Luiz Humberto Gomes

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.

Fermentation and epiphytic microflora dynamics in sugar cane silage

Silagens de cana-de-acucar caracterizam-se pela extensa atividade de leveduras, alto teor de alcool e grandes perdas de materia seca - MS. Conhecer melhor o processo fermentativo e fundamental para o desenvolvimento de tecnicas eficientes de ensilagem da cana. Este trabalho avalia a mudanca temporal na composicao quimica, nas perdas de MS e na microflora epifita nestas silagens. Cana-de-acucar (RB835486) foi colhida manualmente (12 meses de crescimento), picada em picadora estacionaria e ensilada em baldes de plastico de 20 L com valvulas para gases e aparato para colheita de efluentes. Os silos laboratoriais foram mantidos sob temperatura ambiente e amostrados apos ½, 1, 2, 3, 7, 15, 45, 90, 120 e 180 dias. Etanol atingiu 6,4% na MS no 15o dia apos ensilagem, seguido pelo desaparecimento de 71% dos carboidratos soluveis - CHOs. As perdas gasosas e a perda total de MS estabilizaram-se apos 45 dias (16% e 29% da MS). A contagem de leveduras foi maxima no segundo dia (5,05 log ufc g-1). O pH atingiu nivel abaixo de 4,0 no terceiro dia. A producao de efluentes foi insignificante (20,1 kg t-1). O teor de MS da forragem decresceu (35% para 26%) do dia 0 ao 45o dia. O padrao de variacao na concentracao de etanol foi inverso a concentracao de CHOs e a reducao da digestibilidade da silagem. O desenvolvimento de metodos de controle das leveduras, provavelmente com o uso de aditivos, melhorara a eficiencia no uso de silagens de cana-de-acucar pelos pecuaristas.

Presence of the yeast Candida tropicalis in figs infected by the fruit fly Zaprionus indianus (dip.: Drosophilidae)

Nos ultimos anos, a mosca-africana-do-figo Zaprionus indianus tem se tornado a praga mais importante desta cultura no Brasil. Um processo fermentativo esta associado com a infeccao dos frutos por esta mosca. Uma unica especie de levedura, Candida tropicalis, foi identificada nos figos infectados. A presenca de uma unica especie de levedura e a baixa variabilidade genetica (RAPD) dos isolados indica uma relacao muito estreita entre C. tropicalis e Z. indianus.

Production of a monoclonal antibody against Aeromonas hydrophila and its application to bacterial identification

Aims: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces.

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.

Nutritional Quality of Sorghum Seeds: Storage Proteins and Amino Acids

One sorghum commercial genotype (MASSA 03) and nine ICRISAT high-lysine genotypes from India were analyzed for storage protein content, distribution profile, and soluble amino acid concentrations. Storage proteins fraction were extracted and separated by SDS-PAGE. Soluble amino acids contents were determined by HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the sorghum genotypes suggesting genetic variability. Amino acid profile also indicated large variations in the amino acid concentrations. The high lysine and threonine soluble concentrations observed in the seeds of the sorghum genotypes encouraged the use of these genotypes as potential food source due to the better balanced amino acids profile.

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.

Saccharomyces Cerevisiae Used As Probiotic: Strains Characterization And Cell Viability

The assay aimed to evaluate the cell viability of commercial yeast strains for bakery purpose in comparison to a selected strain (Yea-Sacc® 1026 , Alltech®) indicated as a probiotic to be added to animal feed, promising yield gains. The objective of this first study was to check the number of viable cells, once the indicated product to be classified as probiotic needs to provide live cells. We tested the viability of one commercial probiotic strain (Yea-Sacc® 1026 ) in comparison to four bakery commercial yeast. Our results showed a low viability of the probiotic Saccharomyces cerevisiae , which was 7.7 % and the bakery yeasts around 35 % of viable cells. The sustainability calls for alternative sources for animal feeding and the use of microorganisms is very welcome in the livestock chain, provided that the product presented has the characteristics inherent to it.

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).