Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE: Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms

Transposon Mutagenesis in Chlamydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer

Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis. This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis 148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.S.D.L., Z.E.D., K.S.H., S.B., R.J.S., and P.S.H. were funded by NIH (AI126785)J.W. and P.S.H. were supported by NIH AI125929. P.S.H. was also supported by P20GM113117Support for genomic sequencing was supplemented by P20GM10363

The chemical dynamics of XeF₂ and Xe(F₂) reactions with Si(100)

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2004.Vita.Includes bibliographical references.The chemistry of fluorine, F2, and xenon difluoride, XeF2, with clean Si are nearly indistinguishable. Both species react via the atom abstraction mechanism, whereby a surface dangling bond abstracts a F atom from the incident molecule, scattering the complementary F atom or XeF fragment into the gas phase. However, once all dangling bonds are fluorinated and a coverage of 1 ML F is attained, the chemistry of these two species diverges. Further exposure to F2 results in no increase in F coverage, indicating reactions of F2 with the Si-Si -bonds do not occur. In contrast, further exposure to XeF2 results in additional reaction, cleaving the Si-Si bonds and increasing the coverage beyond 1 ML F. Eventually, sufficient fluorination occurs to effect the desorption of the etch product SiF4. The experiments described in this thesis have been designed to probe the origin of the observed difference in reactivity of F2 and XeF2 with a fluorinated Si surface. Two possible explanations are presented that likely account for the difference in reactivity of F2 and XeF2 with fluorinated Si. The first is the reaction of atomic fluorine, which is produced by the gas phase dissociation of XeF following an abstraction event, with the Si. The second is vibrational excitation of the Si lattice, induced by the initial collisions of XeF2, thereby enabling reaction of the F atoms with the Si-Si bonds. The first chapter describes experiments investigating the chemical dynamics of the reaction of a beam of XeF2 with Si(100). The scattered intensities and the time-of-flight distributions of the reaction products XeF2, XeF, and Xe are measured as a function of coverage and scattering angle, as is the intensity of scattered F atoms.(cont.) The F atoms arise from the gas phase dissociation of XeF, which results from partitioning of the exothermicity of the abstraction reaction of XeF2 with Si into the internal energy of XeF. The time-of-flight and angular distributions of F and Xe are simulated by applying conservation of momentum, energy, and flux principles to the measured XeF time-of-flight and angular distributions using only two fitting parameters: the average internal energy partitioned to XeF and the allowed range of molecular orientations of XeF as it is scattered from the surface. These parameters are adjusted to reproduce the measured F atom TOF spectra at three scattering angles for the coverage range 0 - 0.22 ML F. The same parameters are then used to predict the Xe atom TOF distributions resulting from the dissociation of XeF. The intensity of the TOF distributions for scattered Xe is scaled such that one Xe atom is produced for each F atom in the simulation. The simulated TOF spectra result from the analysis of 8,085,000 initial XeF trajectories, which are computed at eight coverage ranges. The simulation is found to reproduce the scattered Xe TOF spectra well, including the intensity, with no fitting of the simulated Xe spectra to the data. This excellent agreement corroborates the XeF dissociation as a gas phase event. The dissociation of XeF is the first observed case of a gas phase dissociation resulting from partitioning of exothermicity to a product of a gas-surface reaction. In addition, the simulation indicates that approximately 10% of the F atoms resulting from XeF dissociation are directed back toward the surface ...by Robert Charles Hefty.Ph.D

Mechanism and dynamics of the reaction of XeF2 with fluorinated Si(100): Possible role of gas phase dissociation of a surface reaction product in plasmaless etching

Xenon difluoride is observed to react with Si–Si σ-dimer and σ-lattice bonds of Si(100)2×1 at 150 K by single and two atom abstraction at F coverages above 1 ML. As in the limit of zero F coverage, a measurable fraction of the scattered, gas phase product of single atom abstraction, XeF, is sufficiently internally excited to dissociate into F and Xe atoms before detection. Using the XeF internal energy and orientation distributions determined in the limit of zero coverage, the laws of conservation of momentum, energy, and mass are applied to the measured F velocity and angular distributions at higher coverage to simulate the Xe atom velocity and angular distributions and their intensities at higher coverage. The simulation predicts the observed Xe atom velocity and angular distributions at high coverage reasonably well, largely because the exothermicity channeled to XeF remains approximately constant as the coverage increases. This constancy is an opportune consequence of the trade-off between the attractiveness of the potential energy surface as the coverage is increased and the dynamics of the XeF product along the potential surface. The energy, momentum, and mass conservation analysis is also used to distinguish between Xe atoms that arise from XeF gas phase dissociation and Xe atoms that are produced by two atom abstraction. This distinction enables the calculation of percentages of the single and two atom abstraction pathways, as well as the percentages of the two pathways available to the Xe atom produced by two atom abstraction, inelastic scattering, and desorption. Finally, the simulation reveals that between 9% and 12% of F atoms produced by gas phase dissociation of XeF are scattered back toward the surface. These F atoms likely react readily with Si to form the higher fluorides that ultimately lead to etching. Gas phase dissociation of the scattered product of a surface reaction is a novel mechanism to explain the unique reactivity of XeF2 to etch Si in the absence of a plasma.National Science Foundation (U.S.) (grant CHE-0517786

Factors affecting particleboard pressing time : interaction with catalyst systems /

Cover title.Includes bibliographical references.Mode of access: Internet

Mixed Adenocarcinomatous and Neuroendocrine Tumor of the Urinary Bladder With Concomitant Carcinoma In Situ: A Case Report With a Comprehensive Immunohistochemical Analysis and Review of the Literature

Mixed adenoneuroendocrine carcinomas are rare and usually occur in the gastrointestinal tract. Although there have been several investigations regarding their developmental mechanism, the molecular origin of these tumors remains unclear. In this article, we present an exceedingly rare case of a mixed tumor of the urinary bladder with an adenocarcinomatous and a neuroendocrine component and a concomitant urothelial carcinoma in situ (CIS). Due to this extraordinary combination of tumor components, our goal was to extensively examine the 3 tumor components with regard to a representable common origin. Therefore, a comprehensive immunohistochemical analysis and review of the literature was performed. Besides expected outcome, our examination also revealed surprising staining results. Urothelial CIS, like the adenocarcinomatous component, showed strong staining for CDX2. In addition, parts of the adenocarcinoma were positive for synaptophysin like the neuroendocrine tumor component. All 3 components showed a significant overexpression of p53 and a moderate to strong membranous and cytoplasmatic staining for β-catenin. To our knowledge, we are the first to describe a case of a mixed tumor of the urinary bladder with an adenocarcinomatous and a neuroendocrine component and a concomitant CIS. The components share striking molecular features that argue for a common clonal origin and a development of the invasive tumor via the urothelial precursor lesion. </jats:p