RESUMO
Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.
Assuntos
Humanos , Raiz Dentária/microbiologia , Candida albicans/genética , DNA Fúngico/genética , Cárie Radicular/microbiologia , Biofilmes/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Candida albicans/crescimento & desenvolvimento , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Regulação para Cima , Análise de Sequência de RNA , Transcriptoma , MorfogêneseRESUMO
Paracoccidioides spp. isolation from environmental samples is rare and hardly reproducible. Molecular techniques have facilitated the fungal detection. However, it can be still difficult. Some strategies to enhance the capacity of DNA detection have been adopted, including the analysis of soil samples belonging to the habitat of animals from which Paracoccidioides spp. have already been isolated, notably armadillo burrows. To date, the detection of Paracoccidioides spp. has not yet been reported from outbreak hotspots. Clusters and outbreaks of acute paracoccidioidomycosis (PCM), usually a more severe clinical form, have currently occurred in urban areas being associated to climate changes, deforestation, and great constructions. These occurrences potentially signalise the fungus' environmental niche, a riddle not yet solved. The authors performed an environmental investigation in a deeply disturbed area, after a highway construction in Rio de Janeiro, Brazil, where a recent outbreak of acute PCM occurred. Specific DNA sequences of Paracoccidioides brasiliensis were detected in shallow soil samples around the highway, reinforcing the association between the road construction and this PCM outbreak.
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Animais , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/microbiologia , Tatus , DNA Fúngico/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/genética , Microbiologia do Solo , Brasil , Sequência de Bases , Análise de Sequência de DNA , EcossistemaRESUMO
Abstract INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.
Assuntos
Humanos , Masculino , Feminino , Adulto , Idoso , Polimorfismo Genético/efeitos dos fármacos , Candida/efeitos dos fármacos , Candida/genética , Fluconazol/farmacologia , Transplante de Rim , Diabetes Mellitus/microbiologia , Antifúngicos/farmacologia , Valores de Referência , Saliva/microbiologia , Candida/isolamento & purificação , DNA Fúngico/genética , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Farmacorresistência Fúngica/genética , Imunocompetência , Pessoa de Meia-Idade , Mutação/efeitos dos fármacosRESUMO
Histoplasmosis is a systemic mycosis infection caused by Histoplasma capsulatum, a heterothallic ascomycete. The sexual reproduction of this fungus is regulated by the mating type (MAT1) locus that contains MAT1-1 and MAT1-2 idiomorphs, which were identified by uniplex polymerase chain reaction (PCR). This study aimed to optimise single-step multiplex PCR for the accurate detection of the distinct mating types of H. capsulatum. Among the 26 isolates tested, 20 had MAT1-1 genotype, while six showed MAT1-2 genotype, in agreement with the uniplex PCR results. These results suggest that multiplex PCR is a fast and specific tool for screening H. capsulatum mating types.
Assuntos
DNA Fúngico/genética , Primers do DNA/genética , Histoplasma/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Reação em Cadeia da Polimerase Multiplex , Genótipo , Histoplasma/classificaçãoRESUMO
Resumen Introducción. La neurocriptococosis es una infección fúngica oportunista que representa un alto costo en vidas humanas y para la economía de los países. Sus agentes causales, las especies del complejo Cryptococcus neoformans/Cryptococcus gattii, tienen una fase sexuada y otra asexuada, cuatro serotipos principales y siete variedades moleculares con diferencias clínico-epidemiológicas, fenotípicas y de sensibilidad a los antifúngicos. Objetivo. Caracterizar molecularmente los aislamientos clínicos de C. neoformans de Guayaquil, Ecuador. Materiales y métodos. Se determinó el tipo de apareamiento, el serotipo y la variedad molecular mediante reacción en cadena de la polimerasa y análisis del polimorfismo de los fragmentos de restricción de 27 aislamientos levaduriformes previamente identificados como C. neoformans mediante métodos convencionales. Los aislamientos fueron recuperados del líquido cefalorraquídeo de pacientes con síndrome neurológico seropositivos para HIV, internados en el Hospital de Infectología "Dr. José Daniel Rodríguez Maridueña", entre diciembre de 2013 y enero de 2015. Resultados. Se demostró el amplio predominio de C. neoformans del serotipo A, MATα y el genotipo VNI entre los aislamientos estudiados. Conclusiones. Estos datos son similares a los obtenidos en otros países y son los primeros de su tipo en Guayaquil, Ecuador, por lo cual constituyen un aporte importante al conocimiento de la criptococosisen esta ciudad.
Abstract Introduction: Neurocryptococcosis is an opportunistic fungal infection that represents a high cost in human lives and for the economy of countries. Its causative agent, the Cryptococcus neoformans/Cryptococcus gattiispecies complex, has a sexual and an asexual phase, four major serotypes and seven molecular varieties with phenotypic, clinical-epidemiological and antifungal susceptibility differences. Objective: To characterize by molecular methods clinicalisolates of C. neoformans from Guayaquil, Ecuador. Materials and methods: We determined mating types, serotypes and molecular varieties by PCR and RFLP in 27 yeast isolates previously identified as C. neoformans by conventional methods. The isolates were recovered from cerebrospinal fluid of HIV seropositive patients with neurological syndrome admitted at "Dr. José Daniel Rodríguez Maridueña" Hospital from December, 2013, to January, 2015. Results: We established a wide prevalence of C. neoformans serotype A, MATαand genotype VNI among the studied isolates. Conclusions: These data are similar to those obtained in other countries and the first identified by molecular characterization in Guayaquil, Ecuador. Therefore, they constitute an important contribution to the knowledge on cryptococcosis in this country.
Assuntos
Humanos , Meningite Criptocócica/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Cryptococcus neoformans/genética , Polimorfismo de Fragmento de Restrição , DNA Fúngico/genética , Sorotipagem , Líquido Cefalorraquidiano/microbiologia , Reação em Cadeia da Polimerase , Prevalência , Estudos Transversais , Estudos Prospectivos , Técnicas de Tipagem Micológica , Meningite Criptocócica/líquido cefalorraquidiano , Meningite Criptocócica/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/classificação , Cryptococcus neoformans/efeitos dos fármacos , Farmacorresistência Fúngica , Equador/epidemiologia , GenótipoRESUMO
BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.
Assuntos
DNA Fúngico/genética , Reação em Cadeia da Polimerase , Enterocytozoon/isolamento & purificação , Enterocytozoon/genética , Filogenia , Brasil , Análise de Sequência , GenótipoRESUMO
ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.
Assuntos
Humanos , Criança , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Candida/genética , Candidíase Bucal/microbiologia , DNA Fúngico/genética , Candida albicans/genética , Candida albicans/isolamento & purificação , Candida/classificação , Candida/isolamento & purificação , Genótipo , Mucosa Bucal/microbiologia , Técnicas de Tipagem Micológica , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
Abstract Phenol and phenolic compounds are environmental pollutants present in industrial wastewaters such as coal tar, oil refineries and petrochemical plants. Phenol removal from industrial effluents is extremely important for the protection of environment. Usually, phenol degradation is carried out by physicochemical methods that are costly and produce hazardous metabolites. Recently, phenol biodegradation has been considered. Yeasts are the most important phenol biodegraders. In this study, the phenol-degrading yeast from environmental samples (soil and wastewater) was isolated from the coking plant of Zarand, Kerman. Then total heterotrophic yeasts were counted. The soil samples had higher rates of yeast degrader, in comparison to wastewater samples. After three passages, four yeasts (K1, K2, K7 and K11) that had the highest growth rate were selected for further study. Also, these yeasts were able to remove phenol measured by Gibbs reagent. The effect of four different concentrations of phenol (50, 125, 200 and 275) mg L−1 was measured and three degradation patterns in these yeasts were observed. The hydrophobicity and emulsification activity were measured in all eleven yeasts. Finally, strong yeasts in phenol degrading yeasts were identified by molecular method using amplification of 18S rRNA gene region. The sequencing results showed that these isolated yeasts belonged to Candida tropicalis strain K1, Pichia guilliermondii strain K2, Meyerozyma guilliermondii strain K7 and C. tropicalis strain K11.
Assuntos
Resíduos Industriais , Fenol/metabolismo , Águas Residuárias/microbiologia , Poluentes Químicos da Água/metabolismo , Leveduras/classificação , Leveduras/metabolismo , Biotransformação , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Irã (Geográfico) , Dados de Sequência Molecular , Filogenia , /genética , Análise de Sequência de DNA , Microbiologia do Solo , Leveduras/genética , Leveduras/isolamento & purificaçãoRESUMO
Abstract Candida species, especially C. albicans, are commensals on human mucosal surfaces, but are increasingly becoming one of the important invasive pathogens as seen by a rise in its prevalence in immunocompromised patients and in antibiotic consumption. Thus, an accurate identification of Candida species in patients with pulmonary symptoms can provide important information for effective treatment. A total of 75 clinical isolates of Candida species were obtained from the bronchoalveolar lavage fluid of both immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures were identified based on nuclear ribosomal Internal Transcribed Spacer (ITS1-ITS2 rDNA) sequence analysis by polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP). Molecular identification indicated that the isolates belonged predominantly to C. albicans (52%), followed by C. tropicalis (24%), C. glabrata (14.7%), C. krusei (5.3%), C. parapsilosis (1.3%), C. kefyr (1.3%) and C. guilliermondii (1.3%). Given the increasing complexity of disease profiles and their management regimens in diverse patients, rapid and accurate identification of Candida species can lead to timely and appropriate antifungal therapy.
Assuntos
Humanos , Líquido da Lavagem Broncoalveolar/microbiologia , Candida/isolamento & purificação , Candidíase/diagnóstico , Pneumopatias Fúngicas/diagnóstico , Candida/classificação , Candida/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Fatores de TempoRESUMO
Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified:
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus niger/enzimologia , Celulose/metabolismo , /metabolismo , Kluyveromyces/enzimologia , Saccharum/microbiologia , Xilosidases/metabolismo , beta-Glucosidase/metabolismo , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/metabolismo , Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , Sequência de Bases , Biomassa , Brasil , DNA Fúngico/genética , DNA Intergênico/genética , Fermentação , Kluyveromyces/isolamento & purificação , Kluyveromyces/metabolismo , Lignina/metabolismo , Tipagem Molecular , Técnicas de Tipagem Micológica , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
Aflatoxin contamination of peanut, due to infection by
Assuntos
Aspergillus flavus , Aflatoxinas/metabolismo , Arachis/microbiologia , Agricultura , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Aspergillus flavus/classificação , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , DNA Fúngico/genética , Ensaio de Imunoadsorção Enzimática , Genes Fúngicos , Variação Genética/genética , Índia , Tipagem Molecular , Técnicas de Tipagem Micológica , Análise de Componente PrincipalRESUMO
Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensisand Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n= 35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1 fg of P. brasiliensisDNA.
Assuntos
Humanos , DNA Fúngico/genética , Proteínas Fúngicas/genética , Paracoccidioides/genética , Paracoccidioidomicose/diagnóstico , Colômbia , Paracoccidioides/isolamento & purificação , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.
Assuntos
Antifúngicos/metabolismo , Endófitos/isolamento & purificação , Endófitos/metabolismo , Fungos/isolamento & purificação , Fungos/metabolismo , Peptídeo Hidrolases/metabolismo , Piper/microbiologia , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Fungos/classificação , Fungos/efeitos dos fármacos , Técnicas Microbiológicas , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Penicillum janthinellum SDX7 was isolated from aged petroleum hydrocarbon-affected soil at the site of Anand, Gujarat, India, and was tested for different pH, temperature, agitation and concentrations for optimal growth of the isolate that was capable of degrading upto 95%, 63% and 58% of 1%, 3% and 5% kerosene, respectively, after a period of 16 days, at optimal growth conditions of pH 6.0, 30 °C and 180 rpm agitation. The GC/MS chromatograms revealed that then-alkane fractions are easily degraded; however, the rate might be lower for branched alkanes, n-alkylaromatics, cyclic alkanes and polynuclear aromatics. The test doses caused a concentration-dependent depletion of carbohydrates of P. janthinellum SDX7 by 3% to 80%, proteins by 4% to 81% and amino acids by 8% to 95% upto 16 days of treatment. The optimal concentration of 3% kerosene resulted in the least reduction of the metabolites of P. janthinellum such as carbohydrates, proteins and amino acids with optimal growth compared to 5% and 1% (v/v) kerosene doses on the 12th and 16th day of exposure. Phenols were found to be mounted by 43% to 66% at lower and higher concentrations during the experimental period. Fungal isolate P. janthinellum SDX7 was also tested for growth on various xenobiotic compounds.
Assuntos
Querosene , Penicillium/crescimento & desenvolvimento , Penicillium/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Xenobióticos/metabolismo , Composição de Bases , Biotransformação , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Cromatografia Gasosa-Espectrometria de Massas , Genes de RNAr , Concentração de Íons de Hidrogênio , Índia , Dados de Sequência Molecular , Penicillium/genética , Penicillium/isolamento & purificação , RNA Fúngico/genética , /genética , Análise de Sequência de DNA , TemperaturaRESUMO
Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g−1) along with low cellulose (2 U g−1) and xylanase (140 U g−1) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes.
Assuntos
Lacase/metabolismo , Trametes/enzimologia , Trametes/crescimento & desenvolvimento , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Estabilidade Enzimática , Lacase/química , Microscopia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Solventes , Temperatura , Trametes/citologia , Trametes/efeitos da radiaçãoRESUMO
Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.
Assuntos
Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lacase/biossíntese , Ativação Transcricional/efeitos dos fármacos , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Cromatografia em Gel , Meios de Cultura/química , DNA Fúngico/genética , Eletroforese em Gel de Poliacrilamida , Resíduos Industriais , Lacase/química , Lacase/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Análise Espectral , Purificação da ÁguaRESUMO
A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.
Assuntos
Bactérias/isolamento & purificação , Bactérias/metabolismo , Celulose/metabolismo , Fungos/isolamento & purificação , Fungos/metabolismo , Microbiologia do Solo , Bactérias/classificação , Bactérias/genética , Colômbia , Celulase/análise , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA de Helmintos/química , DNA de Helmintos/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fungos/classificação , Fungos/genética , Hidrólise , /genética , Análise de Sequência de DNARESUMO
Xylanase (EC 3. 2. 1. 8), hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry) as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7th day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa) using SDS-PAGE.
Assuntos
Microbiologia do Solo , Trichoderma/classificação , Trichoderma/isolamento & purificação , Xilosidases/análise , Cromatografia Líquida , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Técnicas de Tipagem Micológica , Filogenia , /genética , Análise de Sequência de DNA , Trichoderma/enzimologia , Trichoderma/crescimento & desenvolvimento , Xilosidases/química , Xilosidases/isolamento & purificaçãoRESUMO
Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.
Assuntos
Endófitos/isolamento & purificação , Técnicas Microbiológicas/métodos , Esterilização/métodos , Triticum/microbiologia , Eletroforese em Gel de Gradiente Desnaturante , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , DNA de Plantas/química , DNA de Plantas/genética , DNA Espaçador Ribossômico/genética , DNA Espaçador Ribossômico/isolamento & purificação , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Propriedades de Superfície , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/ultraestrutura , Triticum/ultraestruturaRESUMO
Ganoderma includes species of great economic and ecological importance, but taxonomists judge the current nomenclatural situation as chaotic and poorly studied in the neotropics. From this perspective, phylogenetic analyses inferred from ribosomal DNA sequences have aided the clarification of the genus status. In this study, 14 specimens of Ganoderma and two of Tomophagus collected in Brazil were used for DNA extraction, amplification and sequencing of the ITS and LSU regions (rDNA). The phylogenetic delimitation of six neotropical taxa (G. chalceum, G. multiplicatum, G. orbiforme, G. parvulum, G. aff. oerstedtii and Tomophagus colossus) was determined based on these Brazilian specimens and found to be distinct from the laccate Ganoderma from Asia, Europe, North America and from some specimens from Argentina. Phylogenetic reconstructions confirmed that the laccate Ganoderma is distinct from Tomophagus, although they belong to the same group. The use of taxonomic synonyms Ganoderma subamboinense for G. multiplicatum, G. boninense for G. orbiforme and G. chalceum for G. cupreum was not confirmed. However, Ganoderma parvulum was confirmed as the correct name for specimens called G. stipitatum. Furthermore, the name G. lucidum should be used only for European species. The use of valid published names is proposed according to the specimen geographical distribution, their morphological characteristics and rDNA analysis. Rev. Biol. Trop. 62 (3): 1197-1208. Epub 2014 September 01.
Ganoderma incluye especies de gran importancia económica y ecológica, sin embargo, su nomenclatura actual es caótica y poco estudiada en el neotrópico. En este estudio se utilizaron 14 muestras de Ganoderma y dos de Tomophagus recolectados en Brasil para la extracción de ADN, amplificación y secuenciación de las regiones ITS y LSU. La delimitación filogenética de seis táxones neotropicales fue discutida con base en especímenes brasileños y secuencias del GenBank. Estas especies mostraron ser distintas de los Ganoderma lacados de Asia, Europa, América del Norte y de algunos ejemplares de Argentina. Las reconstrucciones filogenéticas confirman que los Ganoderma lacados son distintos de Tomophagus, aunque pertenecen al mismo grupo. No se confirman los sinónimos de G. subamboinense a G. multiplicatum, de G. boninense a G. orbiforme y G. chalceum a G. cupreum. G. parvulum se confirma como el nombre correcto para G. stipitatum. G. lucidum sólo se debe utilizar para especies europeas. Por lo tanto, se propone el uso de nombres publicados válidamente de acuerdo con la distribución geográfica de las muestras, características morfológicas y análisis de ADNr.