Release and regeneration of protoplasts from the fungus Trichothecium roseum

2003 ◽  
Vol 49 (4) ◽  
pp. 263-268 ◽  
Author(s):  
N Balasubramanian ◽  
G Annie Juliet ◽  
P Srikalaivani ◽  
D Lalithakumari
Keyword(s):  
Yeast Extract ◽  
Parent Strain ◽  
Protoplast Isolation ◽  
Lytic Enzyme ◽  
Complete Medium ◽  
Lytic Enzymes ◽  
Trichothecium Roseum ◽  
Osmotic Stabilizer ◽  
Enzyme Combination ◽  
Yeast Extract Agar

A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 × 104 protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose – yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.Key words: Trichothecium roseum, protoplast, isolation, regeneration, lytic enzymes, osmotic stabilizers.

scholarly journals Influence of culture media in viability test of conidia of entomopathogenic fungi

2006 ◽  
Vol 36 (4) ◽  
pp. 1309-1312 ◽  
Author(s):  
Edimara Aparecida Francisco ◽  
Dinalva Alves Mochi ◽  
Antônia do Carmo Barcelos Correia ◽  
Antonio Carlos Monteiro
Keyword(s):  
Yeast Extract ◽  
Culture Medium ◽  
Culture Media ◽  
Complete Medium ◽  
Paecilomyces Fumosoroseus ◽  
Lecanicillium Lecanii ◽  
Extract Agar ◽  
High Germination ◽  
Microscope Slides ◽  
Yeast Extract Agar

This work aimed at investigatimg whether the culture medium used in viability tests affects the conidial germination of Lecanicillium lecanii, Beauveria bassiana and Paecilomyces fumosoroseus isolates. The tests were performed on microscope slides containing one of the culture media: agar-water (AW), minimal medium (MM), potato-dextrose agar (PDA), potato-dextrose-1% yeast extract agar (PDAY), Sabouraud-dextrose-yeast extract agar (SDAY), and complete medium (CM). Three areas per slide were delimited and 0.05ml of a 5.5 x 105 conidia ml-1 suspension was applied to each area. One bioassay was performed for each isolate. Germination was determined after 15 hours of incubation at 26±0.5°C. The culture media influenced the germination of the species studied, verifying within and inter specific variations. CM and PDA provided the highest germination of L. lecanii isolates and the lowest was obtained on SDAY and AW. The germination of B. bassiana isolates was favoured by CM, PDA and PDAY media, a fact not observed in AW and MM. P. fumosoroseus isolates showed the highest germination on CM and PDA media and the lowest on SDAY. However, some isolates presented high germination on nutrient-poor media (AW and MM).

scholarly journals Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

2010 ◽  
Vol 34 (1) ◽  
pp. 74-79 ◽  
Author(s):  
Francine Hiromi Ishikawa ◽  
Quélen de Lima Barcelos ◽  
Elaine Aparecida de Souza ◽  
Eustáquio Souza Dias
Keyword(s):  
Efficient Method ◽  
Incubation Time ◽  
Genetic Manipulation ◽  
Protoplast Isolation ◽  
Colletotrichum Lindemuthianum ◽  
Lytic Enzymes ◽  
Factors Affecting ◽  
Osmotic Stabilizer ◽  
Digestion Mixture ◽  
Protoplast Production

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

A Computational Method for the Identification of Endolysins and Autolysins

2020 ◽  
Vol 27 (4) ◽  
pp. 329-336 ◽  
Author(s):  
Lei Xu ◽  
Guangmin Liang ◽  
Baowen Chen ◽  
Xu Tan ◽  
Huaikun Xiang ◽  
...  
Keyword(s):  
Support Vector Machine ◽  
Cell Wall ◽  
Experimental Results ◽  
Computational Method ◽  
Lytic Enzyme ◽  
Support Vector ◽  
Lytic Enzymes ◽  
Data Set ◽  
Optimal Feature ◽  
Better Than

Background: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. Objective: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. Method: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. Results: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. Conclusion: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.

Comparison of agar-based methods for the isolation and enumeration of heterotrophic bacteria with the new Multidose IDEXX(tm) SimPlate method

2004 ◽  
Vol 50 (1) ◽  
pp. 277-280
Author(s):  
M. Vulindlu ◽  
A. Charlett ◽  
S. Surman ◽  
J.V. Lee
Keyword(s):  
Yeast Extract ◽  
Heterotrophic Bacteria ◽  
Treatment Plant ◽  
Plant Performance ◽  
Extract Agar ◽  
Quality Of Water ◽  
Heterotrophic Microorganisms ◽  
External Sources ◽  
Conventional Methods ◽  
Yeast Extract Agar

Pour and spread plates are the conventional methods of choice for the isolation and enumeration of heterotrophic microorganisms in treated water supplies. The tests are performed at 22°C and 37°C for 72 h and 48 h respectively. Counts at 22°C are associated with pollution of water systems from external sources, while counts at 37°C are used as an indication of treatment plant performance and the deterioration of the general quality of water. Conventional methods using Yeast Extract Agar for a pour plate and R2A agar for a spread plate were compared with the multidose IDEXXTM SimPlate method for the isolation and enumeration of heterotrophic bacteria in water. SimPlate gave a significantly higher count on average than the conventional methods. The R2A method showed the next highest count, being significantly higher than Yeast Extract Agar. In addition, unlike the pour and spread plate methods, SimPlate was easier to use, reduced labour, and the test results were far easier to read.

Nannocystis exedens: A Potential Biocompetitive Agent against Aspergillus flavus and Aspergillus parasiticus

2001 ◽  
Vol 64 (7) ◽  
pp. 1030-1034 ◽  
Author(s):  
WILLIE J. TAYLOR ◽  
FRANCES A. DRAUGHON
Keyword(s):  
Aspergillus Flavus ◽  
Yeast Extract ◽  
Biocontrol Agent ◽  
Aspergillus Parasiticus ◽  
Extract Agar ◽  
Potential Biocontrol Agent ◽  
Aflatoxigenic Fungi ◽  
Close Proximity ◽  
Zones Of Inhibition ◽  
Yeast Extract Agar

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28°C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.

scholarly journals Several Metarhizium Species Produce Ergot Alkaloids in a Condition-Specific Manner

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Caroline E. Leadmon ◽  
Jessi K. Sampson ◽  
Matthew D. Maust ◽  
Angie M. Macias ◽  
Stephen A. Rehner ◽  
...  
Keyword(s):  
Yeast Extract ◽  
Ergot Alkaloid ◽  
Genomic Sequence ◽  
Close Association ◽  
Acid Amide ◽  
Ergot Alkaloids ◽  
Lysergic Acid ◽  
Content Type ◽  
Extract Agar ◽  
Yeast Extract Agar

ABSTRACT Genomic sequence data indicate that certain fungi in the genus Metarhizium have the capacity to produce lysergic acid-derived ergot alkaloids, but accumulation of ergot alkaloids in these fungi has not been demonstrated previously. We assayed several Metarhizium species grown under different conditions for accumulation of ergot alkaloids. Isolates of M. brunneum and M. anisopliae accumulated the lysergic acid amides lysergic acid α-hydroxyethyl amide, ergine, and ergonovine on sucrose-yeast extract agar but not on two other tested media. Isolates of six other Metarhizium species did not accumulate ergot alkaloids on sucrose-yeast extract agar. Conidia of M. brunneum lacked detectable ergot alkaloids, and mycelia of this fungus secreted over 80% of their ergot alkaloid yield into the culture medium. Isolates of M. brunneum, M. flavoviride, M. robertsii, M. acridum, and M. anisopliae produced high concentrations of ergot alkaloids in infected larvae of the model insect Galleria mellonella, but larvae infected with M. pingshaense, M. album, M. majus, and M. guizhouense lacked detectable ergot alkaloids. Alkaloid concentrations were significantly higher when insects were alive (as opposed to killed by freezing or gas) at the time of inoculation with M. brunneum. Roots of corn and beans were inoculated with M. brunneum or M. flavoviride and global metabolomic analyses indicated that the inoculated roots were colonized, though no ergot alkaloids were detected. The data demonstrate that several Metarhizium species produce ergot alkaloids of the lysergic acid amide class and that production of ergot alkaloids is tightly regulated and associated with insect colonization. IMPORTANCE Our discovery of ergot alkaloids in fungi of the genus Metarhizium has agricultural and pharmaceutical implications. Ergot alkaloids produced by other fungi in the family Clavicipitaceae accumulate in forage grasses or grain crops; in this context they are considered toxins, though their presence also may deter or kill insect pests. Our data report ergot alkaloids in Metarhizium species and indicate a close association of ergot alkaloid accumulation with insect colonization. The lack of accumulation of alkaloids in spores of the fungi and in plants colonized by the fungi affirms the safety of using Metarhizium species as biocontrol agents. Ergot alkaloids produced by other fungi have been exploited to produce powerful pharmaceuticals. The class of ergot alkaloids discovered in Metarhizium species (lysergic acid amides) and their secretion into the growth medium make Metarhizium species a potential platform for future studies on ergot alkaloid synthesis and modification.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

scholarly journals Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis

2008 ◽  
Vol 74 (24) ◽  
pp. 7490-7496 ◽  
Author(s):  
Yu Pei Tan ◽  
Philip M. Giffard ◽  
Daniel G. Barry ◽  
Wilhelmina M. Huston ◽  
Mark S. Turner
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Transmembrane Protein ◽  
Random Mutagenesis ◽  
Lytic Enzyme ◽  
Control Strain ◽  
Lytic Enzymes ◽  
Positive Bacterium ◽  
Peptidoglycan Biosynthesis ◽  
Cell Wall Lytic Enzymes

ABSTRACT Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.

Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants

1988 ◽  
Vol 34 (2) ◽  
pp. 157-161 ◽  
Author(s):  
Michel Hébraud ◽  
Michel Fèvre
Keyword(s):  
Cell Fusion ◽  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Lytic Enzymes ◽  
Hebeloma Cylindrosporum ◽  
Regeneration Rate ◽  
Osmotic Stabilizer ◽  
Resistant Mutants ◽  
High Yields ◽  
Protoplast Production

Methods were developed for the reproducible production of high yields of stable protoplasts from monokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum. This is a prerequisite for mutation, cell fusion, and transformation of this mycorrhizal fungus. Protoplasts from Hebeloma cylindrosporum, Hebeloma edurum, Hebeloma sinapizans, and Suillus bellini were released by using cellulase Onozuka R 10 and driselase as lytic enzymes. Production was strongly influenced by the nature and molarity of the osmotic stabilizer and by the mycelial age. In spite of the high percentage of anucleate protoplasts (42%) produced, the regeneration rate averaged 15%. Fungicide-resistant mutants from monokaryotic mycelia of H. cylindrosporum were isolated following ultraviolet irradiation of protoplasts. This indicates that protoplasts will be useful to select physiological mutants from nonsporulating mycelia and to create new strains by mating complementary mycelia or by interspecific cell fusion.

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