Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Alicaligenes faecalis : identification and study of its antagonistic properties against Botrytis cinerea

A Gram negative aerobic flagellated bacterium with fungal growth inhibitory properties was isolated from a culture of Trichoderma harzianum. According to its cultural characteristics and biochemical properties it was identified as a strain of Alcaligenes (aeca/is Castellani and Chalmers. Antisera prepared in Balbc mice injected with live and heat-killed bacterial cells gave strong reactions with the homologous immunogen and with ATCC 15554, the type strain of A. taeca/is, but not with Escherichia coli or Enterobacter aerogens in immunoprecipitation and dot immunobinding assays. Growth of Botrytis cinerea Pers. and several other fungi was significantly affected when co-cultured with A. taeca/is on solid media. Its detrimental effect on germination and growth of B. cinerea has been found to be associated with antifungal substances produced by the bacterium and released into the growth medium. A biotest for the antibiotic substances, based on their inhibitory effect on germination of B. cinerea conidia, was developed. This biotest was used to study the properties of these substances, the conditions in which they are produced, and to monitor the steps of their separation during extraction procedures. It has been found that at least two substances could be involved in the antagonistic interaction. One of these is a basic volatile substance and has been identified as ammonia. The other substance is a nonvolatile, dialysable, heat stable, polar compound released into the growth medium. After separation of growth medium samples by Sephadex G-10 column chromatography a single peak with a molecular weight below 700 Daltons exhibited inhibitory activity. From its behaviour in electrophoretic separation in agarose gels it seems that this is a neutral or slightly positively charged.

Probing cell surfaces of host and nonhost species of a mycoparasite, using FITC-labeled lectins

Cell surfaces of susceptible host species (Mortierella pusllla and Cboanepilora cucurbitarum ), resistant host (Pilascolomyces articulosus ), nonhost (Mortierella candelabrum ) and the mycoparasite (Piptocepilalis virginiana) were examined for sugar distribution patterns using light and fluorescent microscopy techniques. The susceptible host, resistant host and the mycoparasite species exhibited a similar sugar distribution profile; they all showed N-acetyl glucosamine and D-glucose on their cell wall surfaces. The nonhost cell wall surface showed a positive binding reaction to FITClectins specific for N-acetyl glucosamine and also for OI.-fucose, N-acetyl galactosamine and galactose. Treatment of these fungi with mild concentrations of proteinases (both commercial as well as the mycoparasiteproteinase) resulted in the revelation of additional sugars on the fungal cell walls. The susceptible host treated with proteinase expressed higher levels of N-acetyl glucosamine and D-glucose. The susceptible host also showed the presence of OI.-fucose, N-acetyl galactosamine and galactose. The proteinasetreated susceptible host cell walls also showed an increase in the levels of attachment with the mycoparasite. Treatment of the resistant host with proteinases revealed OI.-fucose in addition to N-acetyl glucosamine and D-glucose. Treatment of the nonhost cell wall with proteinase resulted in the exposure of low levels of D-glucose, in addition to sugars found on the untreated nonhost cell wall surface. The mycoparasite treated with proteinase revealed OI.-fucose, N-acetyl galactosamine and galactose on its cell surface in addition to the sugars N-acetyl glucosamine and D-glucose. Protoplasts were isolated from hosts and nonhost fungi and their surfaces were examined for sugar distribution patterns. The susceptible host and nonhost protoplast membranes showed all the sugars (N-acetyl glucosamine, D-glucose, (It.-fucose, N-acetyl galactosamine and galactose) tested for. The resistant host protoplast membrane however, had only N-acetyl glucosamine and D-glucose exposed. This sugar distribution profile resembles that exhibited by the untreated resistant host cell wall, as well as that shown by the untreated mycoparasite cell surface. Only susceptible host protoplasts were successful in attaching to the mycoparasite surface. Resistant host protoplasts did not show any interaction with the i mycoparasite cell surface. Both susceptible as well as resistant host protoplasts were incapable of attaching to agarose beads surface-coated with specific carbohydrates. The mycoparasite however, did attach to agarose beads surface-coated with either N-acetyl glucosamine, D-glucose/Dmannose or o:,- methyl-D-mannose. The relevance of the cell wall and the protoplast membrane in the light of the present results, in reacting appropriately to bring about either a susceptible, a resistant or a nonhost response has been discussed.

Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)

Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the a -Tocopherol Transfer Protein (a-TIP). Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli. outer membrane porin F (OmpF) that co-purified with 6-His-a-hTTP from the metal affinity column as the source of the problem. In order to completely avoid OmpF contamination, a GST -a-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that a-hTTP utilizes a collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s) indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and the packing density of phospholipid head groups within acceptor membranes. Based on the calculated free energy change (dG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of a-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc. The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.