Involvement of TolC protein in the export of siderophore enterobactin in Escherichia coli

Abstract only availableTo acquire the necessary iron against harsh competition in the environment, iron starved bacteria synthesize, excrete and retrieve iron scavenging molecules termed siderophores, one of which is enterobactin. TolC protein may play a vital role in the secretion of enterobactin. Enterobactin molecules destined for secretion must cross both the inner (cytoplasmic) and outer membranes and the intervening periplasmic space, believed to be a distance of at least 130Å across. TolC resembles a trans-periplasmic tunnel embedded in the outer membrane of the cell. It is open to the external environment but is closed at its periplasmic entrance. In order for the cell to export enterobactin, TolC is recruited by substrate specific membrane complexes (translocases) in the periplasmic space and inner membrane. When TolC is recruited, the entrance is opened to allow substrate passage through a continuous machinery spanning the entire cell envelope, from the cytosol to the external environment. PCR primers specific for TolC were designed to amplify the TolC gene. The quality of the PCR product was confirmed using agarose gel electrophoresis. The TolC gene was cloned into a pBAD directional TOPO vector containing an N-terminal His-tag and a gene for kanamycin resistance. The recombinant vector was then transformed into One Shot TOP10 competent Escherichia coli cells. Transformants were selected for by plating on LB medium supplemented with kanamycin. Transformed colonies were analyzed using PCR and restriction digestion. Positive transformants were selected and expression was induced with arabinose. SDS-PAGE assay with His-tag In-gel stain revealed TolC expression. Furthermore, analysis of TolC-null mutations using high performance liquid chromatography (HPLC) reveals that the TolC mutant secretes little, if any, enterobactin. However, some levels of breakdown products 2,3-dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer are observed. These data establish that TolC may be a critical component of the E. coli enterobactin secretion machinery and may represent a type of siderophore export mechanism previously undescribed. TolC family proteins are ubiquitous among gram-negative bacteria, and the conserved apertures present a possible chemotherapeutic target in multidrug-resistant pathogens.Molecular Biology Progra

Provenance and Paleogeography of the 25-17 Ma Rainbow Gardens Formation: Evidence for Tectonic Activity at Ca. 19 Ma and Internal Drainage rather than Throughgoing Paleorivers on the Southwestern Colorado Plateau

The paleogeographic evolution of the Lake Mead region of southern Nevada and northwest Arizona is crucial to understanding the geologic history of the U.S. Southwest, including the evolution of the Colorado Plateau and formation of the Grand Canyon. The ca. 25–17 Ma Rainbow Gardens Formation in the Lake Mead region, the informally named, roughly coeval Jean Conglomerate, and the ca. 24–19 Ma Buck and Doe Conglomerate southeast of Lake Mead hold the only stratigraphic evidence for the Cenozoic pre-extensional geology and paleogeography of this area. Building on prior work, we present new sedimentologic and stratigraphic data, including sandstone provenance and detrital zircon data, to create a more detailed paleogeographic picture of the Lake Mead, Grand Wash Trough, and Hualapai Plateau region from 25 to 18 Ma. These data confirm that sediment was sourced primarily from Paleozoic strata exposed in surrounding Sevier and Laramide uplifts and active volcanic fields to the north. In addition, a distinctive signal of coarse sediment derived from Proterozoic crystalline basement first appeared in the southwestern corner of the basin ca. 25 Ma at the beginning of Rainbow Gardens Formation deposition and then prograded north and east ca. 19 Ma across the southern half of the basin. Regional thermochronologic data suggest that Cretaceous deposits likely blanketed the Lake Mead region by the end of Sevier thrusting. Post-Laramide northward cliff retreat off the Kingman/Mogollon uplifts left a stepped erosion surface with progressively younger strata preserved northward, on which Rainbow Gardens Formation strata were deposited. Deposition of the Rainbow Gardens Formation in general and the 19 Ma progradational pulse in particular may reflect tectonic uplift events just prior to onset of rapid extension at 17 Ma, as supported by both thermochronology and sedimentary data. Data presented here negate the California and Arizona River hypotheses for an “old” Grand Canyon and also negate models wherein the Rainbow Gardens Formation was the depocenter for a 25–18 Ma Little Colorado paleoriver flowing west through East Kaibab paleocanyons. Instead, provenance and paleocurrent data suggest local to regional sources for deposition of the Rainbow Gardens Formation atop a stripped low-relief western Colorado Plateau surface and preclude any significant input from a regional throughgoing paleoriver entering the basin from the east or northeast

The Role of Penning Collisions in Hollow Cathode Helium Cadmium Lasers

The excitation mechanisms leading to the formation of the 5s² ²D5/2 level, the upper level of the 4416 Å transition of Cd+, have been investigated. Experiments were carried out in both a helium cadmium discharge and corresponding afterglow. A self absorption technique was used to measure the variation, with current, pressure and oven temperature, of the densities of selected excited helium levels and the cadmium ion ground state. A comparison of the parametric behaviour of the Penning collision rate with the 4416 Å spontaneous emission provided good evidence that Penning ionization was the dominant mechanism leading to laser oscillation at 4416 Å. Gas temperature effects were found to have a significant influence on the interpretation of the experimental results. Signal averaging techniques were employed to record the pressure, current and oven temperature dependence of the 4416 Å spontaneous decay in the hollow cathode helium cadmium afterglow. The decay was more complex than anticipated but was eventually attributed to the temporal evolution of the helium triplet metastable species in the afterglow. A simplified model of the afterglow was developed and, using the available excited state densities and estimates of the electron and helium ion densities and electron collision rates, the system of coupled differential rate equations was solved and found to be in reasonable agreement with the experimentally observed trends of the 4416 Å decay. Taken as a whole, the results of the study of the helium cadmium d.c. discharge and afterglow show beyond doubt that Penning ionization is the dominant excitation mechanism of the 5s² ²D5/2 level of Cd II

Single-stranded nucleic acid elasticity arises from internal electrostatic tension

Charged, flexible polymers, such as single-stranded nucleic acids (ssNAs), are ubiquitous in biology and technology. Quantitative description of their solution conformation has remained elusive due to the competing effects of polymer configurational freedom and salt-screened electrostatic repulsion between monomers. We investigate this by measuring the elastic response of single ssNA molecules over a range of salt concentrations. The data are well described by a model, inspired by a mean-field approach, in which intrapolymer electrostatic repulsion creates a salt-dependent internal tension whose interplay with the external force determines the elasticity. The internal tension can be related to the polymer’s charge spacing; thus, our results show how mesoscopic polymer conformation emerges from microscopic structure

Characterization of polyoxometalate I as an inhibitor of RNA-dependent RNA polymerase of Foot and Mouth Disease virus [abstract]

Abstract only availableFoot and Mouth Disease (FMD) is a highly contagious disease that affects a variety of domesticated cloven-hoofed animals including cattle, swine, sheep and goats, as well as several wild animal species. FMD outbreaks are currently controlled with mass-extermination of livestock. The financial cost of potential outbreaks would be immense. This disease is caused by foot-and-mouth disease virus (FMDV), a non-enveloped, single-stranded, positive-sense RNA virus. The purpose of our investigation is to identify chemicals that interfere with the replication of FMDV. As part of this effort we have identified a polyoxometalate inhibitor (polyoxometalate I). We have cloned, expressed and purified FMDV RdRp. We use steady-state kinetic experiments and polymerization assays to characterize the inhibitory activity of the polyoxometalate I, determining the precise inhibitory potential and the mechanism of inhibition. Preliminary results show that polyoxometalate I inhibits the FMDV RdRp surprisingly efficiently with an IC50 of 0.5uM. Current experiments are focusing on a detailed kinetic characterization of the mechanism of action for this inhibitor. This research may provide insights that lead to new treatment options to prevent the further spread of FMD to unaffected animals.USD

Alkenones as a promising green alternative for waxes in cosmetics and personal care products

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Cosmetics 5 (2018): 34, doi:10.3390/cosmetics5020034.The move toward green, sustainable, natural products has been growing in the cosmetic and personal care industry. Ingredients derived from marine organisms and algae are present in many cosmetic products. In this study, a new green ingredient, a wax (i.e., long-chain alkenones) derived from Isochyrsis sp., was evaluated as an alternative for cosmetic waxes. First, the melting point was determined (71.1–77.4 °C), then the alkenones’ thickening capability in five emollients was evaluated and compared to microcrystalline wax and ozokerite. Alkenones were compatible with three emollients and thickened the emollients similarly to the other waxes. Then, lipsticks and lip balms were formulated with and without alkenones. All products remained stable at room temperature for 10 weeks. Lipstick formulated with alkenones was the most resistant to high temperature. Finally, alkenones were compared to three cosmetic thickening waxes in creams. Viscosity, rheology, and stability of the creams were evaluated. All creams had a gel-like behavior. Both viscosity and storage modulus increased in the same order: cream with alkenones < cetyl alcohol < stearic acid < glyceryl monostearate. Overall, alkenones’ performance was comparable to the other three waxes. Alkenones can thus offer a potential green choice as a new cosmetic structuring agent.This research was funded by the Washington Research Foundation and a private donor from friends of the Woods Hole Oceanographic Institution, grant number N-126478