The application of STEM and in situ controlled dehydration to bacterial systems using ESEM.

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and ΔmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the ΔmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type.

The application of STEM and in situ controlled dehydration to bacterial systems using ESEM

Transmission imaging with an environmental scanning electron microscope (ESEM) (Wet STEM) is a recent development in the field of electron microscopy, combining the simple preparation inherent to ESEM work with an alternate form of contrast available through a STEM detector. Because the technique is relatively new, there is little information available on how best to apply this technique and which samples it is best suited for. This work is a description of the sample preparation and microscopy employed by the authors for imaging bacteria with Wet STEM (scanning transmission electron microscopy). Three different bacterial samples will be presented in this study: first, used as a model system, is Escherichia coli for which the contrast mechanisms of STEM are demonstrated along with the visual effects of a dehydration-induced collapse. This collapse, although clearly in some sense artifactual, is thought to lead to structurally meaningful morphological information. Second, Wet STEM is applied to two distinct bacterial systems to demonstrate the novel types of information accessible by this approach: the plastic-producing Cupriavidus necator along with wild-type and δmreC knockout mutants of Salmonella enterica serovar Typhimurium. Cupriavidus necator is shown to exhibit clear internal differences between bacteria with and without plastic granules, while the δmreC mutant of S. Typhimurium has an internal morphology distinct from that of the wild type. © 2012 Wiley Periodicals, Inc.

Axial and journal bearings for superconducting flywheel systems

Axial and journal bearings have been investigated for use in superconducting flywheel systems. Our test rig comprises of an Evershed type magnetic bearing used to levitate a 35 kg rotor. The stabilizing forces are provided by superconducting axial and journal bearings. In this study we focus on the vertical stiffness measurements and explore the use of journal bearings. The journal bearing consists of radial magnets with alternating polarities. Our results indicate that this type of journal bearing can effectively stabilize the rotor. Spin-down test shows a linear behavior.

The bacterial cytoskeleton modulates motility, type 3 secretion, and colonization in Salmonella.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.