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1.
Curr Microbiol ; 71(4): 509-16, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202846

RESUMO

Endophytic bacteria play a key role in the biocontrol of phytopathogenic microorganisms. In this study, genotypic diversity was analyzed via repetitive element PCR (rep-PCR) of endophytic isolates of the phylum Actinobacteria that were previously collected from leaves of cultivars of common bean (Phaseolus vulgaris). Considerable variability was observed, which has not been reported previously for this phylum of endophytic bacteria of the common bean. Furthermore, the ethanol extracts from cultures of various isolates inhibited the growth of pathogenic bacteria in vitro, especially Gram-positive pathogens. Extracts from cultures of Microbacterium testaceum BAC1065 and BAC1093, which were both isolated from the 'Talismã' cultivar, strongly inhibited most of the pathogenic bacteria tested. Bean endophytic bacteria were also demonstrated to have the potential to inhibit the quorum sensing of Gram-negative bacteria. This mechanism may regulate the production of virulence factors in pathogens. The ability to inhibit quorum sensing has also not been reported previously for endophytic microorganisms of P. vulgaris. Furthermore, M. testaceum with capacity to inhibit quorum sensing appears to be widespread in common bean. The genomic profiles of M. testaceum were also analyzed via pulsed-field gel electrophoresis, and greater differentiation was observed using this method than rep-PCR; in general, no groups were formed based on the cultivar of origin. This study showed for the first time that endophytic bacteria from common bean plants exhibit high variability and may be useful for the development of strategies for the biological control of diseases in this important legume plant.


Assuntos
Actinobacteria/classificação , Actinobacteria/fisiologia , Antibacterianos/isolamento & purificação , Endófitos/classificação , Endófitos/fisiologia , Phaseolus/microbiologia , Percepção de Quorum , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Antibacterianos/farmacologia , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Endófitos/genética , Endófitos/isolamento & purificação , Variação Genética , Tipagem Molecular , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico/genética
2.
Curr Microbiol ; 68(3): 358-64, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24190756

RESUMO

Pseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.


Assuntos
Ascomicetos/classificação , Ascomicetos/genética , Variação Genética , Tipagem Molecular/métodos , Técnicas de Tipagem Micológica/métodos , Ascomicetos/isolamento & purificação , Brasil , Análise por Conglomerados , Genótipo , Musa/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia
3.
J Ind Microbiol Biotechnol ; 41(10): 1571-80, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25099254

RESUMO

The fungus Penicillium griseoroseum has the potential for application on an industrial scale as a host for the production of homologous and heterologous proteins, mainly because it does not produce some mycotoxins or secrete proteases under the growth conditions for pectinase production. However, for the fungus to be used effectively as an expression heterologous system, an understanding of the organization of its genome, as well as the mechanisms of gene expression and protein production, is required. In the present study, the size of the P. griseoroseum genome was estimated to be 29.8-31.5 Mb, distributed among four chromosomes. An analysis of plg1 and pgg2 pectinolytic genes expression and copy number in recombinant multi-copy strains of P. griseoroseum demonstrated that an increase in the number of gene copies could increase enzyme production, but the transcription could be affected by the gene integration position. Placing a copy of the plg1 gene under the control of the gpd promoter of Aspergillus nidulans yielded a 200-fold increase in transcription levels compared to the endogenous gene, and two copies of the pgg2 gene produced an 1100-fold increase compared with the endogenous gene. These results demonstrated that transcription, translation, and protein secretion in the fungus P. griseoroseum respond to an increased number of gene copies in the genome. The processing capacity and efficiency of protein secretion in P. griseoroseum are consistent with our premise that this fungus can be used for the industrial-scale production of several enzymes.


Assuntos
Proteínas Fúngicas/genética , Penicillium/genética , Poligalacturonase/genética , Polissacarídeo-Liases/genética , Aspergillus nidulans/genética , Sequência de Bases , Proteínas Fúngicas/biossíntese , Dosagem de Genes , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Penicillium/enzimologia , Poligalacturonase/biossíntese , Polissacarídeo-Liases/biossíntese , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Transcrição Gênica
4.
Mycologia ; 106(4): 629-41, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24895425

RESUMO

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen.


Assuntos
Colletotrichum/genética , Elementos de DNA Transponíveis/genética , Genoma Fúngico/genética , Transposases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Brasil , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Marcadores Genéticos/genética , Variação Genética , Sequências Repetidas Invertidas/genética , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Transposases/química
5.
J Basic Microbiol ; 54(2): 133-41, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23553535

RESUMO

A number of parameters, including culture medium pH, affect growth and enzyme production by microorganisms. In the present study, the production and secretion of pectin lyase (PL) and polygalacturonase (PG) by recombinant strains of Penicillium griseoroseum cultured in mineral-buffered media (MBM; initial pH 6.8) and mineral-unbuffered medium (MUM; initial pH 6.3) were evaluated. Under these culture conditions, no change in the transcriptional levels of plg1 and pgg2 was observed. However, the levels of secreted total protein ranged from 7.80 ± 1.1 to 3.25 ± 1.50 µg ml(-1) in MBM and MUM, respectively, and were evaluated by SDS-PAGE. PL and PG enzymatic activities decreased 6.4 and 3.6 times, respectively, when P. griseoroseum was cultivated under acidic pH conditions (MUM). Furthermore, differences were observed in the hypha and mycelium morphology. These findings suggest that acidic growing conditions affect PL and PG secretion, even though the transcription and translation processes are successful. The data obtained in this study will help to establish optimal culture conditions that increase production and secretion of recombinant proteins by filamentous fungi.


Assuntos
Proteínas Fúngicas/metabolismo , Penicillium/metabolismo , Poligalacturonase/metabolismo , Proteínas Fúngicas/biossíntese , Concentração de Íons de Hidrogênio , Organismos Geneticamente Modificados , Penicillium/citologia , Penicillium/genética , Poligalacturonase/biossíntese , Polissacarídeo-Liases/biossíntese , Prostaglandinas G/genética , Prostaglandinas G/metabolismo
6.
J Basic Microbiol ; 54(12): 1358-66, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25080195

RESUMO

The interaction between fungi and plants that form ectomycorrhizae (ECM) promotes alterations in the gene expression profiles of both organisms. Fungal genes expression related to metabolism were evaluated at the pre-symbiotic stage and during the ECM development between Scleroderma laeve and Eucalyptus grandis. Partial sequences of ATP synthase (atp6), translation elongation factor (ef1α), the RAS protein (ras), and the 17S rDNA genes were isolated. The expression of the atp6 and 17S rDNA genes during the pre-symbiotic stage showed an approximately threefold increase compared to the control. During ECM development, the expression of the 17S rDNA gene showed a 4.4-fold increase after 3 days of contact, while the expression of the atp6 gene increased 7.23-fold by the 15th day, suggesting that protein synthesis and respiratory chain activities are increased during the formation of the mantle and the Hartig net. The ras gene transcripts were only detected by RT-PCR 30 days after fungus-plant contact, suggesting that RAS-mediated signal transduction pathways are functional during the establishment of symbiosis. The present study demonstrates that alterations in gene expression occur in response to stimuli released by the plant during ECM association and increases the understanding of the association between S. laeve and E. grandis.


Assuntos
Basidiomycota/metabolismo , DNA Ribossômico/metabolismo , Eucalyptus/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genes ras , Micorrizas/metabolismo , Basidiomycota/genética , DNA Ribossômico/genética , Eucalyptus/genética , Proteínas Fúngicas/genética , Micorrizas/genética , Micorrizas/crescimento & desenvolvimento , Transdução de Sinais
7.
Microbiology (Reading) ; 159(Pt 1): 112-125, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23103978

RESUMO

Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.


Assuntos
Agaricales/genética , Elementos de DNA Transponíveis , Sequência de Aminoácidos , Southern Blotting , Brasil , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , Genótipo , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA
8.
J Basic Microbiol ; 53(10): 823-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23440766

RESUMO

In this study, we demonstrate that ClIRAP primers designed using the transposable element RetroCl1 sequence from Colletotrichum lindemuthianum can be used to generate an efficient IRAP (inter-retrotransposon amplified polymorphism) molecular marker to study intra- and inter-species diversity in fungi. It has been previously demonstrated that primers generated from this TRIM-like element can be used in the Colletotrichum species. We now prove that the RetroCl1 sequence can also be used to analyze diversity in different fungi. IRAP profiles were successfully generated for 27 fungi species from 11 different orders, and intra-species genetic variability was detected in six species. The ClIRAP primers facilitate the use of the IRAP technique for a variety of fungi without prior knowledge of the genome.


Assuntos
Fungos/genética , Variação Genética , Retroelementos , Primers do DNA , Marcadores Genéticos , Genótipo , Filogenia , Polimorfismo Genético , Sequências Repetidas Terminais
9.
Genet Mol Biol ; 36(2): 243-51, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23885207

RESUMO

The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.

10.
Curr Microbiol ; 65(6): 758-63, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22986817

RESUMO

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48 h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Enterobacter cloacae/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Conjugação Genética , Enterobacter cloacae/enzimologia , Enterobacter cloacae/genética , Enterobacter cloacae/metabolismo , Poliestirenos , Proteólise , Aço Inoxidável
11.
World J Microbiol Biotechnol ; 28(3): 1087-95, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22805830

RESUMO

A nonautonomous element of 624 bp, called RetroCl1 (Retroelement Colletotrichum lindemuthianum 1), was identified in the plant pathogenic fungus Colletotrichum lindemuthianum. RetroCl1 contains terminal direct repeats (223 bp) that are surrounded by CTAGT sequences. It has a short internal domain of 178 bp and shows characteristics of terminal-repeat retrotransposon in miniature (TRIM) family. We used RetroCl1 sequence to develop molecular markers for the Colletotrichum genus. IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) markers were used to analyze the genetic diversity of C. lindemuthianum. Fifty-four isolates belonging to different races were used. A total of 45 loci were amplified. The Nei index showed significant differences among the populations divided according to race, indicating that they are structured according to pathotype. No clear correlation between IRAP and REMAP markers with pathogenic characterization was found. C. lindemuthianum has high genetic diversity, and the analysis of molecular variance showed that 51% of variability is found among the populations of different races. The markers were also tested in different Colletotrichum species. In every case, multiple bands were amplified, indicating that these markers can be successfully used in different species belonging to the Colletotrichum genus.


Assuntos
Colletotrichum/classificação , Colletotrichum/genética , DNA Fúngico/genética , Tipagem Molecular/métodos , Técnicas de Tipagem Micológica/métodos , Retroelementos , Sequência de Bases , Análise por Conglomerados , Genótipo , Dados de Sequência Molecular , Polimorfismo Genético
12.
Braz J Microbiol ; 43(4): 1562-75, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24031988

RESUMO

The common bean is one of the most important legumes in the human diet, but little is known about the endophytic bacteria associated with the leaves of this plant. The objective of this study was to characterize the culturable endophytic bacteria of common bean (Phaseolus vulgaris) leaves from three different cultivars (Vermelhinho, Talismã, and Ouro Negro) grown under the same field conditions. The density of endophytic populations varied from 4.5 x 10(2) to 2.8 x 10(3) CFU g(-1) of fresh weight. Of the 158 total isolates, 36.7% belonged to the Proteobacteria, 32.9% to Firmicutes, 29.7% to Actinobacteria, and 0.6% to Bacteroidetes. The three P. vulgaris cultivars showed class distribution differences among Actinobacteria, Alphaproteobacteria and Bacilli. Based on 16S rDNA sequences, 23 different genera were isolated comprising bacteria commonly associated with soil and plants. The genera Bacillus, Delftia, Methylobacterium, Microbacterium, Paenibacillus, Staphylococcus and Stenotrophomonas were isolated from all three cultivars. To access and compare the community structure, diversity indices were calculated. The isolates from the Talismã cultivar were less diverse than the isolates derived from the other two cultivars. The results of this work indicate that the cultivar of the plant may contribute to the structure of the endophytic community associated with the common bean. This is the first report of endophytic bacteria from the leaves of P. vulgaris cultivars. Future studies will determine the potential application of these isolates in biological control, growth promotion and enzyme production for biotechnology.

13.
Braz J Microbiol ; 53(3): 1121-1132, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35821347

RESUMO

Anthracnose, caused by Colletotrichum lindemuthianum, is a disease affecting the common bean plant, Phaseolus vulgaris. To establish infection, the phytopathogen must survive the toxic compounds (phytoanticipins and phytoalexins) that are produced by the plant as a defense mechanism. To study the detoxification and efflux mechanisms in C. lindemuthianum, the abcCl1 gene, which encodes an ABC transporter, was analyzed. The abcCl1 gene (4558 pb) was predicted to encode a 1450-amino acid protein. Structural analysis of 11 genome sequences from Colletotrichum spp. showed that the number of ABC transporters varied from 34 to 64. AbcCl1 was classified in the ABC-G family of transporters, and it appears to be orthologs to ABC1 from Magnaporthe grisea and FcABC1 from Fusarium culmorum, which are involved in pleiotropic drug resistance. A abcT3 (ΔabcCl1) strain showed reduction on aggressivity when inoculated on bean leaves that presented diminishing anthracnose symptoms, which suggests the important role of AbcCl1 as a virulence factor and in fungal resistance to host compounds. The expression of abcCl1 increased in response to different toxic compounds, such as eugenol, hygromycin, and pisatin phytoalexin. Together, these results suggest that AbcCl1 is involved in fungal resistance to the toxic compounds produced by plants or antagonistic microorganisms.


Assuntos
Colletotrichum , Phaseolus , Colletotrichum/genética , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Fatores de Virulência/genética
14.
Can J Microbiol ; 56(10): 831-7, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20962906

RESUMO

Recombinant Penicillium griseoroseum strain 105 overproduces an extracellular pectin lyase (PL) under the transcriptional control of the strong gpdA promoter of Aspergillus nidulans. Our aim was to evaluate PL production by recombinant P. griseoroseum strain 105 in submerged fermentation system bioreactors BioFloIII and BioFloIV using 2 or 10 L working volumes under different growth conditions and to analyze the production of cellulase, polygalacturonase, pectin methylesterase, and protease. PL overproduction by recombinant P. griseoroseum strain 105 was 112 times higher than that of P. griseoroseum PG63 grown in sugarcane juice. Cellulases and proteases were not detected in the culture filtrate, and evaluation for extracellular proteins in the culture medium by SDS-PAGE showed the presence of a 36 kDa predominant band, similar to the molecular mass estimated from the nucleotide sequence of plg1 gene for PL of P. griseoroseum strain 105. This recombinant strain provides the advantage of PL production, which predominates over other extracellular proteins usually present in most commercial pectinase preparations, using sugarcane juice as a substrate of low cost.


Assuntos
Aspergillus nidulans/genética , Penicillium/enzimologia , Penicillium/genética , Polissacarídeo-Liases/biossíntese , Reatores Biológicos , Hidrolases de Éster Carboxílico/biossíntese , Celulase/biossíntese , DNA Recombinante , Eletroforese em Gel de Poliacrilamida , Fermentação/genética , Indústria Alimentícia , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Organismos Geneticamente Modificados , Peptídeo Hidrolases/biossíntese , Poligalacturonase/biossíntese , Especificidade por Substrato , Indústria Têxtil
15.
Mycorrhiza ; 20(8): 531-40, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20177718

RESUMO

The pre-symbiotic phase that precedes physical contact between symbionts is a crucial phase in determining their compatibility, allowing the formation of the ectomycorrhiza. A subtractive cDNA library representing the differentially expressed genes of the fungus Hydnangium sp. in the pre-symbiotic phase was constructed using fungal mycelia obtained through the in vitro mycorrhization technique. The fungus was cultured in the presence of Eucalyptus grandis roots, but with no contact between the hyphae and the root system of the host plant. Genes that code for proteins related to carbohydrate, amino acid, and energy metabolisms, transcription, and protein synthesis, cellular communication, signal transduction, stress response, transposons, and proteins related to the biogenesis of cell components were identified among the 131 expressed sequence tags. Expression of the genes that code for acetyl-CoA acetyltransferase, pyruvate dehydrogenase, ATP synthase, a voltage-dependent protein from the selective ion channel, and hydrophobin was evaluated by the RT-qPCR technique, confirming the activation of these genes in this phase of the association.


Assuntos
Agaricales/genética , Agaricales/metabolismo , Eucalyptus/microbiologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Simbiose , Biblioteca Gênica
16.
Braz J Microbiol ; 51(3): 1009-1020, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32410091

RESUMO

Phytic acid stores 60-90% of the inorganic phosphorus in legumes, oil seeds, and cereals, making it inaccessible for metabolic processes in living systems. In addition, given its negative charge, phytic acid complexes with divalent cations, starch, and proteins. Inorganic phosphorous can be released from phytic acid upon the action of phytases. Phytases are phosphatases produced by animals, plants, and microorganisms, notably Aspergillus niger, and are employed as animal feed additive, in chemical industry and for ethanol production. Given the industrial relevance of phytases produced by filamentous fungi, this work discusses the functional characterization of fungal phytase-coding genes/proteins, highlighting the physicochemical parameters that govern the enzymatic activity, the development of phytase super-producing strains, and key features for industrial applications.


Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , 6-Fitase/química , Ração Animal , Animais , Aspergillus niger/enzimologia , Aspergillus niger/genética , Proteínas Fúngicas/química , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Indústrias , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
17.
Braz J Microbiol ; 40(4): 852-6, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24031433

RESUMO

We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.

18.
Microbiol Res ; 219: 110-122, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30642461

RESUMO

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.


Assuntos
Colletotrichum/enzimologia , Colletotrichum/patogenicidade , Histidina Quinase/genética , Phaseolus/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Sequência de Aminoácidos , Colletotrichum/genética , Enzimas de Restrição do DNA/metabolismo , Histidina Quinase/metabolismo , Mutagênese Insercional , Phaseolus/microbiologia , Doenças das Plantas/terapia , Folhas de Planta/microbiologia , Interferência de RNA , RNA Interferente Pequeno/genética
19.
Microbiol Res ; 170: 205-12, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25023696

RESUMO

The phy gene, which encodes a phytase in Penicillium chrysogenum CCT 1273, was cloned into the vector pAN-52-1-phy and the resulting plasmid was used for the cotransformation of Penicillium griseoroseum PG63 protoplasts. Among the 91 transformants obtained, 23 were cotransformants. From there, the phytase activity of these 23 transformants was evaluated and P. griseoroseum T73 showed the highest. The recombinant strain P. griseoroseum T73 contained the phy gene integrated in at least three sites of the genome and showed a 5.1-fold increase in phytase activity in comparison to the host strain (from 0.56 ± 0.2 to 2.86 ± 0.4 U µg protein(-1)). The deduced PHY protein has 483 amino acids; an isoelectric point (pI) higher than that reported for phytases from filamentous fungi (7.6); higher activity at pH 2.0 (73%), pH 5.0 (100%) and 50 °C; and is stable at pH values 3.0-8.0 and temperatures 70-80 °C. PHY produced by the recombinant strain P. griseoroseum T73 was stable after four weeks of storage at -20, 8 and 25 °C and was effective in releasing Pi, especially from soybeans. The data presented here show that P. griseoroseum is a successful host for expression of heterologous protein and suggest the potential use of PHY in the animal nutrition industry.


Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Clonagem Molecular , Expressão Gênica , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , 6-Fitase/química , Sequência de Aminoácidos , Sequência de Bases , Ativação Enzimática , Dosagem de Genes , Dados de Sequência Molecular , Proteínas Recombinantes , Alinhamento de Sequência , Termodinâmica
20.
J Microbiol ; 51(1): 56-69, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23456713

RESUMO

Fungal endophytes were isolated from the leaves of soybean cultivars in Brazil using two different isolation techniques - fragment plating and the innovative dilution-to-extinction culturing - to increase the species richness, frequency of isolates and diversity. A total of 241 morphospecies were obtained corresponding to 62 taxa that were identified by analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). The Phylum Ascomycota predominated, representing 99% and 95.2% of isolates in the Monsoy and Conquista cultivars, respectively, whereas the Phylum Basidiomycota represented 1% and 4.8% of isolates, respectively. The genera Ampelomyces, Annulohypoxylon, Guignardia, Leptospora, Magnaporthe, Ophiognomonia, Paraconiothyrium, Phaeosphaeriopsis, Rhodotorula, Sporobolomyces, and Xylaria for the first time were isolated from soybean; this suggests that soybean harbours novel and highly diverse fungi. The yeasts genera Rhodotorula and Sporobolomyces (subphylum Pucciniomycotina) represent the Phylum Basidiomycota. The species richness was greater when both isolation techniques were used. The diversity of fungal endophytes was similar in both cultivars when the same isolation technique was used except for Hill's index, N1. The use of ITS region sequences allowed the isolates to be grouped according to Order, Class and Phylum. Ampelomyces, Chaetomium, and Phoma glomerata are endophytic species that may play potential roles in the biological control of soybean pathogens. This study is one of the first to apply extinction-culturing to isolate fungal endophytes in plant leaves, thus contributing to the development and improvement of this technique for future studies.


Assuntos
Biodiversidade , Endófitos/classificação , Endófitos/isolamento & purificação , Fungos/classificação , Fungos/isolamento & purificação , Glycine max/microbiologia , Brasil , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Endófitos/citologia , Endófitos/genética , Fungos/citologia , Fungos/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/microbiologia , Análise de Sequência de DNA
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