Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^

Late Neogene sediment composition and accumulation rates of ODP Hole 120-751A

Through scanning electron microscope analysis of sediment microfabric, we have evaluated variations in high-resolution shipboard physical properties (index properties and shear strength), sediment components (smear slide determinations), and shore-based calcium carbonate and biogenic silica data from Site 751 (Kerguelen Plateau). The stratigraphic section at this site records a change in biogenic ooze composition from predominantly calcareous (nannofossil) to siliceous (diatom) ooze from ~23 Ma to the present, reflecting expansion of Antarctic water masses during the late Neogene. The profound change in physical properties and sediment character at 40.1 mbsf (~5-6 Ma) evidently records the northward movement of the Polar Front and a change in absolute accumulation rates of sediment at this site. Trends in geotechnical properties with depth at Site 751 allowed us to subdivide the sedimentary column into a number of geotechnical units that reflect changes in depositional and postdepositional processes with time. Geotechnical properties are sensitive to changing sedimentary inputs of primarily siliceous and calcareous microfossils. This allows us to study the physical nature of biostratigraphically-identified hiatuses and variations in environmental conditions linked to the migration of the Polar Front across this region. The analysis of geotechnical properties permits a more detailed division of the sedimentary column than is possible from shipboard lithologic descriptions alone. Our study of the sedimentary microfabric indicates that randomly oriented, elongate pennate diatom valves compose the sediments with highest porosity and water content values, and the lowest density values (wet bulk, dry bulk, and grain density). Conversely, sediments composed of nannofossils and disassociated nannofossil crystallites and little or no siliceous remains have the lowest porosity and water content values, and the highest density values. Samples of mixed siliceous/calcareous composition have intermediate physical property values, but these vary according to the nature of the sedimentary matrix and the state of preservation of individual skeletal elements.

(Table 2) Characteristics of influence of cyclonic and anticyclonic activity of water masses on hydrophysical, hydrochemical, and hydrobiological parameters in the southern part of the Black Sea in September 1996

Characteristics of the spatial structure of vertical synoptic currents were calculated from data of the density field surveys in order to estimate their influence on distribution of chlorophyll a concentration. Comparisons of chlorophyll concentration and vertical currents were implemented for two multidisciplinary surveys in the Black Sea carried out in summer, 1991 and in winter, 1994. The results showed qualitative and quantitative indications of coincidence of characteristics cited and, in particular, significant positive values of the correlation coefficient (0.65 for the summer survey and 0.83 for the winter one).

Gill tissue masses and oxygen consumption of Gobionotothen gibberifrons, Notothenia coriiceps and Zoarces viviparus

Mechanisms responsive to hypercapnia (elevated CO2 concentrations) and shaping branchial energy turnover were investigated in isolated perfused gills of two Antarctic Notothenioids (Gobionotothen gibberifrons, Notothenia coriiceps). Branchial oxygen consumption was measured under normo- versus hypercapnic conditions (10,000 ppm CO2) at high extracellular pH values. The fractional costs of ion regulation, protein and RNA synthesis in the energy budgets were determined using specific inhibitors. Overall gill energy turnover was maintained under pH compensated hypercapnia in both Antarctic species as well as in a temperate zoarcid (Zoarces viviparus). However, fractional energy consumption by the examined processes rose drastically in G. gibberifrons (100-180%), and to a lesser extent in N. coriiceps gills (7-56%). In conclusion, high CO2 concentrations under conditions of compensated acidosis induce cost increments in epithelial processes, however, at maintained overall rates of branchial energy turnover.

Properties of water masses, taxa abundance, and isotopic ratios of samples obtained around Svalbard archipelago

The feeding strategies of Calanus hyperboreus, C. glacialis, and C. finmarchicus were investigated in the high-Arctic Svalbard region (77-81 °N) in May, August, and December, including seasons with algal blooms, late- to post-bloom situations, and unproductive winter periods. Stable isotope and fatty acid trophic marker (FATM) techniques were employed together to assess trophic level (TL), carbon sources (phytoplankton vs. ice algae), and diet of the three Calanus species. In addition, population development, distribution, and nutritional state (i.e. storage lipids) were examined to estimate their population status at the time of sampling. In May and August, the vertical distribution of the three Calanus species usually coincided with the maximum algal biomass. Their stable isotope and fatty acid (FA) composition indicated that they all were essentially herbivores in May, when the algal biomass was highest. Their FA composition, however, revealed different food preferences. C. hyperboreus had high proportions of 18:4n3, suggesting that it fed mainly on Phaeocystis, whereas C. glacialis and C. finmarchicus had high proportions of 16:4n1, 16:1n7, and 20:5n3, suggesting diatoms as their major food source. Carbon sources (i.e. phytoplankton vs. ice algae) were not possible to determine solely from FATM techniques since ice-diatoms and pelagic-diatoms were characterised by the same FA. However, the enriched d13C values of C. glacialis and C. finmarchicus in May indicated that they fed both on pelagic- and ice-diatoms. Patterns in absolute FA and fatty alcohol composition revealed that diatoms were the most important food for C. hyperboreus and C. glacialis, followed by Phaeocystis, whereas diatoms, Phaeocystis and other small autotrophic flagellates were equally important food for C. finmarchicus. During periods of lower algal biomass, only C. glacialis exhibited evidence of significant dietary switch, with a TL indicative of omnivory (mean TL=2.4). Large spatial variability was observed in population development, distribution, and lipid store sizes in August. At the northernmost station at the southern margin of the Arctic Ocean, the three Calanus species had similarly low lipid stores as they had in May, suggesting that they ascended later in the year. In December, relatively lipid-rich specimens had TL similar to those during the peak productive season (TL~2.0), suggesting that they were hibernating and not feeding on the available refractory material available at that time of the year. In contrast, lipid-poor specimens in December had substantially high TL (TL=2.5), suggesting that they were active and possibly were feeding.

Phytoplankton production, photosynthetic (P vs E) parameters, and particulate organic carbon and nitrogen within distinct water masses of eastern Australian coastal and shelf waters between 29 °S and 36 °S during spring 2010

The present dataset is part of an interdisciplinary project carried out on board the RV Southern Surveyor off New South Wales (Australia) from the 15th to the 31st October 2010. The main objective of the research voyage was to evaluate how the East Australian Current (EAC) affects the optical, chemical, physical, and biological water properties of the continental shelf and slope off the NSW coast.