Phytase production through response surface methodology and molecular characterization of Aspergillus fumigatus NF191.

Phytase play an important role in phytic acid catalysis that act as a food inhibitor in cereals. Here, we isolated high phytase producing isolates NF191 closely related to Aspergillus fumigatus sp. from piggery soil. DNA was isolated from the fungal culture and amplified the ITS region using ITS1 and ITS4 primer using PCR. The 400-900 bp amplicon was gel eluted and subjected to sequencing. The sequencing results were assembled and compared with NCBI data base which showed the 99% identity of Aspergilllus fumigatus. Different carbon sources viz., fructose, galactose, lactose, dextrose, sucrose, maltose and different nitrogen sources (organic & inorganic) NH4Cl, NH4NO3, (NH4)2SO4, KNO3, NaNO3, urea, yeast extract, peptone, beef extract were tested for optimal production. The 0.3% dextrose, 0.5% NH4NO3 and 96 h incubation time showed the best production and enzyme activity at 45 ºC incubation temperature. The selected parameters, dextrose, ammonium sulphate and incubation time, when employed with statistical optimization approach involving response surface optimization using Box Behnken Design, gave a 1.3 fold increase in phytase production compared to unoptimized condition.

Caracterización de los mecanismos de resistencia a azoles en aislados clínicos chilenos de Candida albicans / Characterization of azole resistance mechanisms in Chilean clinical isolates of Candida albicans

Introduction: The commensal yeast Candida albicans, can cause superficial or systemic candidiasis in susceptible hosts. In Chile, azole antifungals are the most widely used drugs in the treatment of candidiasis. In a previous study performed at our center, 2.1 and 1.6% of clinical isolates of C. albicans were found to be resistant to fluconazole and voriconazole, respectively. Objective: To characterize the resistance mechanisms involved in azoles resistance in Chilean clinical isolates. Methodology: Eight resistant, nine susceptible-dose dependent (SDD) and 10 susceptible strains (n: 27) were selected according to the Clinical Laboratory Standards Institute (CLSI) M27-S3 criteria, from vaginal and urine samples. Mutations in the 408-488 region of the ERG11 gene were studied by sequencing, and the relative expression of ERG11 gene and efflux pump genes CDR1, CDR2 and MDR1, was evaluated by quantitative real-time PCR (q-PCR). Results: No mutations were detected in the ERG11 gene and its overexpression was found only in 12.5% of the resistant strains (1/8). The most prevalent mechanism of resistance was the over-expression of efflux pumps (62.5%; 5/8). Conclusion: The study of the expression of efflux pumps by q-PCR could be a useful diagnostic tool for early detection of azole resistance in C. albicans.

Introducción: Candida albicans es una levadura comensal capaz de causar una infección oportunista en hospederos susceptibles denominada candidiasis, que puede ser superficial o sistémica. En Chile, los antifúngicos más utilizados para el tratamiento de las candidiasis son los azoles. En un estudio previo en nuestro centro, se detectó que 2,1 y 1,6% de cepas clínicas de C. albicans fueron resistentes a fluconazol y voriconazol, respectivamente. Objetivo: Caracterizar los mecanismos de resistencia involucrados en la resistencia a azoles en cepas clínicas chilenas. Metodología: Según los criterios del Clinical Laboratory Standards Institute (CLSI) M27-S3, se seleccionaron ocho cepas resistentes, nueve cepas susceptibles dosis dependiente (SDD) y 10 cepas sensibles (n: 27), aisladas de flujo vaginal y orina. Se evaluó la presencia de mutaciones en la región 408-488 del gen ERG11 por secuenciación y la expresión relativa del gen ERG11 y de los genes de bombas de eflujo CDR1, CDR2 y MDR1 por RPC en tiempo real cuantitativa (q-PCR). Resultados: No se encontraron mutaciones en el gen ERG11 y la sobre-expresión de éste sólo se presentó en 12,5% de las cepas resistentes (1/8). El mecanismo prevalente en la cepas resistentes fue la sobre-expresión de bombas de eflujo encontrándose en 62,5% de las cepas resistentes (5/8). Conclusión: El estudio de la expresión bombas de eflujo por q-PCR podría ser una herramienta diagnóstica útil para la detección temprana de resistencia a azoles en C. albicans.

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.

Functional complementation in Saccharomyces cerevisiae under control of the natural yeast promoter

A novel approach for functional complementation of foreign genes in Saccharomyces cerevisiae is presented. This approach is based on the use of the widely available cognate gene plasmids (e.g. pRS416) of the European Functional Analysis Network (EUROFAN). The functional complementation of the human homolog of YOR159c (SME1 gene) shown here is the first demonstration of complementation using the original yeast promoter, theoretically offering a more natural regulation of protein expression

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

In silico prediction of yeast deletion phenotypes

Analysis of gene deletions is a fundamental approach for investigating gene function. We evaluated an algorithm that uses classification techniques to predict the phenotypic effects of gene deletions in yeast. We used a modified simulated annealing algorithm for feature selection and weighting. The selected features with high weights were phylogenetic conservation scores for bacteria, fungi (excluding Ascomycota), Ascomycota (excluding Saccharomyces cerevisiae), plants, and mammals, degree of paralogy, and number of protein-protein interactions. Classification was performed by weighted k-nearest neighbor and with support vector machine algorithms. To demonstrate how this approach might complement existing experimental procedures, we applied our algorithm to predict essential genes and genes causing morphological alterations in yeast.

Cell cycle, DNA replication, repair, and recombination in the dimorphic human pathogenic fungus Paracoccidioides brasiliensis

DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.

Production of hydrolytic enzymes by Trichoderma isolates with antagonistic activity against Crinipellis perniciosa, the causal agent of witches' broom of cocoa

Two isolates of Trichoderma, which reduce the incidence of witches'broom disease caused in cocoa by Crinipellis perniciosa, were evaluated for their potential to produce hydrolases in liquid medium. Very low or no hydrolytic activity was produced in the absence of any substrate. The activities of chitinase, N-acetylglucosaminidase, beta-1,3-glucanase, total cellulase, endoglucanase, aryl- beta-glucosidase, beta-glucosidase, protease and amylase increased dramatically within 72-120 h of growth in the presence of specific substrates. Except for N-acetylglucosaminidase and beta-glucosidase Trichoderma harzianum isolate 1051 produced the largest amounts of hydrolases. The possible involvement of these enzymes in the antagonistic interaction between Trichoderma and C. perniciosa is discussed.

Pathogenicity characteristics of filamentous fungi strains isolated from processed oat

Nineteen strains of filamentous fungi isolated from processed oat were tested for pathogenecity factores, based on three parameters: growth at 37ºC, production of phospholipase and urease. "Aspergillus niveus", "Oidiidendron gryseum and "Spotothrix cyanescens" were positive for the three parameters. The other species were positive only for one or two of them