Searching for Moniliophthora perniciosa pathogenicity genes.

The basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as "Defence genes, Virulence, and Cell response" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen's own survival in the hostile environment.

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production.

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Obtaining and selection of hexokinases-less strains of Saccharomyces cerevisiae for production of ethanol and fructose from sucrose.

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract-peptone-fructose. In fermentations with a medium containing sucrose (180.3 g L(-1)) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L(-1)) and ethanol production (until 42.3 g L(-1)) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.

Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6.

The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1,10-phenanthroline characterized Q1 enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca(2+). ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum.

Monoclonal antibodies to identify tomato mosaic tobamovirus (TOMV)

Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV) isolated in Brazil. One antibody (8G7G2) isotyped as IgG2b (k light chain) showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV). It can be used in identification of tomato mosaic virus (ToMV).

Minisatellite probes in yeast DNA fingerprinting

As sequências 33.6, 33.15, M13 e R18.1 foram utilizadas como sondas para a análise do parentesco de linhagens de leveduras. A sonda R18.1 originária de um banco genômico bovino mostrou intensa hidridizaçäo, produzindo perfis com alto grau de polimorfismo do DNA em Saccharomyces cerevisae e Kluyveromyces marxianus. Nas comparaçöes intra e interespecíficas, os perfis polimórficos permitiram identificaçäo de todas as linhagens, mesmo aquelas altamente relacionadas

Atividade da inulinase em leveduras e fermentaçöes de extratos de Helianthus tuberosus L / Inulinase activity in yeasts and fermentations of Helranthus tuberosus L extracts

Devido às concentraçöes de inulina em H. tuberosus, leveduras com boa produçäo de inulinase e alto potencial fermentativo säo imprescindíveis na viabilizaçäo industrial deste vegetal. Selecionando-se leveduras com estas propriedades, dentre 21 espécies avaliadas, encontram-se 11 linhagens com boa capacidade fermentativa. As leveduras com maior rendimento de produçäo de álcool foram Kluyveromyces marxianus, K. fragilis, Candida pseudotropicalis e Torulopsis coelliculosa. Em fermentaçöes sucessivas conduzidas a 30-C, com 15,6% de ART e por 48 horas, chegou-se a obter o rendimento médio de 85% em relaçäo ao teórico. Nestas condiçöes, após 10 horas de fermentaçäo encontraram-se rendimentos de 74,4% e uma taxa de fermentaçäo de 5,9g/1/h**-1. A atividade máxima da inulinase foi de 3,6micronM (equivalente frutose)/ml/min e verificou-se ocorrer repressäo catabólica da inulinase por frutose em K. marxianus e K. fragilis. Com estes resultados conclui-se ser viável a fermentaçäo de extratos sem prévia hidrólise, o que facilitará o processamento industrial de extratos de H. tuberosus