DNA fingerprinting of <Emphasis Type="Italic">Flavobacterium columnare</Emphasis> using RAPD-PCR

In the present study, DNA fingerprinting of eight strains of Flavobacterium columnare was done by random amplification of polymorphic DNA (RAPD) fingerprinting method. The strains were collected from Fish Health Management Division, Central Institute of Freshwater Aquaculture, Bhubaneswar, India. A total number of 160 primers were screened for RAPD-PCR, of which 10 primers yielded amplification with all the strains. The molecular weight of amplified bands varied from 0.29–2.63 Kb. The number of bands varied from 1 to 8. Unique band was seen with primer OPY-15 with molecular weight 0.75 Kb that can be used for epidemiological study. Genetic variability was investigated using NTSYS software. Highest genetic similarity was found between MS1 and MS3 followed by MS5 and MS7. Minimum genetic similarity was found between MS2 and MS8. Phylogenetic tree was constructed using UPGMA and neighbor joining methods.     

Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis

Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an α-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways.     

Regulation of Morphogenesis and Biocontrol Properties in Trichoderma virens by a VELVET Protein,Vel1

Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.Trichoderma-based formulation products account for about 60% of the biofungicide market (35). Despite the use of Trichoderma-based biofungicides as an alternative and additive to chemical fungicides, the applications of these preparations are limited because their efficacy is lower than that of fungicides. A lack of understanding of the regulation of biocontrol has limited progress in enhancing the competitiveness of these fungi through genetic manipulation of desired traits. The success of a biocontrol agent also depends on the ability of researchers to develop an effective formulation based on active propagules that survive under the conditions that occur in nature and are effective against the target pathogens. Trichoderma spp. produce two types of propagules, conidia during solid-state fermentation and chlamydospores during liquid fermentation. Both types are used in commercial formulations depending on the growth conditions (17, 35). Thus, understanding how the two sporulation pathways are controlled is critical for obtaining an improved, balanced formulation product. Identification of a global regulator of morphogenesis and biocontrol properties (such as antibiosis and mycoparasitism) would provide an opportunity to manipulate the morphogenetic and antagonistic traits, leading to wider commercial acceptance of Trichoderma spp. in the long run.Trichoderma virens is a commercially formulated biocontrol agent that is effective against soilborne plant pathogens, such as Rhizoctonia solani, Sclerotium rolfsii, and Pythium spp.; its major direct mode of action is antibiosis and mycoparasitism (20, 36). This species has also been used as a model system for studies of biocontrol mechanisms, and the genome has recently been sequenced (http://genome.jgi-psf.org/Trive1). The role of beta-glucanases, chitinases, and proteases in biocontrol has been reported previously (2, 8, 29). Some strains of T. virens (designated Q strains) produce copious amounts of the antibiotic gliotoxin that is involved in biocontrol (10, 12, 39). In an attempt to identify regulators of biocontrol properties, the role of a mitogen-activated protein kinase (MAPK) pathway was studied previously (22, 24). Deletion of the TmkA/Tvk1 MAPK gene resulted in derepressed conidiation and different biocontrol behavior for two strains of T. virens; Mukherjee et al. (24) noted the reduced ability of these mutants to parasitize the sclerotia of S. rolfsii and R. solani, while Mendoza-Mendoza et al. (22) found that deletion of this MAPK gene improved the biocontrol activity of T. virens against R solani and P. ultimum. The production of secondary metabolites was not affected by deletion of this gene. To date, no gene that regulates the balance between conidiation or chlamydospore formation, secondary metabolism, and antagonistic or biocontrol properties has been identified in any Trichoderma sp.The Vel1 VELVET protein has been shown to be a regulator of morphogenesis and secondary metabolism in some filamentous fungi (6). In Aspergillus nidulans, VeA physically interacts with VelB and the regulator of secondary metabolism LaeA to form a complex that regulates secondary metabolism and sexual reproduction (3). Deletion of the VeA gene leads to an increase in asexual development (conidiation in the dark) and reduced biosynthesis of sterigmatocystin (the product of a polyketide synthetase [PKS]) and penicillin (the product of a nonribosomal peptide synthetase [NRPS]), while it reduces and delays sexual reproduction (15, 16). VeA is also required for the production of sclerotia and for aflatoxin biosynthesis in Aspergillus parasiticus (7). Deletion of the VeA gene in Neurospora crassa, like deletion of the VeA gene in A. nidulans, results in deregulated conidiation, while in Acremonium chrysogenum, loss of VeA leads to increased hyphal fragmentation and reduced cephalosporin production (4, 9). Deletion of the VeA gene in Fusarium verticilliodes resulted in a loss of hydrophobicity and an increased macroconidium-to-microconidium ratio; these defects could be restored by growing the organism on osmotically stabilized media (18). The mutants were also defective in production of the mycotoxins fumonisin and fusarin (25).To test the hypothesis that Vel1 is a global regulator of gene expression in T. virens, we examined the functions of Vel1 in this organism by using gene knockout and complementation. Here we report that in addition to a role in conidiation and secondary metabolism, Vel1 also regulates conidiophore aggregation, chlamydosporogenesis, mycoparasitism, and biocontrol efficacy in T. virens. Thus, we identified the first master regulator of morphogenesis and antagonistic properties in this economically important fungus.     

Alternative sigma factors in the free state are equilibrium mixtures of open and compact conformations

Conformational switching upon core RNA polymerase binding is an integral part of functioning of bacterial sigma factors. Here, we have studied dynamical features of two alternative sigma factors. A study of fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli σ(32) suggest a compact shape like those found in complex with anti-sigma factors. On the other hand, the fluorescence anisotropy of probes attached to different regions of the protein and previous hydrogen exchange measurements suggest significant internal flexibility, particularly in the C-terminal half and region 1. In a homologous sigma factor, σ(F) of Mycobacterium tuberculosis, emission spectra and fluorescence resonance energy transfer between the single tryptophan (W112) and probes placed in different regions suggest a compact conformation for a major part of the N-terminal half encompassing region 2 and the flexible C-terminal half. Fluorescence anisotropy measurements suggest significant flexibility in the C-terminal half and region 1, as well. Thus, free alternative sigma factors may be in equilibrium between two conformations: a compact one in which the promoter interacting motifs are trapped in the wrong conformation and another less abundant one with a more open and flexible conformation. Such flexibility may be important for promoter recognition and interaction with many partner proteins.     

Genetic diversity in important members of Cucurbitaceae using isozyme, RAPD and ISSR markers

Biochemical and molecular markers have been used on eleven species of Cucurbitaceae collected from lower Gangetic plains. Six enzyme systems were selected. Among 40 primers examined, 14 random amplified polymorphic DNA (RAPD) and 10 inter-simple sequence repeat (ISSR) primers were selected for the analysis. Generated RAPD (100) and ISSR (100) fragments showed high variations among the species. Jaccard similarity coefficients were used for the evaluation of pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate interspecific diversity. Further, principal coordinate analysis was performed to evaluate the resolving power of the three marker systems to differenciate among the species.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Genome Re-duplication and Irregular Segregation Occur During the Cell Cycle of Entamoeba histolytica

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.