Etude du polymorphisme intraclonal chez le Verticillium albo-atrum, forme à microsclérotes. I. La morphogénèse des thalles à partir des microconidies et ses variations

1980 ◽  
Vol 58 (22) ◽  
pp. 2407-2419
Author(s):  
Claude Boisson ◽  
Houria Lahlou
Keyword(s):  
Aerial Mycelium ◽  
Point Of View ◽  
Wild Type ◽  
Phenotypic Characteristics ◽  
Morphological Aspects ◽  
Inoculation Techniques ◽  
Experimental Inoculation ◽  
Morphological Variants ◽  
Verticillium Albo Atrum

In vitro thallus regeneration from microconidia was studied to define the stages of morphogenesis of some Moroccan strains of Verticillium albo-atrum Reinke and Berthold, microsclerotial forms: germination of microconidia, initiation and growth of vegetative mycelium, formation of microconidia and microsclerotia. Wild-type phenotypic characteristics such as rate of radial growth, length of time for the appearance of microsclerotia, and morphological aspects of each strain were evaluated.The morphogenesis of clones obtained from isolated microconidia showed however wide variations in percentage of regeneration and in growth and phenotype of the thallus formed from the microconidia. These variations seem to depend in part on the age of the culture from which the microconidia are taken and generally involve modifications in the regeneration potentiality of microconidia and also the appearance of stable morphological variants of which the most frequent is the hyaline type forming no microsclerotia and covered with an abundant white cotton-like aerial mycelium. The consequences of these variations are considered from a phytopathological point of view and especially in relation to experimental inoculation techniques using microconidia.

scholarly journals ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (16) ◽  
pp. 4606-4616 ◽  
Author(s):  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Mark J. Buttner
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Streptomyces Coelicolor ◽  
Aerial Mycelium ◽  
Sigma Factors ◽  
Colony Development ◽  
Null Mutant ◽  
Wild Type

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-inducedwhi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed thatwhiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficientbld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC,bldF, bldK, or bldJ or onbldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended onbldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro.

scholarly journals DevA, a GntR-Like Transcriptional Regulator Required for Development in Streptomyces coelicolor

2006 ◽  
Vol 188 (14) ◽  
pp. 5014-5023 ◽  
Author(s):  
Paul A. Hoskisson ◽  
Sebastien Rigali ◽  
Kay Fowler ◽  
Kim C. Findlay ◽  
Mark J. Buttner
Keyword(s):  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Aerial Mycelium ◽  
Developmental Cycle ◽  
Wild Type ◽  
Mobility Shift ◽  
S1 Nuclease ◽  
Aerial Hyphae ◽  
In The Wild

ABSTRACT The gram-positive filamentous bacterium Streptomyces coelicolor has a complex developmental cycle with three distinct phases: growth of the substrate mycelium, development of reproductive structures called aerial hyphae, and differentiation of these aerial filaments into long chains of exospores. During a transposon mutagenesis screen, we identified a novel gene (devA) required for proper development. The devA mutant produced only rare aerial hyphae, and those that were produced developed aberrant spore chains that were much shorter than wild-type chains and had misplaced septa. devA encodes a member of the GntR superfamily, a class of transcriptional regulators that typically respond to metabolite effector molecules. devA forms an operon with the downstream gene devB, which encodes a putative hydrolase that is also required for aerial mycelium formation on R5 medium. S1 nuclease protection analysis showed that transcription from the single devA promoter was temporally associated with vegetative growth, and enhanced green fluorescent protein transcriptional fusions showed that transcription was spatially confined to the substrate hyphae in the wild type. In contrast, devAB transcript levels were dramatically upregulated in a devA mutant and the devA promoter was also active in aerial hyphae and spores in this background, suggesting that DevA might negatively regulate its own production. This suggestion was confirmed by gel mobility shift assays that showed that DevA binds its own promoter region in vitro.

Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability

Biologia ◽  
2010 ◽  
Vol 65 (3) ◽  
Author(s):  
Andrej Godány ◽  
Katarína Majzlová ◽  
Viera Horváthová ◽  
Barbora Vidová ◽  
Štefan Janeček
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Thermotoga Maritima ◽  
Amino Acid Sequences ◽  
Point Of View ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Ph Optimum ◽  
Tim Barrel

AbstractThe presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.

Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

2020 ◽  
Vol 27 (5) ◽  
pp. 400-410
Author(s):  
Valentina De Luca ◽  
Luigi Mandrich
Keyword(s):  
Gene Evolution ◽  
Sequence Divergence ◽  
High Specificity ◽  
Industrial Applications ◽  
Point Of View ◽  
Enzyme Promiscuity ◽  
Primary Activity ◽  
Starting Point ◽  
Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

Insecticidal Active Rotenoids from Plant Parts and Callus Culture of Medicago sativa L. from a Semiarid Region of India (Rajasthan)

2020 ◽  
Vol 16 (6) ◽  
pp. 937-941
Author(s):  
Sharad Vats ◽  
Preeti Mehra
Keyword(s):  
Medicago Sativa ◽  
Callus Culture ◽  
Point Of View ◽  
Semiarid Region ◽  
Disease Vectors ◽  
Vector Borne Diseases ◽  
Plant Parts ◽  
Resistant Varieties ◽  
Medicago Sativa L

Background: Vector-borne diseases are quite prevalent globally and are one of the major causes of deaths due to infectious diseases. There is an availability of synthetic insecticides, however, their excessive and indiscriminate use have resulted in the emergence of resistant varieties of insects. Thus, a search for novel biopesticide has become inevitable. Methods: Rotenoids were isolated and identified from different parts of Medicago sativa L. This group of metabolites was also identified in the callus culture, and the rotenoid content was monitored during subculturing for a period of 10 months. Enhancement of the rotenoid content was evaluated by feeding precursors in a tissue culture medium. Results: Four rotenoids (elliptone, deguelin, rotenone and Dehydrorotenone) were identified, which were confirmed using spectral and chromatographic techniques. The maximum rotenoid content was found in the seeds (0.33±0.01%), followed by roots (0.31±0.01%) and minimum in the aerial parts (0.20±0.05%). A gradual decrease in the rotenoid content was observed with the ageing of subcultured tissue maintained for 10 months. The production of rotenoids was enhanced up to 2 folds in the callus culture using amino acids, Phenylalanine and Methionine as precursors as compared to the control. The LC50 value of the rotenoids was found to be 91 ppm and 162 ppm against disease vectors of malaria and Dracunculiasis, respectively. Conclusion: The study projects M. sativa as a novel source of biopesticide against the disease vectors of malaria and Dracunculiasis. The use of precursors to enhance the rotenoid content in vitro can be an effective venture from a commercial point of view.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

2020 ◽  
Vol 117 (32) ◽  
pp. 19245-19253 ◽  
Author(s):  
Soumyadip Sahu ◽  
Zhenzhen Wang ◽  
Xinfu Jiao ◽  
Chunfang Gu ◽  
Nikolaus Jork ◽  
...  
Keyword(s):  
Stress Responses ◽  
Messenger Rna ◽  
Control Process ◽  
Stem Cell Differentiation ◽  
Wild Type ◽  
Inositol Pyrophosphate ◽  
Body Dynamics ◽  
Processing Body ◽  
The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

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