Microbial Phototrophic, Heterotrophic, and Diazotrophic Activities Associated with Aggregates in the Permanent Ice Cover of Lake Bonney, Antarctica

Abstract The McMurdo Dry Valley lakes, Antarctica, one of the Earth's southernmost ecosystems containing liquid water, harbor some of the most environmentally extreme (cold, nutrient-deprived) conditions on the planet. Lake Bonney has a permanent ice cover that supports a unique microbial habitat, provided by soil particles blown onto the lake surface from the surrounding, ice-free valley floor. During continuous sunlight summers (Nov.-Feb.), the dark soil particles are heated by solar radiation and melt their way into the ice matrix. Layers and patches of aggregates and liquid water are formed. Aggregates contain a complex cyanobacterial-bacterial community, concurrently conducting photosynthesis (CO2 fixation), nitrogen (N2) fixation, decomposition, and biogeochemical zonation needed to complete essential nutrient cycles. Aggregate-associated CO2- and N2-fixation rates were low and confined to liquid water (i.e., no detectable activities in the ice phase). CO2 fixation was mediated by cyanobacteria; both cyanobacteria and eubacteria appeared responsible for N2 fixation. CO2 fixation was stimulated primarily by nitrogen (NO3-), but also by phosphorus (PO43-). PO43- and iron (FeCl3 + EDTA) enrichment stimulated of N2 fixation. Microautoradiographic and physiological studies indicate a morphologically and metabolically diverse microbial community, exhibiting different cell-specific photosynthetic and heterotrophic activities. The microbial community is involved in physical (particle aggregation) and chemical (establishing redox gradients) modification of a nutrient- and organic matter-enriched microbial "oasis," embedded in the desertlike (i.e., nutrient depleted) lake ice cover. Aggregate-associated production and nutrient cycling represent microbial self-sustenance in a microenvironment supporting "life at the edge," as it is known on Earth.     

Yield reduction and mycotoxin contamination of wheat due to<Emphasis Type="Italic">Fusarium culmorum</Emphasis>

The inoculation of wheat ears with 27 isolates ofFusarium culmorum in growth stage 65 reduced 1000-grain weights by 14 to 61%. For the phytopathological characterisation of isolates the virulence on primary wheat leaves and the growth rate an potato-dextrose-agar were assessed. Deoxynivalenol-producing isolates ofF. culmorum reduced the 1000-grain weight more than nivalenol-producing isolates.     

New species and new record of Tetranychus Dufour from Australia, with a key to the major groups in this genus based on females (Acari: Prostigmata: Tetranychidae)

A new species of spider mite, Tetranychus bunda sp. n., is described and illustrated from Australia. It was found damaging the foliage of Desmodium tortuosum (Sw.) DC. (Fabaceae) in Darwin, Northern Territory. In addition, the geographical range of Tetranychus fijiensis Hirst is extended to include Australia. This species was found in the Northern Territory feeding on frangipani ( Plumeria sp., Apocynaceae), betel palm ( Areca catechu L., Arecaceae) and Macarthur feather palm ( Ptychosperma macarthurii [H. Wendl. ex Veitch] (H. Wendl. ex Hook. f., Arecaceae)). Details of the biology of T. bunda sp. n. and T. fijiensis are given. A key to the major groups of Tetranychus Dufour of the world, based on females, is presented and species known to occur in Australia are outlined.     

The murine gammaherpesvirus 68 v-cyclin gene is an oncogene that promotes cell cycle progression in primary lymphocytes

Several gammaherpesviruses contain open reading frames encoding proteins homologous to mammalian D-type cyclins. In this study, we analyzed the expression and function of the murine gammaherpesvirus 68 (gammaHV68) viral cyclin (v-cyclin). The gammaHV68 v-cyclin gene was expressed in lytically infected fibroblasts as a leaky-late mRNA of approximately 0.9 kb encoding a protein of approximately 25 kDa. To evaluate the effect of the gammaHV68 v-cyclin on cell cycle progression in primary lymphocytes and to determine if the gammaHV68 v-cyclin gene is an oncogene, we generated transgenic mice by using the lck proximal promoter to express the gammaHV68 v-cyclin in early T cells. Expression of the gammaHV68 v-cyclin significantly increased the number of thymocytes in cell culture, as determined by measuring both DNA content and incorporation of 5-bromo-2-deoxyuridine following in vivo pulse-labeling. Expression of the gammaHV68 v-cyclin interfered with normal thymocyte maturation, as shown by increased numbers of CD4(+) CD8(+) double-positive thymocytes and decreased numbers of CD4(+) or CD8(+) single-positive and T-cell-receptor-bright thymocytes and splenocytes in transgenic mice. Despite increased numbers of cycling thymocytes, gammaHV68-v-cyclin-transgenic mice did not have proportionately increased thymocyte numbers, and staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling demonstrated increased apoptosis in the thymi of v-cyclin-transgenic mice. Fifteen of 38 gammaHV68-v-cyclin-transgenic mice developed high-grade lymphoblastic lymphoma between 3 and 12 months of age. We conclude that (i) the gammaHV68 v-cyclin is expressed as a leaky-late gene in lytically infected cells, (ii) expression of the gammaHV68 v-cyclin in thymocytes promotes cell cycle progression and inhibits normal T-cell differentiation, and (iii) the gammaHV68 v-cyclin gene is an oncogene.     

Ligand-induced conformational changes in a thermophilic ribose-binding protein

Background

Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments.

Results

Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation ( ^app T _m value is 108°C) than the mesophilic Escherichia coli homolog (ecRBP) ( ^app T _m value is 56°C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved.

Conclusion

Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.     

Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase

A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.     

The hydrophobic core of Escherichia coli thioredoxin shows a high tolerance to nonconservative single amino acid substitutions.

A set of single amino acid substitutions has been constructed at positions Leu42 and Leu78 in the hydrophobic core of Escherichia coli thioredoxin. This protein is required for the in vivo assembly of filamentous bacteriophages such as M13. Almost all the mutants retain this activity regardless of the change in size, hydrophobic nature, or charge of the substitution. Determination of the free energies of unfolding of the mutants containing charged residues shows that these are significantly destabilized as would be expected from simple considerations of the hydrophobic effect. Thioredoxin therefore represents a class of proteins where the often observed correlation between a particular biological activity and thermodynamic stability is not evident for single mutants in the all-or-none assay used. Native thioredoxin is very stable. Thus, structurally single mutants may not perturb the folding equilibrium or the dynamic behavior sufficiently for the effects to be sensed in vivo.     

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.     

Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein-protein interactions directly from DNA

Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein-protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody-affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed.