Isolation and characterization ofCryphonectria parasiticamutants that mimic a specific effect of hypovirulence-associated dsRNA on laccase activity

1995 ◽  
Vol 73 (10) ◽  
pp. 1655-1661 ◽  
Author(s):  
Daniel Rigling
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Laccase Activity ◽  
Specific Effect ◽  
Chestnut Blight ◽  
Malt Extract ◽  
Wild Type ◽  
Fungal Virulence ◽  
Isolation And Characterization ◽  
Endothia Parasitica

Hypovirulent, double-stranded (ds) RNA-containing strains of the chestnut blight fungus Cryphonectria parasitica were previously shown to produce less laccase activity than virulent strains when grown on malt extract agar containing tannic acid (Bavendamm reaction). Three mutants that lacked this specific laccase activity were selected after UV mutagenesis of a dsRNA-free, wild-type strain. Complementation tests and sexual crosses showed that all mutations were recessive, two were allelic (lacR1-1 and lacR1-2), and one (lacR2) was nonallelic. No linkage was detected between the two loci. None of the known C. parasitica laccases (LAC1, LAC2, and LAC3) was substantially reduced in the lacR1 mutants. The lacR2 mutant, in contrast, produced about 10-fold less extracellular LAC1 and LAC3 activities than the wild-type strain. Intracellular LAC2 was reduced to about 50% in this mutant. These results suggest a role for both LAC1 and LAC3 in the Bavendamm reaction. The three mutations had no significant effect on fungal virulence, pigmentation, and sporulation, all phenotypes that were suppressed in an isogenic dsRNA-containing strain. Key words: Endothia parasitica, genetics, hypovirulence, dsRNA, chestnut blight.

scholarly journals Mutation of waaC, Encoding Heptosyltransferase I in Campylobacter jejuni 81-176, Affects the Structure of both Lipooligosaccharide and Capsular Carbohydrate

2006 ◽  
Vol 188 (9) ◽  
pp. 3273-3279 ◽  
Author(s):  
Margaret I. Kanipes ◽  
Erzsebet Papp-Szabo ◽  
Patricia Guerry ◽  
Mario A. Monteiro
Keyword(s):  
Campylobacter Jejuni ◽  
Type Strain ◽  
Insertional Mutagenesis ◽  
Lipid A ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Resonance Spectroscopy ◽  
Gas Liquid Chromatography ◽  
Wild Type ◽  
Isolation And Characterization

ABSTRACT Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first l-glycero-d-manno-heptose residue to 3-deoxy-d-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

scholarly journals Isolation and Characterization of Methanobacterium thermoautotrophicum ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions

1998 ◽  
Vol 180 (10) ◽  
pp. 2676-2681 ◽  
Author(s):  
Jeroen L. A. Pennings ◽  
Jan T. Keltjens ◽  
Godfried D. Vogels
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Methanobacterium Thermoautotrophicum ◽  
Mrna Levels ◽  
Wild Type ◽  
Physiological Characterization ◽  
Isolation And Characterization ◽  
Hydrogen Supply ◽  
In The Wild

ABSTRACT By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F390, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.

scholarly journals Integration of Insecticidal Protein Vip3Aa1 into Beauveria bassiana Enhances Fungal Virulence to Spodoptera litura Larvae by Cuticle and PerOs Infection

2010 ◽  
Vol 76 (14) ◽  
pp. 4611-4618 ◽  
Author(s):  
Yi Qin ◽  
Sheng-Hua Ying ◽  
Ying Chen ◽  
Zhi-Cheng Shen ◽  
Ming-Guang Feng
Keyword(s):  
Beauveria Bassiana ◽  
Type Strain ◽  
Spodoptera Litura ◽  
Wild Type Strain ◽  
Insect Pests ◽  
Wild Type ◽  
Median Lethal Concentration ◽  
Fungal Virulence ◽  
Lepidopteran Pests ◽  
Oriental Leafworm Moth

ABSTRACT The entomopathogenic fungus Beauveria bassiana acts slowly on insect pests through cuticle infection. Vegetative insecticidal proteins (Vip3A) of Bacillus thuringiensis kill lepidopteran pests rapidly, via per os infection, but their use for pest control is restricted to integration into transgenic plants. A transgenic B. bassiana strain (BbV28) expressing Vip3Aa1 (a Vip3A toxin) was thus created to infect the larvae of the oriental leafworm moth Spodoptera litura through conidial ingestion and cuticle adhesion. Vip3Aa1 (∼88 kDa) was highly expressed in the conidial cytoplasm of BbV28 and was detected as a digested form (∼62 kDa) in the larval midgut 18 and 36 h after conidial ingestion. The median lethal concentration (LC50) of BbV28 against the second-instar larvae feeding on cabbage leaves sprayed with conidial suspensions was 26.2-fold lower than that of the wild-type strain on day 3 and 1.1-fold lower on day 7. The same sprays applied to both larvae and leaves for their feeding reduced the LC50 of the transformant 17.2- and 1.3-fold on days 3 and 7, respectively. Median lethal times (LT50s) of BbV28 were shortened by 23 to 35%, declining with conidial concentrations. The larvae infected by ingestion of BbV28 conidia showed typical symptoms of Vip3A action, i.e., shrinkage and palsy. However, neither LC50 nor LT50 trends differed between BbV28 and its parental strain if the infection occurred through the cuticle only. Our findings indicate that fungal conidia can be used as vectors for spreading the highly insecticidal Vip3A protein for control of foliage feeders such as S. litura.

scholarly journals Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene.

1993 ◽  
Vol 13 (12) ◽  
pp. 7782-7792 ◽  
Author(s):  
L Zhang ◽  
A C Churchill ◽  
P Kazmierczak ◽  
D H Kim ◽  
N K Van Alfen
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Cryphonectria Parasitica ◽  
Apple Fruit ◽  
Host Gene ◽  
Fruiting Bodies ◽  
Null Mutant ◽  
Wild Type ◽  
Fungal Virulence ◽  
Double Stranded Rna

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.

scholarly journals Biochemical genetics of Neurospora nuclease I: Isolation and characterization of nuclease (nuc) mutants

1983 ◽  
Vol 41 (3) ◽  
pp. 271-286 ◽  
Author(s):  
A. M. Forsthoefel ◽  
N. C. Mishra
Keyword(s):  
Nucleic Acids ◽  
Type Strain ◽  
Wild Type Strain ◽  
Extracellular Enzymes ◽  
Spontaneous Mutation ◽  
Subsequent Paper ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Phosphorus Source

SUMMARYIsolation and characterization of five new nuclease (nuc) deficient mutants ofNeurosporahave been described. The new mutants are unable to utilize nucleic acids as the sole phosphorus source and possess growth characteristics similar to thosenuc(nuc-1andnuc-2) mutants described previously. Two new mutants (nuc-4andnuc-5) were able to use RNA or predigested DNA (but not intact DNA) as phosphorus source and showed temperature sensitive growth at 37 °C. Based on the data from complementation and genetic analyses the five new nuc mutants (nuc-3, nuc-4, nuc-5, nuc-6andnuc-7) were found nonallelic to each other and to previously describednuc(nuc-1andnuc-2) mutants; the newnucmutants mapped to the right ofarg-12on linkage group II. On biochemical analyses, thesenucmutants were found to possess a lower level of extracellular nucleases and alkaline phosphatase as compared to the wild type strain. The ds DNase activity of the new mutants was only about 2–12% of that of the wild type strain; thus, the low level of these extracellular enzymes in thenucmutants causes their inability to utilize nucleic acids as the sole phosphorus source. Wild type levels of these enzymes were restored in the complementing heterokaryons capable of full growth on the DNA medium. Data from intercrosses, mutagen sensitivity and spontaneous mutation-frequency studies (as discussed in a subsequent paper) indicated the involvement of thenucgenes in DNA repair and recombination.

scholarly journals Overexpression of the Victoriocin Gene in Helminthosporium (Cochliobolus) victoriae Enhances the Antifungal Activity of Culture Filtrates

2010 ◽  
Vol 100 (9) ◽  
pp. 890-896 ◽  
Author(s):  
Patricia B. de Sá ◽  
Hua Li ◽  
Wendy M. Havens ◽  
Mark L. Farman ◽  
Said A. Ghabrial
Keyword(s):  
Antifungal Activity ◽  
Type Strain ◽  
Wild Type Strain ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
Wild Type Virus ◽  
Wild Type ◽  
Cochliobolus Victoriae ◽  
Isolation And Characterization ◽  
Culture Filtrates

We have previously reported the isolation and characterization of the broad-spectrum antifungal protein, victoriocin, from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium (teleomorph: Cochliobolus) victoriae. We predicted that the 10-kDa mature victoriocin is derived in vivo from a preprotoxin precursor that is processed by a signal peptidase and kexin-like endopeptidase. We also presented evidence that the victoriocin precursor is encoded by a host gene, designated the victoriocin (vin) gene. In the present study, an H. victoriae genomic DNA library was constructed in the cosmid vector pMLF-2, and a cosmid clone carrying the vin gene and flanking sequences was isolated and used to generate constructs for transformation of virus-free and virus-infected H. victoriae isolates with the vin gene. Culture filtrates of the virus-free vin transformants exhibited high levels of antifungal activity compared with that revealed by the nontransformed virus-free wild-type strain, which exhibited little or no antifungal activity. Moreover, transformation of the wild-type virus-infected H. victoriae strain with the vin gene resulted in still higher production of victoriocin and higher antifungal activity in the culture filtrates of the vin transformants compared with the virus-infected wild-type strain. As previously predicted, the presence in the vin transformants of the preprovictoriocin and its post-translationally generated products, the provictoriocin and the mature victoriocin, was clearly demonstrated. Processing of the victoriocin preprotoxin requires eukaryotic host factors because no processing occurred in an in vitro translation system or in bacteria. It is of interest that some of the virus-free isolates transformed with the vin gene exhibited some features of the virus-induced disease phenotype, including moderate stunting and sectoring. Present data suggests that victoriocin may play an indirect role in disease development. Taken together, these results indicate that victoriocin is the primary protein responsible for the antifungal activity in culture filtrates of virus-infected H. victoriae isolates and that virus infection upregulates the expression of victoriocin. Overproduction of victoriocin may give the slower-growing virus-infected fungal strains some competitive advantage by inhibiting the growth of other fungi.

scholarly journals Cryptococcus neoformanssecretes small molecules that inhibit IL-1β inflammasome-dependent secretion

2019 ◽  
Author(s):  
Pedro Henrique Bürgel ◽  
Clara Luna Marina ◽  
Pedro H. V. Saavedra ◽  
Patrícia Albuquerque ◽  
Paulo Henrique Holanda ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Cryptococcus Neoformans ◽  
Type Strain ◽  
Wild Type Strain ◽  
Conditioned Media ◽  
Inflammasome Activation ◽  
Wild Type ◽  
Fungal Virulence ◽  
The Impact

AbstractCryptococcus neoformansis an encapsulated yeast that causes disease mainly in immunosuppressed hosts. It is considered a facultative intracellular pathogen because of its capacity to survive and replicate inside phagocytes, especially macrophages. This capacity is heavily dependent on various virulence factors, particularly the glucuronoxylomannan (GXM) component of the polysaccharide capsule, that render the non- or poorly-activated macrophage ineffective against phagocytosed yeast. Strategies utilized by macrophages to prevent this scenario include pyroptosis (a rapid highly inflammatory cell death) and vomocytosis (the expulsion of the pathogen from the intracellular environment without lysis). Inflammasome activation in phagocytes is usually protective against fungal infections, including cryptococcosis. Nevertheless, recognition ofC. neoformansby inflammasome receptors requires specific changes in morphology or the opsonization of the yeast, impairing a proper inflammasome function. In this context, we analyzed the impact of molecules secreted byC. neoformansB3501 strain and its acapsular mutantΔcap67in an inflammasome activationin vitromodel. Our results showed that conditioned media derived from B3501 was capable of inhibiting inflammasome dependent events (i. e. IL-1β secretion and LDH release via pyroptosis) more strongly than conditioned media fromΔcap67, regardless of GXM presence. We also demonstrated that macrophages treated with conditioned media were less responsive against infection with the virulent strain H99, exhibiting lower rates of phagocytosis, increased fungal burdens and enhanced vomocytosis. Moreover, we showed that the aromatic metabolite DL-Indole-3-lactic acid (ILA) was present in B3501’s conditioned media and that this fungal metabolite is involved in the regulation of inflammasome activation byC. neoformans. Overall, the results presented show that conditioned media from a wild-type strain can inhibit an important recognition pathway and subsequent fungicidal functions of macrophages, contributing to fungal survivalin vitroand suggesting that this serves as an important role for secreted molecules during cryptococcal infections.Author’s SummaryCryptococcus neoformansis the agent of cryptococcal meningitis, a disease that can be life-threatening in immunocompromised hosts such as those infected with HIV. The infection thrives in hosts that poorly activate their immune system, mainly because of the yeast’s ability to survive inside macrophages and migrate towards the central nervous system. Emerging data indicate that cryptococci modulate the host immune response, but the underlying mechanisms remain largely uncharacterized. Here we show that secreted molecules from a wild-type strain ofC. neoformansimpair inflammatory responses driven by inflammasome activation, which in turn impact the macrophage antifungal activity. We further show that this inhibition does not involve GXM, the main constituent of the fungal capsule, but rather is partially dependent on DL-Indole-3-lactic acid (ILA), a metabolite not previously implicated in fungal virulence.

scholarly journals Isolation and Characterization of a Vibrio vulnificus Mutant Deficient in Both Extracellular Metalloprotease and Cytolysin

2001 ◽  
Vol 69 (9) ◽  
pp. 5943-5948 ◽  
Author(s):  
Jong-Jin Fan ◽  
Chung-Ping Shao ◽  
Ya-Chi Ho ◽  
Chun-Keung Yu ◽  
Lien-I Hor
Keyword(s):  
Type Strain ◽  
Alimentary Tract ◽  
Wild Type Strain ◽  
Vibrio Vulnificus ◽  
Wild Type ◽  
Allelic Exchange ◽  
Isolation And Characterization

ABSTRACT We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.

scholarly journals Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene

1993 ◽  
Vol 13 (12) ◽  
pp. 7782-7792
Author(s):  
L Zhang ◽  
A C Churchill ◽  
P Kazmierczak ◽  
D H Kim ◽  
N K Van Alfen
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Cryphonectria Parasitica ◽  
Apple Fruit ◽  
Host Gene ◽  
Fruiting Bodies ◽  
Null Mutant ◽  
Wild Type ◽  
Fungal Virulence ◽  
Double Stranded Rna

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.

Isolation and characterization of a mutator strain of Streptomyces ambofaciens ATCC23877 exhibiting an increased level of genetic instability

1996 ◽  
Vol 42 (6) ◽  
pp. 562-570 ◽  
Author(s):  
Dominique Vandewiele ◽  
Jean-Nicolas Volff ◽  
Bertrand Aigle ◽  
Jean-Marc Simonet ◽  
Bernard Decaris
Keyword(s):  
Genetic Instability ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Analysis ◽  
Wild Type ◽  
Mutator Strain ◽  
Isolation And Characterization ◽  
Streptomyces Ambofaciens ◽  
In The Wild

In Streptomyces ambofaciens ATCC23877, 0.7% of pigment-defective mutants (Pig−) can be observed in the progeny of wild-type colonies. A mutator (Mut−) strain was isolated from the offspring of the wild-type strain. The Mut− strain produced colonies that sported nonpigmented papillae. Furthermore, the frequency of Pig− colonies obtained in the progeny of this strain was fivefold higher than in the wild-type strain. This strain showed the same level of sensitivity to ultraviolet light and mitomycin C as the wild-type strain. This Mut− phenotype was found to be reversible at high frequency (3 × 10−3). Genomic analysis using pulsed-field gel electrophoresis (PFGE) showed that the Pig− mutants arisen from the Mut− strain were less frequently rearranged (32% were deleted) compared with the mutants arising from the wild type (59% were deleted). Moreover, the Pig− papillae mutants possessed no visible rearrangement as revealed by PFGE analyses.Key words: Streptomyces, genetic instability, mutator strain, papillae.

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