Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
Navegar
27 resultados
Resultados da Pesquisa
Item Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum(Brazilian Journal of Microbiology, 2007-01) Queiroz, Marisa Vieira de; Lana, Taís Guimarães; Gonçalves, Daniel Bonoto; Araújo, Elza Fernandes de; Brito, Admilson Toscano Ribeiro de; Varavallo, Maurilio AntonioProtoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold).Item The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao(Genetics and Molecular Biology, 2009) Lima, Juliana O.; Pereira, Jorge F.; Rincones, Johana; Barau, Joan G.; Araújo, Elza F.; Pereira, Gonçalo A. G.; Queiroz, Marisa V.This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.Item PCR amplification and sequence analyses of Reverse Transcriptase-like genes in Crinipellis perniciosa isolates(Fitopatologia Brasileira, 2007-09) Pereira, Jorge F.; Ignacchiti, Mariana D. C.; Araújo, Elza F.; Brommonschenkel, Sérgio H.; Cascardo, Júlio C. M.; Pereira, Gonçalo A. G.; Queiroz, Marisa V.Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from the gypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.Item Biocontrole de Listeria monocytogenes por Pediococcus acidilactici em couve minimamente processada(Food Science and Technology, 2009-10) Costa, Wanessa Altimiras; Vanetti, Maria Cristina Dantas; Puschamann, RolfEste estudo avaliou um sistema de biocontrole para inibição de Listeria monocytogenes em couve minimamente processada, objetivando sua segurança durante estocagem sob refrigeração e em condições de abuso de temperatura. O potencial inibitório de bactérias láticas tolerantes ao sal e psicrotróficas contaminantes naturais da couve e Lactobacillus plantarum, Lactobacillus delbrueckii ATCC 9649 e Lactobacillus casei CCT 1465 foram avaliadas contra L. monocytogenes. O isolado de couve identificado como P. acidilactici CCA3 inibiu L. monocytogenes a 10 e 15 °C em ágar MRS e foi selecionado como possível agente de biocontrole. O número de L. monocytogenes na couve minimamente processada aumentou 3,7 e 4,7 ciclos logarítmicos a 5 e 10 °C, respectivamente, após 20 dias de armazenamento e 4,6 ciclos logarítmicos após oito dias a 15 °C. Entretanto, quando 108 UFC.g-1 de P. acidilactici CCA3 foram inoculados no produto processado, o crescimento de L. monocytogenes reduziu 2,3 ciclos logarítmicos sob temperatura abusiva de 15 °C. A acidez titulável e as características sensoriais da couve não foram alteradas pela presença de CCA3 ao longo do período de vida útil. Estes resultados sugerem o potencial de aplicação dos bioconservantes na couve minimamente processada, que necessitam estar associados à refrigeração e sanitização para garantir segurança.Item Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao(Brazilian Archives of Biology and Technology, 2008-01) Lopes, Francis Julio Fagundes; Queiroz, Marisa Vieira de; Lima, Juliana Oliveira; Silva, Viviane Aline Oliveira; Araújo, Elza Fernandes deThe presence of restriction enzymes in the transformation mixture improved the efficiency of transformation in Moniliophthora perniciosa. The influence of the vector shape (linear or circular), the patterns of plasmid integration in genomic sites and the influence of the promoter used to express the gene marker were also analyzed. The addition of BamHI or NotI increased the number of transformants by 3-10-fold and 3-fold, respectively, over the control without added enzyme. The use of pre-linearized plasmid did not increase the transformation efficiency in comparison with the circular plasmid. However, the frequency of multi-copy transformants increased significantly. The transformation procedure here reported resulted in better production of protoplasts and transformation efficiency. In addition, the time necessary for the detection of the first transformants and the number of insertions were reduced.Item A pH signaling mechanism involved in the spatial distribution of calcium and anion fluxes in ectomycorrhizal roots(New Phytologist, 2009-01) Ramos, Alessandro C.; Lima, Pedro T.; Dias, Pedro N.; Kasuya, Maria Catarina M.; Feijó, José A.Mycorrhization is a typical example of a host–pathogen symbiotic interaction where the pathogen cell biology and the host immune response coevolved several functional links. Here, the role played by ion fluxes across the root concerning nutrient uptake, osmoregulation, growth and signaling events is addressed. An ion‐selective vibrating probe system was used to determine the net fluxes of protons (H+), calcium (Ca2+) and anions (A−) along nonmycorrhizal and ectomycorrhizal (ECM) roots of Eucalyptus globulus colonized by Pisolithus sp. These data show that, from five root zones analyzed, the main effect of fungal colonization was localized to the elongation zone. Here, strong changes in ion dynamics and rhizosphere acidification capacity were observed. Additionally, ion fluxes exhibited periodic fluctuations. To verify whether these fluctuations corresponded to sustained oscillations, continuous wavelet time spectrum analysis was applied and it was determined that H+ and A− fluxes from ECM roots had longer periods than nonmycorrhizal roots. By contrast, Ca2+ oscillations were completely abolished following fungal interaction. These results are interpreted in the light of a working model in which nutrient uptake and stimulation of growth are mediated by ECM fungi and may be pH‐dependent. Furthermore, the variations detected in ECM roots for H+ and A− fluxes suggest a main contribution from the plant, while the results obtained for Ca2+ point to a significant involvement of the fungus.Item The activity of β-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate(Journal of Applied Microbiology, 2007-11-27) Ornelas, A. P.; Silveira, W. B.; Sampaio, F. C.; Passos, F. M. LKluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β-galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h−1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h−1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples.The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min−1 g−1 at a dilution rate of 0·24 h−1.The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.Item Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum(Journal of Industrial Microbiology & Biotechnology, 2007-11-12) Teixeira, Janaina Aparecida; Ribeiro, João Batista; Teixeira, Janaina Aparecida; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes deThe pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml−1 respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.Item Physiological implications of trehalose in the ectomycorrhizal fungus Pisolithus sp. under thermal stress(Journal of Thermal Biology, 2007-01) Tótola, Marcos R.; Ferreira, Adão S.; Borges, Arnaldo C.The trehalose cycle in filamentous fungi has been suggested to be an important mechanism of tolerance against adverse stress conditions, particularly in thermal stress. Here, we demonstrate that trehalose and trehalase activity can be involved as an additional mechanism in development of thermotolerance in Pisolithus sp. In response to heat shock at 42 °C, an accumulation of intracellular trehalose and an increase in trehalase activity in the mycelial mass of the fungus was observed, when compared to the control treatment (28 °C). In vitro, assays showed that trehalose had a protective effect on β-glucosidase activity under thermal stress. Therefore, trehalose production may be an important mechanism of protection in ectomycorrhizal fungi, and this capacity could be used in the selection of isolates with greater capacity for adaptation to environmental stress.Item Mobilisation of bacteria in a fine-grained residual soil by electrophoresis(Journal of Hazardous Materials, 2009-01-15) Rocha, Ulisses Nunes Da; Tótola, Marcos Rogério; Pessoa, Denise Maria Mano; Araruna Júnior, José Tavares; Neves, Júlio César Lima; Borges, Arnaldo ChaerAn investigation of electrokinetic bacterial mobilisation in a residual soil from gneiss is presented here. The experimental program aimed at assessing the efficacy of electrophoresis against the electro-osmotic flow to transport endospores of Bacillus subtilis LBBMA 155 and nitrogen-starved cells of Pseudomonas sp. LBBMA 81. Electrokinesis was performed on a low hydraulic reconstituted clayey soil column submitted to a 5 mA electrical current for 24 h. Cells were coccoid-shaped and characterised as possessing low surface hydrophobicity and less than 1 μm in diameter. Distribution coefficient for B. subtilis in the soil was between 16.8 and 19.9 times higher than that for Pseudomonas sp. Distribution coefficient for B. subtilis between eluate and anionic exchange column was 11.8 times higher than that for Pseudomonas sp. After the electrokinesis, it was shown that cells and endospores were distributed hyperbolically through the soil probe and moved against the electro-osmotic flow; however, endospores were transported throughout all soil core and starved cells only till half of its length. The higher transport efficiency of B. subtilis endospores was attributed to their higher negative charge on cell surface. These results demonstrate that electrokinesis can be used for bacteria transport in soils with low hydraulic conductivity, even against the electro-osmotic flow.
- «
- 1 (current)
- 2
- 3
- »