protocol for high enrichment of nuclei by sucrose density-gradient centrifugation. Moreover, we describe how to use these isolated nuclei as specimens for immunofluorescence and immune-electron microscopy of ultrathin sections. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Extrachromosomal Inducible Expression
Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled expression in. Two types of inducible plasmids are presented, in which transcription is induced by either adding or removing doxycycline, respectively. Detailed protocols are provided for the construction of the plasmids and the inducible expression of the target protein. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
The Application of the Cre-loxP System for Generating Multiple Knock-out and Knock-in Targeted Loci
Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We previously described a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both, knock-out and knock-in gene targ...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Measuring Cheating, Fitness, and Segregation in Dictyostelium discoideum
Dictyostelium has become a model organism for the study of social evolution because of the stage in its life cycle where thousands of independent amoebae together form a fruiting body. Some individuals die to form a stalk that holds aloft the remaining cells for dispersal to new environments as spores. Different genotypes can aggregate together, creating opportunities for exploitation by cheaters that contribute a smaller proportion of cells to the stalk. Clustering of genotypes into separate fruiting bodies reduces the opportunities for cheating. Some genotypes achieve this by segregating after aggregation. Here we descri...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
N-Glycomic and N-Glycoproteomic Studies in the Social Amoebae
N-glycans modify the great majority of all secreted and plasma membrane proteins, which themselves constitute one-third to one-half of the proteome. The ultimate definition of the glycoproteome would be the identification of all the N-glycans attached to all the modified asparaginyl sites of all the proteins, but glycosylation heterogeneity makes this an unachievable goal. However, mass spectrometry in combination with other methods does have the power to deeply mine the N-glycome of Dictyostelium, and characterize glycan profiles at individual sites of glycoproteins. Recent studies from our laboratories using mass spectro...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Pharmacogenetics of Resistance to Cisplatin and Other Anticancer Drugs and the Role of Sphingolipid Metabolism
Dictyostelium discoideum has proven to be a useful lead genetic system for identifying novel genes and pathways responsible for the regulation of sensitivity to the widely used anticancer drug cisplatin. Resistance to cisplatin is a major factor limiting the efficacy of the drug in treating many types of cancer. Studies using unbiased insertional mutagenesis in D. discoideum have identified the pathway of sphingolipid metabolism as a key regulator in controlling sensitivity to cisplatin. Using the genetic tools including directed homologous recombination and ectopic gene expression available with D. discoideum has shown ho...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Assessment of Multidrug Efflux Assemblies by Surface Plasmon Resonance
Surface plasmon resonance (SPR) is a powerful tool for kinetic analyses of protein–protein interactions. Here we describe the application of this method to study interactions of membrane proteins involved in multidrug efflux in Escherichia coli.These so-called multidrug efflux pumps comprise an inner membrane transporter, a periplasmic membrane fusion protein and an outer membrane channel. The three components are assembled into a protein conduit that enables bacteria to expel multiple drugs directly into the external medium bypassing the periplasm. The “across-two-membranes” transport mechanism makes tri...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Analysis of Exopolysaccharides in Myxococcus xanthus Using Confocal Laser Scanning Microscopy
Confocal laser scanning microscopy (CLSM) is a useful research tool to explore the exopolysaccharides (EPS) in bacterial biofilms. Here, we describe the analysis of different biofilms of Myxococcus xanthusformed in a modified chamber slide system with CLSM. In conjunction with several specific fluorescent probes, the EPS within M. xanthusbiofilms can be visualized and analyzed in situ. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Chromatographic Analysis of the Escherichia coli Polysialic Acid Capsule
Polysialic acid capsules are the major virulence factors in Escherichia coliK1, K92, and groups B and C meningococci. The sialic acid monomers (2-keto-3-deoxy-5-acetamido-7,8,9-d-glycero-d-galacto-nonulosonic acids) comprising these homopolymeric polysaccharide chains can be selectively modified with 1,2-diamino-4,5-methylenedioxy-benzene to produce highly fluorescent quinoxalinone derivatives distinguished by their elution times during reverse phase chromatography. Here, we describe methods to release the constituent capsular polysialic acid monomers, and to detect and quantify them by sensitive fluorometry. There are rel...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Single Cell Microfluidic Studies of Bacterial Motility
A large number of bacterial species move smoothly on solid surfaces in the absence of extracellular organelles. In the deltaproteobacterium Myxococcus xanthus, this surface motion, termed gliding motility, involves a novel macromolecular machinery Agl-Glt. During the motility process, the Agl-Glt system, an integral envelope protein complex, is assembled on the ventral side of the cell. Doing so, the complex couples surface adhesion to the activity of the Agl motility motor. On the cytosolic side, the Agl-Glt system is linked to the bacterial actin cytoskeleton MreB. It is proposed that motility is produced when surfa...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Visualization of Gram-positive Bacterial Pili
Pili or fimbriae are recognized as essential virulence determinants assembled on the bacterial surface. Gram-positive bacteria produce covalently linked pilus structures that are distinct from gram-negative counterparts. In this chapter, we describe three commonly used techniques to extract, detect, and visualize pili from gram-positive bacteria: (1) Western blot analysis, (2) Immuno-Electron Microscopy, and (3) Atomic Force Microscopy. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Experimental Manipulation of the Microbial Functional Amyloid Called Curli
Curli are proteinaceous fibrous structures produced on the surface of many gram-negative bacteria. As a major constituent of the extracellular matrix, curli mediate interactions between the bacteria and its environment, and as such, curli play a critical role in biofilm formation. Curli fibers share biophysical properties with a growing number of remarkably stable and ordered protein aggregates called amyloid. Here we describe experimental methods to study the biogenesis and assembly of curli by exploiting their amyloid properties. We also present methods to analyze curli-mediated biofilm formation. These approaches are st...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Purification of the Outer Membrane Usher Protein and Periplasmic Chaperone-Subunit Complexes from the P and Type 1 Pilus Systems
Understanding molecular mechanisms of protein secretion by bacteria requires the purification of secretion machinery components and the isolation of complexes between the secretion machinery and substrate proteins. Here, we describe methods for the purification of proteins from the chaperone/usher pathway, which is a conserved secretion pathway dedicated to the assembly of polymeric surface fibers termed pili or fimbriae in gram-negative bacteria. Specifically, we describe the isolation of the PapC and FimD usher proteins from the bacterial outer membrane, and the purification of PapD-PapG and FimC-FimH chaperone-subu...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
Visualizing the Bacterial Cell Surface: An Overview
The ultrastructure of bacteria is only accessible by electron microscopy. Our insights into the architecture of cells and cellular compartments such as the envelope and appendages is thus dependent on the progress of preparative and imaging techniques in electron microscopy. Here, I give a short overview of the development and characteristics of methods applied for imaging (components of) the bacterial surface and refer to key investigations and exemplary results. In the beginning of electron microscopy, fixation of biological material and staining for contrast enhancement were the standard techniques. The results from fre...
Source: Springer protocols feed by Microbiology - January 10, 2013 Category: Microbiology Source Type: news
