Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin

In non-irrigated agricultural fields in tropical zones, high temperature and water stress prevail during the main cropping season. Natural epizootics of Beauveria bassiana on lepidopteran pests occur during winter. Application of B. bassiana during hot months when pest populations are at their climax may prove an effective management strategy. Therefore, 29 isolates of B. bassiana were tested for their ability to germinate and grow in temperature and water availability conditions prevailing during the pest season in these fields. The effect of temperature cycles with 8 h duration of high temperature fluctuating with 16 h duration of lower temperature (similar to field conditions); low water availability; and a combination of these two stress conditions was studied. Germination and growth assays were done at fluctuating temperature cycles of 32, 35, 38, and 42+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and in media with water stress created by 10, 20, 30, and 40% polyethylene glycol (PEG 6000). Assays set at a continuous temperature of 25+/-1 degrees C with no PEG in the medium served as controls. Stress was assessed as percentage germination or as growth relative to control. Isolates showing 90% growth relative to the control at temperature cycles including high temperatures of 35 and 38+/-1 degrees C were identified. One isolate (ARSEF 2860) had a thermal threshold above 43 degrees C. At 25 degrees C, all but one isolate of B. bassiana showed >90% growth relative to the control in 10% PEG (-0.45 MPa). Some isolates were found with >90% growth relative to control in medium having 30% PEG with water availability (1.33 MPa), nearly equivalent to that in soils which induce permanent wilting point of plants. When isolates that showed >90% growth relative to the control at both stress conditions, were stressed simultaneously, a decrease in growth was observed. Growth was reduced by approximately 20% at 35+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG and was affected to a greater degree in combinations of harsher stress conditions. The isolate ARSEF 2860 with a thermal threshold of >43 degrees C showed approximately 80% relative growth at a combined stress of 38+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG. These findings will aid the selection of isolates for use in field trials in hot or dry agricultural climates.     

Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.     

Substitutions at the Asp-473 latch residue of chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO(2)/O(2) specificity

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO(2) and O(2). Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V(max)/K(m)) and approximately 16% decreases in CO(2)/O(2) specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO(2)/O(2) specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.     

Structure and transport mechanism of the bacterial oxalate transporter OxlT

Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, "closed" state of OxlT at 6.5 A determined by cryo-electron microscopy with a model of the "open" state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter.     

Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.     

In vitro antibacterial activity in seed extracts of Manilkara zapota, Anona squamosa, and Tamarindus indica

Extracts prepared from seeds of Manilkara zapota, Anona squamosa, and Tamarindus indica were screened for their antibacterial activity by disc diffusion and broth dilution methods. Acetone and methanol extracts of T. indica seeds were found active against both gram-positive and gram-negative organisms. MIC values of potent extracts against susceptible organisms ranged from 53-380 μg/mL. Methanol extract of T. indica and acetone extract of M. zapota seeds were found to be bactericidal.     

CD44 differentially activates mouse NK T cells and conventional T cells

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.     

Novel Mutations that Enhance or Repress the Aggregation Potential of SOD1

Mutations in SOD1 cause FALS by a gain of function likely related to protein misfolding and aggregation. SOD1 mutations encompass virtually every domain of the molecule, making it difficult to identify motifs important in SOD1 aggregation. Zinc binding to SOD1 is important for structural integrity, and is hypothesized to play a role in mutant SOD1 aggregation. To address this question, we mutated the unique zinc binding sites of SOD1 and examined whether these changes would influence SOD1 aggregation. We generated single and multiple mutations in SOD1 zinc binding residues (H71, H80 and D83) either alone or in combination with an aggregate forming mutation (A4V) known to cause disease. These SOD1 mutants were assayed for their ability to form aggregates.Using an in vitro cellular SOD1 aggregation assay, we show that combining A4V with mutations in non-zinc binding domains (G37R or G85R) increases SOD1 aggregation potential. Mutations at two zinc binding residues (H71G and D83G) also increase SOD1 aggregation potential. However, an H80G mutation at the third zinc binding residue decreases SOD1 aggregation potential even in the context of other aggregate forming SOD1 mutations. These results demonstrate that various mutations have different effects on SOD1 aggregation potential and that the H80G mutation appears to uniquely act as a dominant inhibitor of SOD1 aggregation.