Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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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 Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene(Brazilian Journal of Microbiology, 2005-04) Queiroz, Marisa Vieira de; Pereira, Jorge Fernando; Ribeiro, Ronney Adriano; Soares, Marcos Antônio; Ribeiro, João Batista; Araújo, Elza Fernandes de; Varavallo, Maurílio AntônioPenicillium brevicompactum is a filamentous fungus that presents a potential for industrial use due its efficient pectinase production. A heterologous transformation system was developed for P. brevicompactum based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. One mutant named 4457-18X was chosen for the transformation experiments with the pNH24 vector containing de Fusarium oxysporum nitrate reductase gene. A frequency of approximately 3 transformants/µg DNA was obtained using the circular vector pNH24. This frequency was multiplied about 10 fold using the linearized vector with the Xba I restriction enzyme. Southern analysis of the transformants showed a tendency of the linearized vector to diminish the number of integrations compared to the use of the circular vector. The integration was random and stable in the analyzed transformants. The establishment of a transformation system for P. brevicompactum is fundamental for genetic manipulation of this microorganism.Item Padrão de integração de pAN7-1 em mutantes de Magnaporthe grisea com patogenicidade alterada em arroz(Summa Phytopathologica, 2010-01) Queiroz, Marisa Vieira de; Marchi, Carlos Eduardo; Brommonschenkel, Sérgio Hermínio; Borges, Mírian de Freitas; Mizubuti, Eduardo Seiti G.Ensaios foram conduzidos para verificar a presença, o número de cópias e de sítios de integração de pAN7-1 no genoma de mutantes de M. grisea I-22 com patogenicidade alterada em arroz. Foram analisados T41, T93, T251 (gerados por mutagênese REMI) e T108 (oriundo de mutagênese convencional), os quais exibiram diferentes fenótipos mutantes. O DNA total desses mutantes foi submetido à reação em cadeia de polimerase (PCR) e às análises de hibridização com o vetor (Southern blot). A presença de pAN7-1 no genoma de todos os mutantes foi confirmada por PCR. Segundo as análises de Southern blot, T41 exibiu duas integrações do vetor, ambas na forma de cópia única. No genoma de T93 também foram detectados dois sítios de inserção de pAN7-1, um dos quais envolvendo múltiplas cópias do vetor. Os resultados indicaram a presença de apenas uma cópia do vetor em um único sítio nos genomas de T108 e T251. O padrão de integração em T251 foi o único a sugerir a ocorrência de evento REMI. As diferenças quanto ao tamanho dos fragmentos com homologia a pAN7-1 refletiram a possível aleatoriedade dos eventos de integração no genoma de M. grisea. Os resultados evidenciaram o potencial de REMI para a mutagênese insercional de M. grisea, quando conduzida com pAN7-1 e HindIIIItem 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 Pulsed field gel electrophoresis reveals chromosome length and number differences in Brazilian strains of Metarhizium Anisopliae(Brazilian Archives of Biology and Technology, 2005-01) Queiroz, Marisa Vieira de; Kava-Cordeiro, Vanessa; Pizzirani-Kleiner, Aline Aparecida; Azevedo, João LúcioElectrophoretic karyotypes of eight wild-type strains of Metarhizium anisopliae var. anisopliae were obtained by pulsed-field gel electrophoresis. These strains were isolated from insects of six different Brazilian states. The chromosomal DNA molecules of three strains were separated into seven bands and of five strains into eight bands. Chromosome length polymorphisms were also observed. The size of the chromosomal DNA of all strains varied between 7.7 and 0.9 Mb using the Aspergillus nidulans chromosomes as size standards. The total genome size of these strains was estimated in at least 29.7 Mb. Some correlations between differences in karyotype and occurrence of parasexual cycle likewise the host specificity were discussed.Item Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.(Brazilian Archives of Biology and Technology, 2010-01) Coelho, Irene da Silva; Queiroz, Marisa Vieira de; Costa, Maurício Dutra; Kasuya, Maria Catarina Megumi; Araújo, Elza Fernandes deThe aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 106 protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 107 protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.Item The repertoire of effector candidates in Colletotrichum lindemuthianum reveals important information about Colletotrichum genus lifestyle(Applied Microbiology and Biotechnology, 2019) Queiroz, Casley Borges de; Correia, Hilberty L. Nunes; Santana, Mateus Ferreira; Batista, Diego Silva; Vidigal, Pedro M. Pereira; Brommonschenkel, Sérgio Hermínio; Queiroz, Marisa Vieira deThe fungus Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean (Phaseolus vulgaris), and anthracnose is one of the most devastating diseases of this plant species. However, little is known about the proteins that are essential for the fungus-plant interactions. Knowledge of the fungus’ arsenal of effector proteins is of great importance for understanding this pathosystem. In this work, we analyzed for the first time the arsenal of Colletotrichum lindemuthianum effector candidates (ClECs) and compared them with effector proteins from other species of the genus Colletotrichum, providing a valuable resource for studying the infection mechanisms of these pathogens in their hosts. Isolates of two physiological races (83.501 and 89 A2 2-3) of C. lindemuthianum were used to predict 353 and 349 ClECs, respectively. Of these ClECs, 63% were found to be rich in cysteine, have repetitive sequences of amino acids, and/or possess nuclear localization sequences. Several conserved domains were found between the ClECs. We also applied the effector prediction to nine species in the genus Colletotrichum, and the results ranged from 247 predicted effectors in Colletotrichum graminicola to 446 in Colletotrichum orbiculare. Twelve conserved domains were predicted in the effector candidates of all analyzed species of Colletotrichum. An expression analysis of the eight genes encoding the effector candidates in C. lindemuthianum revealed their induction during the biotrophic phase of the fungus on the bean.Item Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation(Canadian Journal Of Microbiology, 2004) Pereira, Jorge Fernando; Queiroz, Marisa Vieira de; Lopes, Francis Júlio Fagundes; Rocha, Rodrigo Barros; Daboussi, Marie-Josée; Araújo, Elza Fernandes dePenicillium griseoroseum has been studied because of its efficient pectinases production. In this work, the Penicillium griseoroseum nitrate reductase gene was characterized, transcriptionally analyzed in different nitrogen sources, and used to create a phylogenetic tree and to develop a homologous transformation system. The regulatory region contained consensus signals involved in nitrogen metabolism and the structural region was possibly interrupted by 6 introns coding for a deduced protein with 864 amino acids. RT-PCR analysis revealed high amounts of niaD transcript in the presence of nitrate. Transcription was repressed by ammonium, urea, and glutamine showing an efficient turnover of the niaD mRNA. Phylogenetics analysis showed distinct groups clearly separated in accordance with the classical taxonomy. A mutant with a 122-bp deletion was used in homologous transformation experiments and showed a transformation frequency of 14 transformants/microg DNA. All analyzed transformants showed that both single- and double-crossover recombination occurred at the niaD locus. The establishment of this homologous transformation system is an essential step for the improvement of pectinase production in Penicillium griseoroseum.Item Mycovirus in Pseudocercospora griseola, the causal agent of angular leaf spot in common bean(Canadian Journal of Microbiology, 2010) Lima, Swiany Silveira; Abadio, Ana Karina Rodrigues; Araújo, Elza Fernandes; Kitajima, Elliot Watanabe; Watanabe, Elliot; Queiroz, Marisa Vieira dePseudocercospora griseola (Sacc.) Crous & Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800–4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S1. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1–6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.Item Purification and characterization of two new cell-bound bioactive compounds produced by wild Lactococcus lactis strain(FEMS Microbiology Letters, 2017-06) Saraiva, Margarete Alice Fontes; Brede, Dag Anders; Nes, Ingolf Figved; Baracat- Pereira, Maria Cristina; Queiroz, Marisa Vieira de; Moraes, Celia Alencar deNovel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID1.5 were isolated and partially characterized. Following purification by cationic exchange and a solid-phase C18 column, antimicrobial activity was recovered after three runs of RPC using 60% (v/v) and 100% (v/v) of 2-propanol for elution, suggesting that more than one antimicrobial compound were produced by L. lactis ID1.5, which were in this study called compounds AI and AII. The mass spectrum of AI and AII showed major intensity ions at m/z 1070.05 and 955.9 Da, respectively. The compound AI showed a spectrum of antimicrobial activity mainly against L. lactis species, while the organisms most sensitive to compound AII were Bacillus subtilis, Listeria innocua, Streptococcus pneumoniae and Pseudomonas aeruginosa. The antimicrobial activity of both compounds was suppressed by treatment with Tween 80. Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L. lactis, and have a significant inhibitory effect against two clinically important respiratory pathogens.