Flavio Cesar Almeida Tavares

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.

Evaluation of biochemical and serological methods to identify and clustering yeast cells of oral Candida species by CHROMagar test, SDS-PAGE and ELISA

The purpose of this work was to evaluate biochemical and serological methods to characterize and identify Candida species from the oral cavity. The strains used were five Candida species previously identified: C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. tropicalis, and Kluyveromyces marxianus, as a negative control. The analyses were conducted through the SDS-PAGE associated with statistical analysis using software, chromogenic medium, and CHROMagar Candida (CA), as a differential medium for the isolation and presumptive identification of clinically important yeasts and an enzyme-linked immunoabsorbent assay (ELISA), using antisera produced against antigens from two C. albicans strains. This method enabled the screening of the three Candida species: C. albicans, C. tropicalis, and C. krusei, with 100% of specificity. The ELISA using purified immunoglobulin G showed a high level of cross-reaction against protein extracts of Candida species. The SDS-PAGE method allowed the clustering of species-specific isolates using the Simple Matching coefficient, S(SM) = 1.0. The protein profile analysis by SDS-PAGE increases what is known about the taxonomic relationships among oral yeasts. This methodology showed good reproducibility and allows collection of useful information for numerical analysis on information relevant to clinical application, and epidemiological and systematical studies.

Produção e qualidade de frutos de diferentes cultivares de morangueiro em ensaios conduzidos em Atibaia e Piracicaba

Field experiments were carried out in Sao Paulo State (Brazil), in Atibaia, Cfb climate and Piracicaba, Cwa climate, in 1996. The experimental design was of randomized blocks with four replications and sixteeen plants per plot using the cultivars Campinas, Dover, Guarani, Princesa Isabel and AGF 080. The presence of neck, shape, yield, number and average weight, amount of soluble solids, pH, external and internal color, texture and components of yield and of the fruits were evaluated. Atibaia experiment resulted in greater production of fruits (468,30 g/plant), greater number of fruits (46,75 fruits/plant) and greater fruit mean weight (10,06 g) in relation to Piracicaba. Characteristics of the strawberry fruit and plant were not influenced by local effects. The internal color measures, texture and neck presence indicate the cv. Guarani suitable for industrial purposes. In function of the weight measures, amount of soluble solids, pH and texture, the Campinas, Agf 80 and Princesa Isabel cvs. are the most suitable for the fresh market while Guarani and Dover are inadequate. Guarani, Dover and Princesa Isabel are the most resistant cvs. to transportation and Campinas and Agf 80 are the less resistant.

A simple method for DNA isolation from Xanthomonas spp.

A simple DNA isolation method was developed with routine chemicals that yields high quality and integrity preparations when compared to some of the most well known protocols. The method described does not require the use of lysing enzymes, water bath and the DNA was obtained within 40 minutes The amount of nucleic acid extracted (measured in terms of absorbancy at 260 nm) from strains of Xanthomonas spp., Pseudomonas spp. and Erwinia spp. was two to five times higher than that of the most commonly used method.

Imobilização da inulinase de Kluyveromyces marxianus para a hidrólise de extratos de Helianthus tuberosus L.

This experiment studied the immobilization of inulinase from Kluyveromyces marxianus in different supports to bioconvert the inulin from Helianthus tuberosus. Inulin was extracted from H. tuberosus tubers, desproteinized and concentrated to 25% total reducing sugars. Inulinase from K. marxianus was concentrated in a rotative evaporator and immobilized onto chitin (with and without glutaraldehyde), sodium alginate (2 and 4%), pectin, a dialysis membrane or controlled-porosity silicate. On chitin, with or without glutaraldehyde, the imobilization rate was 73 and 48 U g-1 respectively. However the hydrolysys of 1g L-1 inulin was very low in both treatments (2.4 % per hour). In sodium alginate gel of 2% and 4% concentration, the conversion was 12% and 26% per hour, respectively. Immobilization onto pectin was not possible due to a high activity pectinase in the enzyme extract. Binding of the enzyme onto dialysis membrane provided recovery of 50% total reducing sugars (42g) in 6h of operation. The controlled-porosity silicate showed an imobilization rate of 43 U g-1 silicate, hydrolyzing 43% of substrate per hour. This activity was, however, exhausted quickly during the process.

SDS-Page and numerical analysis of Candida albicans from human oral cavity and other anatomical sites

O objetivo da presente pesquisa foi analisar os graus de polimorfismos proteicos entre isolados de C. albicans provenientes de diversos sitios anatomicos de quarenta e dois pacientes clinicos, atraves do emprego da eletroforese em gel de poliacrilamida (SDS-PAGE) e analise numerica, a fim de se identificar subespecies e suas similaridades nos diversos nichos infecciosos. Culturas celulares foram desenvolvidas em meio YEPD, coletadas por centrifugacao e lavadas com solucao salina gelada. As proteinas celulares totais, foram extraidas por rompimento celular, usando perolas de vidro e submetidas a tecnica de SDS-PAGE. Apos a eletroforese, as bandas de proteinas foram coradas com coomassie-blue e analisadas pelo conjunto de programas estatistico NTSYS-pc versao 1,70. Matrizes de similaridade e dendrogramas foram gerados pela aplicacao do coeficiente de similaridade simple-matching e do algoritmo UPGMA, respectivamente. Os resultados obtidos revelaram varios subtipos de C. albicans e seus graus de similaridade (80% a 100%). Tais dados permitiram demonstrar que, certos pacientes podem estar infectados com dois ou mais subtipos de C. albicans em determinados sitios anatomicos (i.e. apenas na cavidade oral de pacientes imunocomprometidos, sangue ou secrecao traqueal), ou ainda, dois ou mais pacientes podem estar infectados em sitios anatomicos identicos (i.e. apenas em lavagem bronquica, urina, cavidade oral, secrecao traqueal, secrecao vaginal ou saliva saudavel) com um mesmo subtipo de C. albicans. No entanto, dois ou mais pacientes tambem podem apresentar infeccoes em sitios correspondentes (i.e. apenas na cavidade oral de pacientes imunocomprometidos, sangue, secrecao orofaringea, cavidade oral, secrecao traqueal, secrecao vaginal e saliva saudavel) por diferentes subtipos de C. albicans. Alem disso, dois ou mais pacientes tambem podem estar infectados com subtipos identicos ou nao de C. albicans em diferentes sitios anatomicos (i.e.1. identicos subtipos na secrecao vaginal, secrecao traqueal e urina; secrecao abdominal e escarro; drenagem e cavidade oral; cateter e saliva saudavel - i.e.2. diferentes subtipos em lavagem bronquica, secrecao orofaringea, secrecao pulmonar, cavidade oral de pacientes imunocomprometidos e sangue). Dados complementares envolvendo amostras de C. albicans isoladas de varios sitios anatomicos de pacientes imunocompetentes ou imunocomprometidos (antes, durante e apos terapias especificas) e seus familiares ou trabalhadores hospitalares, deverao ser obtidos a fim de se estabelecer as possiveis fontes de colonizacao por esses microrganismos. De modo geral, os perfis de proteinas totais obtidos por SDS-PAGE associados com analise numerica computadorizada, permitem a obtencao de criterios adicionais para os estudos epidemiologicos e taxonomicos de C. albicans.

Control of spoilage yeasts in fuel ethanol production

A selective process has been developed to control contaminant wild yeasts in industrial ethanol production. A genetically improved yeast resistant to nystatin is essential and the economic use of this antibiotic is described. The process strain starts at the stage of pure culture development and if necessary, after cycles of continuous cultures or fed batches, with no need of special equipment or operational process modifications. The principle can be adopted in other fermentations.SummaryA selective process has been developed to control contaminant wild yeasts in industrial ethanol production. A genetically improved yeast resistant to nystatin is essential and the economic use of this antibiotic is described. The process strain starts at the stage of pure culture development and if necessary, after cycles of continuous cultures or fed batches, with no need of special equipment or operational process modifications. The principle can be adopted in other fermentations.

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).

A vector carrying the GFP gene (Green fluorescent protein) as a yeast marker for fermentation processes

Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3). Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.

Isolation and characterization of Metolachlor-resistant mutants of Saccharomyces cerevisiae

The herbicide Metolachlor (α-chloroacetamide group) inhibits the growth of Saccharomyces cerevisiae on complete, minimal, and non-fermentative media. Spontaneous and induced resistant mutants showed monogenic segregation patterns. Among the resistant clones, 70% were recessives, 16.4% were partially dominants and 13.4% were dominants. The spontaneous partially dominant mutation Mtc1 was mapped on linkage group XV at 33.3 cM from ade2 and 31.7 cM from his3, in a region that is characterized by the presence of several resistant genes. The recessive mutation mtc2 was located on chromosome IV. Although all the mutants had the ability to grow in the presence of the herbicide, they remained affected in their respiration efficiency, indicating two different mechanism of action of Metholachor on yeast cells.