Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (approximately 14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.     

The genes gmdA, encoding an amidase, and bzuA, encoding a cytochrome P450, are required for benzamide utilization in Aspergillus nidulans

Two unlinked loci, gmdA and bzuA, have previously been identified as being required for the utilization of benzamide as the sole nitrogen source by Aspergillus nidulans. We have cloned each of these genes via direct complementation. The gmdA gene encodes a predicted product belonging to the amidase signature sequence family that displays similarity to AmdS from A. nidulans. However, identity is significantly higher to the amdS gene from Aspergillus niger. The bzuA gene encodes a protein belonging to the cytochrome P450 superfamily and is orthologous to the benzoate para-hydroxylase-encoding gene bphA of A. niger. The bzuA1 mutation prevents the use of benzoate as a carbon source and intracellular accumulation of benzoate results in growth inhibition on benzamide. Northern blot analysis has shown that gmdA expression is subject solely to AreA-dependent nitrogen metabolite repression while bzuA is strongly benzoate inducible and subject to CreA-mediated carbon catabolite repression and a probable inactivation of benzoate induction by glucose. Fluorescence microscopy of a fusion of the N-terminal end of BzuA to green fluorescent protein revealed that this protein localizes to the endoplasmic reticulum.     

Effects of LETS glycoprotein on cell motility

Addition of LETS glycoprotein to normal or transformed cells produces increased migration of the cells, as determined by formation of phagokinetic tracks on gold particle-coated coverslips. These tracks arise by a combination of phagocytosis of the gold particles and cellular migration. Increased motility is also evident on plastic in the absence of gold particles. The added LETS protein attaches to the cells in a fibrillar network, and binding is greater to normal than to transformed cells. The effects of LETS protein on migration are consistent with its effects on cell adhesion, morphology and cytoskeleton, and have potential implications for the determination of cellular migration in vivo.     

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P. marneffei, albeit to differing degrees. Based on this functional characterisation and high degree of sequence conservation with other fungal GATA factors, P. marneffei areA represents an orthologue of Aspergillus nidulans areA and Neurospora crassa NIT2. Based on this study it is proposed that AreA is likely to contribute to the pathogenicity of P. marneffei by facilitating growth in the host environment and regulating the expression of potential virulence factors such as extracellular proteases.     

The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico "matrisome" defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.     

Overview of the matrisome--an inventory of extracellular matrix constituents and functions

Completion of genome sequences for many organisms allows a reasonably complete definition of the complement of extracellular matrix (ECM) proteins. In mammals this "core matrisome" comprises ～300 proteins. In addition there are large numbers of ECM-modifying enzymes, ECM-binding growth factors, and other ECM-associated proteins. These different categories of ECM and ECM-associated proteins cooperate to assemble and remodel extracellular matrices and bind to cells through ECM receptors. Together with receptors for ECM-bound growth factors, they provide multiple inputs into cells to control survival, proliferation, differentiation, shape, polarity, and motility of cells. The evolution of ECM proteins was key in the transition to multicellularity, the arrangement of cells into tissue layers, and the elaboration of novel structures during vertebrate evolution. This key role of ECM is reflected in the diversity of ECM proteins and the modular domain structures of ECM proteins both allow their multiple interactions and, during evolution, development of novel protein architectures.     

Insecticidal activities of Ginkgo biloba seed coat extracts against Spodoptera exigua (Lepidoptera: Noctuidae)

The present study relates to a methanol extract of the seed coat of Ginkgo biloba, and tested particularly on the third instar larvae of Spodoptera exigua. The extract was found to have an inhibitory effect on the growth of the larvae besides bringing a change in the nutrient reserves in the body of the insect. Topical application of five different doses of the methanol extract resulted in a mortal effect to third instar larvae of S. exigua that is very much dependent on the dose as well as duration of exposure. Lower doses revealed lower mortality after 24 h of application. At doses of 1.00, 2.00, 4.00, 8.00 and 16.00 ng/larva, mortalities were 9.25, 26.07, 50.32, 56.28 and 92.44%, respectively. The dose for 50% mortality (LD₅₀) of methanol extracts by applied by a topical method with 1 µL of acetone solution was 1.92 ng/larva. Nutrient reserves like protein, glycogen and lipid are known to regulate pupation and adult emergence. These reserves have been found to be lower in treated larvae, indicating the insecticidal role of methanol extracts from G. biloba against third instar larvae of S. exigua.     

ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth

Saccharomyces cerevisiae yeast cells containing the chaperonin CCT (chaperonin-containing t-complex polypeptide 1 (TCP-1)) with the G345D mutation in subunit CCT4 (anc2-1) are temperature-sensitive for growth and display defects in organization of actin structure, budding and cell shape. In this first structure-function analysis of CCT, we show that this mutation abolishes both intra- and inter-ring cooperativity in ATP binding by CCT. The finding that a single mutation in only one subunit in each CCT ring has such drastic effects highlights the importance of allostery for its in vivo function. These results, together with other kinetic data for wild-type CCT reported in this study, provide support for the sequential model for ATP-dependent allosteric transitions in CCT.