Aline Aparecida Pizzirani-Kleiner

Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a β-1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5–8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6–8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50–60°C are required over long incubation periods.

3-Hydroxypropionic Acid as an Antibacterial Agent from Endophytic Fungi Diaporthe phaseolorum

Endophytic fungi are considered a rich source of active compounds resulting from their secondary metabolism. Fungi from marine environment grow in a habitat with unique conditions that can contribute to the activation of metabolic pathways of synthesis of different unknown molecules. The production of these compounds may support the adaptation and survival of the fungi in the marine ecosystem. Mangroves are ecosystems situated between land and sea. They are frequently found in tropical and subtropical areas and enclose approximately 18.1 million hectares of the planet. The great biodiversity found in these ecosystems shows the importance of researching them, including studies regarding new compounds derived from the endophytic fungi that inhabit these ecosystems. 3-hydroxypropionic acid (3-HPA) has been isolated from the mangrove endophytic fungus Diaporthe phaseolorum, which was obtained from branches of Laguncularia racemosa. The structure of this compound was elucidated by spectroscopic methods, mainly 1D and 2D NMR. In bioassays, 3-HPA showed antimicrobial activities against both Staphylococcus aureus and Salmonella typhi. The structure of this antibiotic was modified by the chemical reaction of Fischer-Speier esterification to evaluate the biologic activity of its chemical analog. The esterified product, 3-hydroxypropanoic ethyl ester, did not exhibit antibiotic activity, suggesting that the free carboxylic acid group is important to the pharmacological activity. The antibiotic-producing strain was identified with internal transcribed spacer sequence data. To the best of our knowledge, this is the first report of antibacterial activity by 3-HPA against the growth of medically important pathogens.

Species diversity of culturable endophytic fungi from Brazilian mangrove forests

This study aimed to perform a comparative analysis of the diversity of endophytic fungal communities isolated from the leaves and branches of Rhizophora mangle, Avicennia schaueriana and Laguncularia racemosa trees inhabiting two mangroves in the state of São Paulo, Brazil [Cananeia and Bertioga (oil spill-affected and unaffected)] in the summer and winter. Three hundred and forty-three fungi were identified by sequencing the ITS1-5.8S-ITS2 region of rDNA. Differences were observed in the frequencies of fungi isolated from the leaves and branches of these three different plant species sampled from the Bertioga oil spill-affected and the oil-unaffected mangrove sites in the summer and winter; these differences indicate a potential impact on fungal diversity in the study area due to the oil spill. The molecular identification of the fungi showed that the fungal community associated with these mangroves is composed of at least 34 different genera, the most frequent of which were Diaporthe,Colletotrichum, Fusarium, Trichoderma and Xylaria. The Shannon and the Chao1 indices [H′(95xa0%)xa0=xa04.00, H′(97xa0%)xa0=xa04.22, Chao1(95xa0%)xa0=xa0204 and Chao1(97xa0%)xa0=xa0603] indicated that the mangrove fungal community possesses a vast diversity and richness of endophytic fungi. The data generated in this study revealed a large reservoir of fungal genetic diversity inhabiting these Brazilian mangrove forests and highlighted substantial differences between the fungal communities associated with distinct plant tissues, plant species, impacted sites and sampling seasons.

Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines

The diversity of endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane plants and its isoline was evaluated by cultivation followed by amplified rDNA restriction analysis (ARDRA) of randomly selected strains. Transgenic and non-transgenic cultivars and their crop management (herbicide application or manual weed control) were used to assess the possible non-target effects of genetically modified sugarcane on the fungal endophytic community. A total of 14 ARDRA haplotypes were identified in the endophytic community of sugarcane. Internal transcribed spacer (ITS) sequencing revealed a rich community represented by 12 different families from the Ascomycota phylum. Some isolates had a high sequence similarity with genera that are common endophytes in tropical climates, such as Cladosporium, Epicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of molecular variance indicated that fluctuations in fungal population were related to both transgenic plants and herbicide application. While herbicide applications quickly induced transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. These results represent the first draft on composition of endophytic filamentous fungi associated with sugarcane plants. They are an important step in understanding the possible effects of transgenic plants and their crop management on the fungal endophytic community.

Specific plant induced biofilm formation in Methylobacterium species

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes.

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

The Selection Exerted by Oil Contamination on Mangrove Fungal Communities

Mangrove ecosystems are tropical environments that are characterized by the interaction between the land and the sea. As such, this ecosystem is vulnerable to oil spills. Here, we show a culture-independent survey of fungal communities that are found in the sediments of the following two mangroves that are located on the coast of Sao Paulo State (Brazil): (1) an oil-spill-affected mangrove and (2) a nearby unaffected mangrove. Samples were collected from each mangrove forest at three distinct locations (transect from sea to land), and the samples were analyzed by quantitative PCR and internal transcribed spacer (ITS)-based PCR-DGGE analysis. The abundance of fungi was found to be higher in the oil-affected mangrove. Visual observation and correspondence analysis (CA) of the ITS-based PCR-DGGE profiles revealed differences in the fungal communities between the sampled areas. Remarkably, the oil-spilled area was quite distinct from the unaffected sampling areas. On the basis of the ITS sequences, fungi that are associated with the Basidiomycota and Ascomycota taxa were most common and belonged primarily to the genera Epicoccum, Nigrospora, and Cladosporium. Moreover, the Nigrospora fungal species were shown to be sensitive to oil, whereas a group that was described as “uncultured Basidiomycota” was found more frequently in oil-contaminated areas. Our results showed an increase in fungal abundance in the oil-polluted mangrove regions, and these data indicated potential fungal candidates for remediation of the oil-affected mangroves.

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.

Electrophoretic characterization of Aspergillus nidulans strains with chromosomal duplications

Abstract Pulsed-field gel electrophoresis was used to characterize strains of Aspergillus nidulans with a chromosomal duplication Dp(I-II).Morphologically deteriorated and improved variants of these strains were also analyzed. The electrophoretic karyotype demonstra tedthat in two duplicated strains (A and B) the 4.2 Mb band, which corresponds to chromosome II, was absent and a new band wasobserved. Hybridization studies using the uapA (chromosome I) and wA (chromosome II) genes demonstrated that the new bandcorresponded to chromosome II plus the duplicated segment of chromosome I. The size of the chromosomal duplication was approxi-mately 1.0 Mb. Analysis of the chromosomal bands of a morphologically improved strain showed that the duplicated segment of chr omo-some I was completely lost. The morphologically deteriorated variants V9 and V17 had the same karyotype as the duplicated strai ns.However, the deteriorated variant V5 lost part of chromosome I and had a rearrangement involving chromosome V. This rearrangeme ntmay have resulted from the mutagenic treatment used to obtain the genetic markers. Pulsed-field gel electrophoresis was found t o be anexcellent tool for locating chromosomal rearrangements.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic β-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP’s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable β-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.