Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

玫烟色棒束孢Pr1酶活力、Pr1酶基因表达量与其毒力的相关性

丝氨酸弹性凝乳蛋白酶Pr1是一类能高效降解昆虫体壁蛋白的重要酶,其活力与虫生真菌的毒力有很大的关系。探索玫烟色棒束孢不同菌株Pr1酶活力、Pr1蛋白酶基因表达量与毒力的相关性对该菌的应用具有重要的意义。文中采用专一性短肽底物Suc-Ala-Ala-Pro-Phe-pNA和荧光定量PCR分别测定了玫烟色棒束孢不同菌株的Pr1酶活和Pr1基因的表达量,并采用坡塔喷雾法测定了供试菌株对桃蚜的毒力。结果表明:不同供试菌株Pr1蛋白酶活力与其毒力的线性回归方程为y=3.64x+0.62,R~2=0.432,两者呈正相关;供试菌株Pr1酶活力、Pr1基因表达量与毒力的回归方程为y=0.236+10.833x_1–0.039x_2 (x_1=Pr1酶活力,x_2=Pr1基因表达量),R~2=0.568,说明线性拟合方程能很好地反映原始数据;序列相关系数D-W为2.444,在0.05水平上相关显著,表明Pr1酶活力、Pr1基因表达量对毒力有显著影响;VIF=12.705表明Pr1酶活力、Pr1基因表达量存在中度多重共线性。因此建议将Pr1蛋白酶的酶活力和Pr1基因表达量作为菌株毒力筛选时的重要指标。     

Phytochromes from Agrobacterium tumefaciens: difference spectroscopy with extracts of wild type and knockout mutants

Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.     

Cuticle-Degrading Proteases Produced by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Their Induction in Different Media

Protease production by fourteen M. anisopliae isolates differing in geographical origin and host insect were investigated. Highest protease activity was observed during 4–8 days of culture incubation. Pr1 and Pr2 activity was investigated in various media containing different carbon and nitrogen source to evaluate the induction mechanism of these enzymes. Basal levels of Pr1 and Pr2 activity were observed in minimal medium suggesting constitutive production. Casein (1%) as an exogenous protein supplement was not able to induce significant release of Pr1 and Pr2 enzymes, whereas high levels of activity were observed in the medium containing colloidal chitin (2%) as sole carbon and nitrogen source. The pH, ammonia and oxalic acid production in in vitro conditions was also investigated and the alteration in pH for protease production was not significant in the different media used except for the medium containing casein (1%) as a supplement.     

Triclinic structural and magnetic properties of praseodymium(III) picrate triethylene glycol complex

A study on the structure and magnetic properties of [Pr(NO₃)(Pic)(H₂O)₂(EO3)](Pic) complex, where EO3 = triethylene glycol and Pic = picrate anion was conducted and characterized by single crystal X-ray structure analysis. Magnetic susceptibility (χ_M) was carried out from 2 to 300 K under both field-cooled (FC) and zero-field-cooled (ZFC) measurements with an applied magnetic field of 2000 Oe. The complex is crystallized in triclinic with space group . The coordination geometry around the Pr(III) ion was a tetradecahedron with a ten-coordination number. In the crystal, the molecular structure was stabilized by moderate and weak hydrogen bonding interactions between the cation [Pr(NO₃)(Pic)(H₂O)₂(EO3)]⁺ moiety and [Pic]⁻ as counter-anion that led to the formation of a one-dimensional network. The temperature dependence of the magnetic susceptibility of [Pr(NO₃)(Pic)(H₂O)₂(EO3)](Pic) shows the presence of weak antiferromagnetic interactions between the Pr(III) centers. The magnetic susceptibility for complex also obeys the Curie-Weiss law and is effective at high temperatures. Some factors that influence the photoluminescence intensity were also reported.     

Ionic complexation properties of per(3,6-anhydro)cyclodextrin derivatives towards lanthanides

Using per(3,6-anhydro)cyclodextrin derivatives [per(3,6-anhydro)CD], it was possible to produce new lanthanide chelates by careful choice of the size and functional groups. Heptakis(3,6-anhydro-2-O-methyl)cyclomaltoheptaose fulfils the best criteria for complexation of lanthanide ions. Nuclear magnetic resonance was used to derive the association constants and the stoichiometries of these new complexes. Finally, a three-dimensional structure of these complexes consistent with the NMR data is proposed, to ascertain the position of lanthanide in the cavity of the per(3,6-anhydro)CD. For the present purposes, heptakis(2-O-acetyl-3,6-anhydro)cyclomaltoheptaose, octakis(2-O-acetyl-3,6-anhydro)cyclomaltooctaose, heptakis(3,6-anhydro-2-O-methyl)cyclomaltoheptaose and octakis(3,6-anhydro-2-O-methyl)cyclomaltooctaose have been synthesized and purified.     

Metal-mediated and metal-catalyzed hydrolysis of nitriles

The essential goals of this review are the following: (i) to verify various factors which affect the metal-mediated hydrolysis of organonitriles; (ii) to draw attention to unusual conversions of RCN species, yet underdeveloped and non-systematic, which involve hydrolysis and lead to compounds of synthetic and/or pharmacological significance. The metal-mediated and/or metal-catalyzed reactions of RCN species are surveyed and the experimental material on metal-mediated hydration of RCN species at diverse metal centers along the Periodic Table is summarized in a tabular form.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

Electronic and fluorescent studies on the interaction of DNA and BSA with a new ternary praseodymium complex containing 2,9-dimethyl 1,10-phenanthroline and antibacterial activities testing

In this study, fluorescence emission spectra, UV–vis absorption spectra, ethidium bromide (EB)-competition experiment, and iodide quenching experiment were used for the interaction study of the Fish salmon DNA (FS-DNA) with [Pr(dmp)2Cl3(OH2)] where dmp is 2,9-dimethyl 1,10-phenanthroline. The binding constant and the number of binding sites of the complex with FS-DNA were 6.09?±?0.04 M^?1 and 1.18, respectively. The free energy, enthalpy, and entropy changes (ΔG°, ΔH°, and ΔS°) in the binding process of the Pr(III) complex with FS-DNA were –8.02?kcal mol^?1, +39.44?kcal mol^?1, and +159.56?cal mol^?1 K^?1, respectively. Based on these results, the interaction process between FS-DNA with [Pr(dmp)2Cl3(OH2)] was spontaneous and the main binding interaction force was groove binding mode. Also, Fluorescence and electronic absorption spectroscopy were used in order to evaluate the binding characteristics, stoichiometry, and interaction mode of praseodymium(III) (Pr(III)) complex with bovine serum albumin (BSA). Title complex showed good binding propensity to BSA presenting moderately high Kb values. The fluorescence quenching of BSA by Pr(III) complex has been observed to be the static process. The positive ΔH° and ΔS° values showed that the hydrophobic interaction is the main force in the binding of Pr(III) complex and BSA. Eventually, the average aggregation number, <J>, of BSA potentially induced by title complex confirmed the 1:1 stoichiometry for title complex-BSA adducts. In vitro, antimicrobial activity of title complex was indicated that the complex is more active against both Escherichia coli and Enterococcus faecalis bacterial strains than Staphylococcus aureus, and Pseudomonas aeruginosa.

Communicated by Ramaswamy H. Sarma