Physiological and enzymatic studies of respiration in Dehalococcoides species strain CBDB1

Die Dissertation beschreibt die physiologischen und enzymatischen Fähigkeiten von Dehalococcoides sp Stamm CBDB1. Wachstum des Stammes CBDB1 auf Grundlage von Dehalorespiration mit HCB und PeCB konnte gezeigt werden. Diese Ergebnisse stellen ein neues System für die Kultivierung von Stamm CBDB1 mit hochchlorierten Benzolen als Elektronenakzeptoren zur Verfügung. Hydrogenase und Dehalogenase sind die Schlüsselenzyme bei der Dehalorespiration. Beide Enzyme sind membrangebunden mit den katalytischen Zentren nach außen. Hydrogenaseaktivität konnte auch im Cytoplasma gemessen werden. Die Hydrogenase von Stamm CBDB1 ist sehr empfindlich gegenüber Sauerstoff, so verloren die Zellen sofort ihre Enzymaktivität, wenn sie der Luft ausgesetzt waren. Die Aktivität der reduktiven Dehalogenasen in Kulturen von Stamm CBDB1 angezüchet auf verschiedenen Chlorbenzolen weisen darauf hin, dass verschiedene Elektronenakzeptoren unter Umständen unterschiedliche reduktive Dehalogenasen induzieren. Die Dehalogenaseaktivität gemessen an ganzen Zellen mit artifiziellen Elektrondonoren lassen vermuten, dass ein Redoxpotential von ?-360 mV für die Reduktion von Chlorbenzolen benötigt wird. Auch sterische Effekte haben einem Einfluss auf die Dehalogenaseaktivität. So war die Dehalogenaseaktivität, die mit Methylviologen ( E0´=-450 mV) gemessen wurde, höher als die mit Ethylviologen ( E0´=-480 mV). Chinone scheinen als physiologische Elektronenmediatoren in der Transportkette von Stamm CBDB1 auszuscheiden, weil durch HONOQ, einen Inhibitor von Chinon abhängigen Redoxreaktionen, die Dechlorierung in intakten Zellen mit Wasserstoff als Elektronendonor nicht gehemmt werden konnte. Bei Dechlorierungsversuchen mit intakten Zellen und Wasserstoff als Elektronendonor in Gegenwart des Protonophors TCS stellte sich heraus, dass Stamm CBDB1 nicht auf einen reversen Elektronentransport angewiesen ist. 1,2,3,4-TeCB inhibierte die Dechlorierung in Zellkulturen mit Wasserstoff als Elektronendonor. Der genaue Mechanismus der Inhibition ist unbekannt. Es wird vermutet, dass 1,2,3,4-TeCB den Elektronentransport unterbricht, ohne mit der Hydrogenase oder Dehalogenase zu interagieren. Ein putatives Gencluster, bestehend aus den Strukturgenen hupS und hupL, die membrangebundene Gruppe 1 [Ni-Fe] Hydrogenasen codieren, wurde aus Stamm CBDB1 amplifiziert und sequenziert. Das Cluster enthielt außerdem hupD, ein Gen, das ein akzessorisches Protein für die Reifung der Hydrogenase codiert. Die Amplifizierung der drei Gene aus mRNA über RT-PCR bestätigte, dass das Gencluster als polycistronischer Messenger transkribiert wird. Dem amplifizierten Operon fehlte ein Gen, das für Cytochrom b codiert. Dieses Gen existiert in allen bisher bekannten membrangebundenen [Ni-Fe] Hydrogenasen, nicht aber in löslichen Hydrogenasen. Die Hydrogenaseaktivität wurde in der Membranfraktion gemessen. Ein einzigartiges hydrophobes Segment in der kleinen Untereinheit (HupS) der Hydrogenase könnte für das Anhaften an der Membran verantwortlich sein. An diesem Punkt unterscheidet sich das Hydrogenaseoperon von Stamm CBDB1 von den bisher in der Literatur beschriebenen.The thesis describes physiological properties of Dehalococcoides species strain DBDB1 and enzymes involved in dehalorespiration. Growth of strain CBDB1 with HCB and PeCB provided an efficient system for the cultivation with highly chlorinated benzenes as electron acceptors. The key enzymes, hydrogenase and dehalogenase activities, were membrane associated with catalytic sites oriented towards the outside. Hydrogenase activity was also detected in the cytoplasm. Hydrogenase of strain CBDB1 was found to be highly sensitive to oxygen. Reductive dehalogenase activity detected in cells of strain CBDB1 pregrown with different chlorobenzene congeners as electron acceptors indicated that the different electron acceptors might induce different reductive dehalogenases. Dehalogenase activity of whole cells detected with artificial electron donors indicated that a redox potential of £-360 mV is needed for the chlorobenzenes reduction. However, steric effects also influenced dehalogenase activity because a higher reaction rate of dehalogenase activity was measured with methyl viologen (Eo´= -450 mV) compared to ethyl viologen (Eo´= -480 mV). Quinones seem not to be physiological electron mediators in the electron transport of strain CBDB1, because HONOQ, an inhibitor of quinone dependent redox reactions, did not inhibit the reductive dechlorination reaction by intact cells with hydrogen as electron donor. Dechlorination by intact cells with hydrogen as electron donor in the presence of a protonophore, TCS, revealed that strain CBDB1 does not require reverse electron transport. 1,2,3,4-TeCB strongly inhibited the dechlorination by whole cells with hydrogen as electron donor. The precise mechanism of inhibition by 1,2,3,4-TeCB is unknown. However, 1,2,3,4-TeCB is believed to interfere somehow with a step in the electron transport of strain CBDB1 without inhibiting hydrogenase or dehalogenase activity. The putative gene cluster consisting of structural genes hupS and hupL coding for a membrane bound group-1 [Ni-Fe] hydrogenase was amplified and sequenced. The gene cluster also contained hupD, a gene encoding an accessory protein for hydrogenase maturation. Amplification of all three genes by RT-PCR from mRNA confirms that the investigated gene cluster is transcribed as a polycistronic messenger. The amplified operon lacks a gene coding for cytochrome b that is found in all other membrane bound [Ni-Fe] hydrogenases known so far, but is not present in soluble hydrogenases. However, based on the hydrogenase activity in the membrane fraction, a unique hydrophobic segment found in the small subunit (HupS) of the hydrogenase could be responsible for attaching the complex to the membrane. In this aspect, the hydrogenase operon of strain CBDB1 differs from all other membrane bound hydrogenase operons described so far

Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment

Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity1. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM)1. PCM assembly is a tightly regulated, critical step that determines a centrosome’s size and capability2–4. Here, we report a role for tubulin in regulating PCM recruitment via the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins5,6. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, tubulin’s regulation of PCM recruitment depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules7. Based on its guanine bound state, tubulin can act as a molecular switch in PCM recruitment

Marine Ecosystems Challenges and Opportunities (MECOS 09) Book of Abstracts

Marine ecosystems contain several unique qualities that set them apart from other ecosystems. Of the 89 elements occurring in nature, the presence of 80 has been confirmed in seawater. It is perhaps true that the remaining 9 elements are also present, but in concentrations too small to be detected. This wide range of substances dissolved in seawater has placed the marine organisms in a more advantageous position than their freshwater counterparts. These elements provide the essential materials required for the synthesis of all the basic nourishments of the body including the skeletal support of marine animals. In the terrestrial ecosystems, the physical boundaries are well marked and environmental variabilities are rather wide. The terrestrial organisms and ecosystems have developed internal mechanisms to cope up with variabilities. In contrast, in the marine ecosystems, the physical variability is small and extends over very long time scales due to the large thermal capacity of the oceans and the long periods of exchange between deep and near shore waters. Consequently, the marine ecosystems are more vulnerable to large-scale environmental changes because they do not have the internal adaptability inherent in the terrestrial systems

Explicit incremental matrices for the postbuckling analysis of thin plates with small initial curvature

The postbuckling behaviour of thin plates is an important phenomenon in the design of thin plated structures. In reality plates possess small imperfections and the behaviour of such imperfect plates is of great interest. To numerically study the postbuckling behaviour of imperfect plates explicit incremental or secant matrices have been presented in this paper. These matrices can be used in combination with any thin plate element. The secant matrices are shown to be very accurate in tracing the postbuckling behaviour of thin plates

Explicit incremental matrices for the postbuckling analysis of thin plates with small initial curvature

The postbuckling behaviour of thin plates is an important phenomenon in the design of thin plated structures. In reality plates possess small imperfections and the behaviour of such imperfect plates is of great interest. To numerically study the postbuckling behaviour of imperfect plates explicit incremental or secant matrices have been presented in this paper. These matrices can be used in combination with any thin plate element. The secant matrices are shown to be very accurate in tracing the postbuckling behaviour of thin plates

Improved secant matrices for the postbuckling analysis of thin composite plates

To study the postbuckling behavior of imperfect laminated composite plates, improved incremental or secant matrices are presented in this paper using what is called additional displacement formulation (ADF). These secant matrices are derived using the Marguerre's shell theory and they can be used in combination with any thin plate finite element. The advantage of the present formulation is that it involves no numerical approximation in forming the initial imperfection matrices as opposed to earlier secant matrices published in the literature using total displacement formulation. With the addition of shear stiffness matrix and little modification, the present incremental matrices could be extended to model postbuckling behavior of plates using the first-order shear deformation theory. The secant matrices presented in this study are shown to be very accurate in tracing the postbuckling behavior of thin isotropic and laminated composite plates with general initial imperfections

Preparation and characterization of electron beam evaporated WO3 thin films

Thin film of tungsten oxide (WO3) has been extensively studied as an electrochromic material and has numerous applications in electrochromic devices, smart windows, gas sensors and optical windows. In order to explore the possibility of using this in electrochromic devices, a preliminary and thorough study of the optical properties of the host material is an important step. Based on the above criterion, the effect of annealing temperature on the structural, surface morphological and optical properties of WO3 films has been studied in the present work. The host material, WO3 films, has been prepared by the physical vapor deposition method of electron beam evaporation (PVD:EBE) technique under a pressure of 1 · 10�5 mbar. The single phase nature, monoclinic structure and textured nature of the films have been confirmed by the X-ray diffraction analysis. The needle-like crystallites have been observed from surface morphological studies. The evaluated crystallite size is in the nanometer range. The shift in absorption edge towards the higher wavelength region observed from optical studies may be due to the coloration effect on the films

Characterization on electron beam evaporated a-MoO3 thin films by the influence of substrate temperature

Electrochromic molybdenum oxide (MoO3) thin films were prepared by electron beam evaporation technique using the dry MoO3 pellets. The films were deposited on glass and fluorine doped tin oxide (SnO2:F or FTO) coated glass substrates at different substrate temperatures like room temperature (RT, 30 C), 100 C and 200 C. The influence of substrate temperature on the structural, surface morphological and optical properties of the films has been studied. The X-ray diffraction analysis showed that the films are having orthorhombic phase MoO3 (a-MoO3) with h110i preferred orientation. The laser Raman scattering spectrum shows the polycrystalline nature of MoO3 films deposited at 200 C. The Raman-active band at 993 cm-1 is corresponding to Mo–O stretching mode that is associated with the unique character of the layered structure of orthorhombic MoO3. Needle—like morphology was observed from the SEM analysis. The energy band gap of MoO3 films was evaluated which lies between 2.8 and 2.3 eV depending on the substrate temperature and substrates. The decrease in band gap value with increasing substrate temperature is owing to the oxygen-ion vacancies. The absorption edge shift shows the coloration effect on the films

Investigation of x-ray photoelectron spectroscopic (XPS), cyclic voltammetric analyses ofWO3 films and their electrochromic response in FTO/WO3/electrolyte/FTO cells

Electrochromic thin films of tungsten oxide (WO3) were prepared on transparent conducting oxide substrates, i.e., fluorine doped tin oxide coated (FTO or SnO2:F) glass and microscopic glass substrates by the electron beam evaporation technique using pure WO3 (99.99%) pellets at various substrate temperatures (i.e., Tsub = room temperature (RT, 30 ◦C), 100 ◦C and 200 ◦C). The films were prepared under vacuum of the order of 1 × 10−5 mbar. The room temperature prepared films were further post-heat-treated (Tanne) at 200 and 300 ◦C for about 1 h in the vacuum environment. The prepared films are in monoclinic phase. The chemical composition has been characterized by using the XPS technique. The W 4f and O 1s core levels of WO3 films have been studied on the samples. The obtained core level binding energies revealed the WO3 films contained six-valent tungsten (W6+). The electrochemical nature of the films was studied by a three-electrode electrochemical cell in the configuration of FTO/WO3/H2SO4/Pt, SCE, using the cyclic voltammetry (CV) technique. Electrochromic devices (ECDs) of the general type FTO/WO3/electrolyte/FTO were studied. The films produced at higher substrate temperature show smaller modulation of the visible spectrum, compared with the films produced at lower temperatures. The significant chemical bonding nature associated with the coloring/bleaching process which follows the H+ ion incorporation in the film is studied by FTIR analysis. The W–O–W framework peak was observed at 563 cm−1 and confirms the stability of the films in the electrochemical analysis. The results obtained from cyclic voltammetry technique and ECD cell characterization are used to emphasize the suitability for some applications of the solar control systems