Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^

Riego por goteo en Mendoza, Argentina : : evaluación de la uniformidad del riego y del incremento de salinidad, sodicidad e iones cloruro en el suelo

El presente trabajo tiene por objeto evaluar la uniformidad del riego por goteo en las zonas de regadío de las cuencas de los ríos Mendoza y Tunuyán (zonas Alta y Baja), de la provincia de Mendoza, Argentina. Las evaluaciones -en 17 propiedades- permitieron determinar: coeficientes de uniformidad, salinidad del agua de riego y del suelo en cabeza, medio y pie de la subunidad de riego, y en el bulbo húmedo e interfilar en dos estratos del perfil del suelo (0,10‑0,30 m y 0,30-0,50 m). Se determinó, además, la textura del suelo y su posible relación con los niveles de salinidad de la rizósfera. Se estimó que el 18% de las subunidades de riego evaluadas presentan un coeficiente de uniformidad por debajo del rango recomendable y que en el 94% de las propiedades existen diferencias significativas entre caudales medios registrados entre subunidades y entre sectores de operación de riego. Se encontraron diferencias significativas en la salinidad del extracto de saturación, en la sodicidad y en la concentración del anión cloruro, al comparar el suelo extraído del bulbo de mojado respecto del interfilar. Asimismo, los resultados muestran que no existen diferencias significativas de esas variables en las distintas profundidades de suelo analizadas. El aumento de la salinidad resultó en función del lugar de muestreo (bulbo o interfilar), la calidad del agua de riego y la textura del suelo. La variación de sodicidad, en cambio, dependió del lugar de muestreo (bulbo o interfilar) y del contenido de bicarbonatos en el suelo. Los resultados indican la importancia de realizar evaluaciones rutinarias del comportamiento de los sistemas de riego en términos de uniformidad y salinización inducida.

Stable oxygen isotopes of Globigerinoides ruber and biostratigraphic datums at ODP Site 165-1002

Ocean Drilling Program Site 1002 in the Cariaco Basin was drilled in the final two days of Leg 165 with only a short transit remaining to the final port of San Juan, Puerto Rico. Because of severe time constraints, cores from only the first of the three long replicate holes (Hole 1002C) were opened at sea for visual description, and the shipboard sampling was restricted to the biostratigraphic examination of core catchers. The limited sampling and general scarcity of biostratigraphic datums within the late Quaternary interval covered by this greatly expanded hemipelagic sequence resulted in a very poorly defined age model for Site 1002 as reported in the Leg 165 Initial Reports volume of the Proceedings of the Ocean Drilling Program. Here, we present for the first time a new integrated stratigraphy for Site 1002 based on the standard of late Quaternary oxygen-isotope variations linked to a suite of refined biostratigraphic datums. These new data show that the sediment sequence recovered by Leg 165 in the Cariaco Basin is continuous and spans the time interval from 0 to ~580 ka, with a basal age roughly twice as old as initially suspected from the tentative shipboard identification of a single biostratigraphic datum. Lithologic subunits recognized at Site 1002 are here tied into this new stratigraphic framework, and temporal variations in major sediment components are reported. The biogenic carbonate, opal, and organic carbon contents of sediments in the Cariaco Basin tend to be high during interglacials, whereas the terrigenous contents of the sediments increase during glacials. Glacioeustatic variations in sea level are likely to exert a dominant control on these first-order variations in lithology, with glacial surface productivity and the nutrient content of waters in the Cariaco Basin affected by shoaling glacial sill depths, and glacial terrigenous inputs affected by narrowing of the inner shelf and increased proximity of direct riverine sources during sea-level lowstands.

The nitrogen costs of photosynthesis in a diatom under current and future pCO2

With each cellular generation, oxygenic photoautotrophs must accumulate abundant protein complexes that mediate light capture, photosynthetic electron transport and carbon fixation. In addition to this net synthesis, oxygenic photoautotrophs must counter the light-dependent photoinactivation of Photosystem II (PSII), using metabolically expensive proteolysis, disassembly, resynthesis and re-assembly of protein subunits. We used growth rates, elemental analyses and protein quantitations to estimate the nitrogen (N) metabolism costs to both accumulate the photosynthetic system and to maintain PSII function in the diatom Thalassiosira pseudonana, growing at two pCO2 levels across a range of light levels. The photosynthetic system contains c. 15-25% of total cellular N. Under low growth light, N (re)cycling through PSII repair is only c. 1% of the cellular N assimilation rate. As growth light increases to inhibitory levels, N metabolite cycling through PSII repair increases to c. 14% of the cellular N assimilation rate. Cells growing under the assumed future 750 ppmv pCO2 show higher growth rates under optimal light, coinciding with a lowered N metabolic cost to maintain photosynthesis, but then suffer greater photoinhibition of growth under excess light, coincident with rising costs to maintain photosynthesis. We predict this quantitative trait response to light will vary across taxa.

Diatom events and Neogene nannofossils of ODP Hole 201-1225A

Shipboard investigation of magnetostratigraphy and shore-based investigation of diatoms and calcareous nannofossils were used to identify datum events in sedimentary successions collected at Ocean Drilling Program (ODP) Leg 201 Site 1225. The goal was to extend the magnetic record previously studied at the same site, ODP Leg 138 Site 851, and provide a comprehensive age model for Site 1225. Two high-magnetic intensity zones at 0-70 and 200-255 meters below seafloor (mbsf) were correlated with lithologic Subunits IA and IC in Hole 1225A. Subunit IA (0-70 mbsf) contains the magnetic reversal record until the Cochiti Subchronozone (3.8 Ma) and has a sedimentation rate of 1.7 cm/k.y. This agrees with previous work done at Site 851. Subunit IC (200-255 mbsf) was not sampled at Site 851. Diatom and nannofossil biostratigraphy constrained this subunit, and we found it to contain the magnetic reversal record between Subchrons C4n.2r and C5n.2n (8.6-9.7 Ma), yielding a sedimentation rate of 2.7 cm/k.y. Biostratigraphy was used to establish the sedimentation rates within Subunits IB and ID (70-200 mbsf and 255-300 mbsf, respectively). These subunits had higher sedimentation rates (~3.4 cm/k.y.) and coincide with the late Miocene-early Pliocene biogenic bloom event (4.5-7 Ma) and the Miocene global cooling trend (10-15 Ma). High biogenic productivity associated with these subunits resulted in the pyritization of the magnetic signal. In lithologic Subunit ID, basement flow is another factor that may be altering the magnetic signal; however, the good correlation between the biostratigraphy and magnetostratigraphy indicates that the magnetic record was locked-in near the seafloor and suggests the age model is robust.

Geochemistry of the volcanic basement of ODP Hole 134-831B

The basement of Bougainville Guyot drilled at Site 831 consists of andesitic hyalobreccias derived from a submarine arc volcano. The volcanic sequence has been dated by K/Ar at approximately 37 Ma. The 121 m of andesitic hyalobreccias drilled in Hole 831B have been divided into five subunits of two types: one appears to be primary, and the other contains evidence of reworking and a subaerial clastic input. Variations are attributed to fluctuations in water depth. The distinctive hyalobreccias consist of andesitic blebs with chilled margins and peripheral fractures set in a chaotic greenish matrix that is mainly altered glass, with crystals similar to those in the blebs or clasts. Their formation is attributed to violent reaction of andesitic magma discharged into seawater, in perhaps the submarine equivalent of fire-fountaining. There was limited reworking by currents and debris flows on the flanks of the submarine volcano. The andesite shows no significant compositional variation in phenocryst phases throughout the drilled sequence and contains phenocrysts of plagioclase (An88-43), clinopyroxene (Ca44Mg46Fe10-Ca41Mg40Fe19), orthopyroxene (Ca4Mg79Fe17-Ca3Mg58Fe39), and titanomagnetite. There is a systematic change in volcanic composition with height in the section, from more mafic andesites at the base, to overlying more acid andesites, and strong evidence exists that magma mixing may have played a significant role in the genesis of these lavas. The andesites have affinities with the low-K arc tholeiite series. Trace element and isotopic systematics for these rocks indicate very minor involvement of a LILE- and 87Sr-enriched slab-derived fluid in their petrogenesis. This accords with the previous suggestion that Bougainville Guyot forms part of an Eocene proto-island arc developed along the southern side of the d'Entrecasteaux Zone, above a southward-dipping subduction zone.

Paleomagnetic of ODP Hole 133-823A

Hole 823A covers the upper 120 m (Subunits IA and IB) of Site 823 at the bottom of the Queensland Trough. This hole contains an abundance of gravity-flow deposits, but is thought to have a monotonic age sequence. Above 32 mbsf, a strong, stable (normal) magnetic remanence having a relatively small viscous remanent magnetization (VRM) is seen. Below 32 mbsf, the sediments are subject to widespread VRM, which appears to obliterate the primary magnetization and precludes identification of the Brunhes/Matuyama boundary. Progressive alternating field (AF) demagnetization is limited to low fields (typically <400 Oe) by the weak magnetization in these sediments. As a consequence, the possibility of a high-coercivity component of primary magnetization cannot be ruled out. Lowrie-Fuller tests indicate that this VRM overprinting does not have a multidomain origin. An approximately linear relationship exists between median destructive field (MDF) and the logarithm of the natural remanent magnetization (NRM). Carbonate dilution does not appear to be a dominant factor in controlling variations in concentration-dependent magnetic parameters, such as magnetic susceptibility. The sedimentological distinction between Subunits IA and IB does not show up in the magnetic record. However, a sharp change in magnetic properties does occur at 32 mbsf, with low background magnetizations below this level and high background magnetizations above it. The boundary coincides with a change from thick (>10 cm thick) to thin (<10 cm thick) turbidite deposition, and is also near the boundary separating the sulfate-reduction zone in the upper part of the sequence from the sulfate-free zone beneath. The abrupt nature of the magnetic boundary is evidence that nannofossil subzone CN14b is not condensed, but is missing in a hiatus at 32 mbsf. Nine peaks have been identified in the susceptibility (K) record that are superimposed on ôbackgroundö signals. ARM/K ratios are uniformly low for the background sediments below 32 mbsf, intermediate for strong susceptibility peaks, and high for background sediments above 32 mbsf and weak susceptibility peaks. Comparisons with results from Site 820 suggest that (1) the background sediments above 32 mbsf and the weak susceptibility peaks carry a stable single-domain magnetization, and (2) the high susceptibility peaks are caused by the addition of a superparamagnetic contribution. Expectations are that the distinctive features of the Hole 823A magnetic record are linked to major environmental changes.

Major oxides for tektites and for conglomerate clast of the Upper Eocene ejecta-bearing layer of ODP Hole 150-904A (Table 1)

Evidence for the Chesapeake Bay Crater as the source for New Jersey continental margin ejecta is provided by fine-grained tektites and coarse-grained unmelted ejecta. The Upper Eocene ejecta deposit, now demonstrated to be part of the North American strewn field, occurs on the New Jersey continental margin at Ocean Drilling Program (ODP) Sites 904 and 903. The mineralogy, major oxide composition of the ejecta materials, and biostratigraphic age of the enclosing sediments link the origin of these ejecta to the recently recognized Chesapeake Bay impact crater, located only 330 km away. Sediments associated with the ejecta provide information about the dynamics of impact events. The 35-cm-thick ejecta-bearing layer can be subdivided into three subunits that indicate a sequence of events. Bottom subunit III documents sediment failure and deposition of gravel-sized fragments, middle subunit II records deposition of abundant sand-sized ejecta by gravity settling, and upper subunit I contains a 12-cm-thick sedimentary deposit containing rare silt-sized tektites and evidence of waning currents. These events are interpreted by linking sediment deposition to seismic ground motion and subsequent tsunami waves triggered by both the Chesapeake Bay impact and slope failures.

Physical properties and AMS characteristics of ODP Site 185-1149 sediments

During Ocean Drilling Program Leg 185, we studied progressive changes of microfabrics of unconsolidated pelagic and hemipelagic sediments in Holes 1149A and 1149B in the northwest Pacific at 5818 m water depth. We paid particular attention to the early consolidation and diagenetic processes without tectonic deformation before the Pacific plate subduction at the Izu-Bonin Trench. Shape, size, and arrangement of pores were analyzed by scanning electron microscope (SEM) and were compared to anisotropy of magnetic susceptibility (AMS) data. The microfabric in Unit I is nondirectional fabric and is characterized by large peds of ~10-100 µm diameter, which are made up of clay platelets (mainly illite) and siliceous biogenic fragments. They are ovoid in shape and are mechanically packed by benthic animals. Porosity decreases from 0 to 60 meters below seafloor (mbsf) in Unit I (from 60% to 50%) in association with macropore size decreases. The microfabric of coarser grain particles other than clay in Unit II is characterized by horizontal preferred orientation because of depositional processes in Subunit IIA and burial compaction in Subunit IIB. On the other hand, small peds, which are probably made of fragments of fecal pellets and are composed of smectite and illite (3-30 µm diameter), are characterized by random orientation of clay platelets. The clay platelets in the small peds in Subunit IIA are in low-angle edge-to-face (EF) or face-to-face (FF) contact. These peds are electrostatically connected by long-chained clay platelets, which are interconnected by high-angle EF contact. Breaking of these long chains by overburden pressure diminishes the macropores, and the clay platelets in the peds become FF in contact, resulting in decreases in the volume of the micropores between clay platelets. Thus, porosity in Subunits IIA and IIB decreases remarkably downward. The AMS indicates random fabric and horizontal preferred orientation fabric in Units I and II, respectively. This result corresponds to that of SEM microfabric observations.In Subunit IIB, pressure solutions around radiolarian tests and clinoptilolite veins with normal displacement sense are seen distinctively below ~170 mbsf, probably in correspondence to the transition zone from opal-A to opal-CT.

Physical properties and abundance of diatoms of the ANDRILL AND1-1B drill core

In the austral summer of 2006/7 the ANDRILL MIS (ANtarctic geological DRILLing- McMurdo Ice Shelf) project recovered a 1285 m sediment core from beneath the Ross Ice Shelf near Hut Point Peninsula, Ross Island, Antarctica in a flexural moat associated with the volcanic loading of Ross Island. Contained within the upper ~600 m of this core are sediments recording 38 glacial to interglacial cycles of Early Pliocene to Pleistocene time, including 13 discrete diatomite units (DU). The longest of these, DU XI, is ~76 m thick, contains two distinct unconformities marked by layers of volcanic brecciated sands, and has been assigned an Early to Mid-Pliocene age (5-3 Ma). A detailed record (avg. sample spacing of 33 cm) of the siliceous microfossil assemblages have been generated for DU XI and used in conjunction with geochemical and sedimentological data to subdivide DU XI into four discrete subunits of continuous sedimentation. Within each unit, changes in diatom assemblages have been correlated with the d18O record, providing a temporal resolution as high as 600 yr, and allowing for the construction of a detailed age model and calculation of associated sediment accumulation rates within DU XI. Results indicate a productivity-dominated sedimentary record with higher sediment accumulation rates containing a greater proportion of hemipelagic mud occurring during relatively cool periods and reduced accumulation during warmer intervals. This implies that even during periods of substantial warmth, Milankovitch-paced changes in Antarctic ice volume can be linked to ecological changes recorded as shifts in diatom assemblages.

Geotechnical properties of sediments at DSDP Site 75-532

During Leg 75 of the Deep Sea Drilling Project (DSDP) from the D/V Glomar Challenger, a 200-m deep hole was drilled at Hole 532A on the eastern side of Walvis Ridge at a water depth of 1331 m. Sediment cores were obtained by means of a hydraulic piston corer. All of the cores from this boring were designated for geotechnical studies and were distributed among eight institutions. The results of laboratory studies on these sediment cores were compiled and analyzed. Sediment properties, including physical characteristics, strength, consolidation, and permeability were studied to evaluate changes as a function of depth of burial. It was concluded that the sediment profile to the explored depth of 200 m at Walvis Ridge consists of approximately 50 m of foram-nannofossil marl (Subunit 1a) over 64 m of diatom-nannofossil marl (Subunit 1b) over nannofossil marl (Subunit 1c) to the depth explored. All three sediment units appear to be normally consolidated, although some anomalies seem to exist to a depth of 120 m. No distinct differences were found among the sediment properties of the three subunits (1a, 1b, and 1c) identified at this site.

Chemistry of bulk-rocks and minerals of ODP Hole 120-748C

Five hundred meters of a unique Upper Cretaceous Cr-rich glauconitic sequence (Unit III) that overlies a 3-m-thick alkali-basalt flow with underlying epiclastic volcanogenic sediments was drilled at ODP Leg 120 Site 748. The Cr-rich glauconitic sequence is lithostratigraphically and biostratigraphically divided into three subunits (IIIA, IIIB, IIIC) that can also be recognized by the Cr concentration of the bulk sediment, which is low (<200 ppm) in Subunits IIIC and IIIA and high (400-800 ppm) in Subunit IIIB. The Cr enrichment is caused by Cr-spinel, which is the only significant heavy mineral component beside Fe-Ti ores. Other Cr-bearing components are glauconite pellets and possibly some other clay minerals. The glauconitic sequence of Subunit IIIB was formed by reworking of glauconite and volcanogenic components that were transported restricted distances and redeposited downslope by mass-transportation processes. The site of formation was a nearshore, shallow inner shelf environment, and final deposition may have been on the outer part of a narrow shelf, at the slope toward the restricted, probably synsedimentary, faulted Raggatt Basin. The volcanic edifices uncovered on land were tholeiitic basalts (T-MORB), alkali-basaltic (OIB) and (?)silicic volcanic complexes, and ultramafic rocks. The latter were the ultimate source for the Cr-spinel contribution. Terrestrial aqueous solutions carried Fe, K, Cr, Si, and probably Al into the marine environment, where, depending on the redox conditions of microenvironments in the sediment, green (Fe- and K-rich) or brown (Al-rich) glauconite pellets formed. The Upper Cretaceous glauconitic sequence at Site 748 on the Southern Kerguelen Plateau constitutes the transition in space and time from terrestrial to marine, from magmatically active subaerial to magmatically passive submarine conditions, and from a tranquil platform to active rifting conditions.

Seawater carbonate chemistry and photosystem II photoinactivation of the coastal diatom Thalassiosira pseudonana in a laboratory experiment

We studied the interactive effects of pCO2 and growth light on the coastal marine diatom Thalassiosira pseudonana CCMP 1335 growing under ambient and expected end-of-the-century pCO2 (750 ppmv), and a range of growth light from 30 to 380 µmol photons/m**2/s. Elevated pCO2 significantly stimulated the growth of T. pseudonana under sub-saturating growth light, but not under saturating to super-saturating growth light. Under ambient pCO2 susceptibility to photoinactivation of photosystem II (sigma i) increased with increasing growth rate, but cells growing under elevated pCO2 showed no dependence between growth rate and sigma i, so under high growth light cells under elevated pCO2 were less susceptible to photoinactivation of photosystem II, and thus incurred a lower running cost to maintain photosystem II function. Growth light altered the contents of RbcL (RUBISCO) and PsaC (PSI) protein subunits, and the ratios among the subunits, but there were only limited effects on these and other protein pools between cells grown under ambient and elevated pCO2.

Texture description and distribution of components in ODP Hole 103-639D (Figure 2)

A detailed analysis of the texture, matrix, and elements of the microfacies from the carbonate sequence recovered in ODP Hole 639D resulted in a typological classification of 10 major microfacies types and their variants. The variations in distribution and succession of type microfacies allowed us to divide the carbonate sequence into 12 facies-defined subunits. Based on the analyzed characteristics and their relations, we also propose a paleoenvironmental interpretation involving a mixed carbonate/terrigenous ramp model instead of the previous, classical zoned carbonate platform.