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 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.

Action of a slowly hydrolysable cyclic AMP analogue on developing cells of Dictyostelium discoideum

1979 ◽  
Vol 35 (1) ◽  
pp. 321-338
Author(s):  
C. Rossier ◽  
G. Gerisch ◽  
D. Malchow
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Agar Plate ◽  
Cell Aggregation ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Camp Analogue ◽  
Order Of Magnitude

Adenosine 3′,5′-cyclic phosphorothioate (cAMP-S) is a cyclic AMP (cAMP) analogue which is only slowly hydrolysed by phosphodiesterases of Dictyostelium discoideum. The affinity of cAMP-S to cAMP receptors at the cell surface is only one order of magnitude lower than that of cAMP. cAMP-S can replace cAMP as a stimulant with respect to all receptor-mediated responses tested, including chemotaxis and the induction of cAMP pulses. cAMP-S does not affect growth of D. discoideum but it blocks cell aggregation at a uniform concentration of 5 × 10(−7) M in agar plate cultures of strain NC-4 as well as its axenically growing derivative, Ax-2. Another wild-type strain of D. discoideum, v-12, is able to aggregate on agar plates supplemented with 1 mM cAMP-S. The development of Polysphondylium pallidum and P. violaceum is also highly cAMP-S resistant. In Ax-2 both differentiation from the growth phase to the aggregation-competent stage and chemotaxis are cAMP-S sensitive, whereas in v-12 only chemotaxis is inhibited. v-12 can still form streams of cohering cells and fruiting bodies when chemotaxis is inhibited by cAMP-S. Whereas cAMP induces differentiation into stalk cells at concentrations of 10(−3) or 10(−4) M, cAMP-S has the same effect in strain v-12 at the much lower concentration of 10(−6) M.

scholarly journals Bradyrhizobium elkanii rtxC Gene Is Required for Expression of Symbiotic Phenotypes in the Final Step of Rhizobitoxine Biosynthesis

2004 ◽  
Vol 70 (1) ◽  
pp. 535-541 ◽  
Author(s):  
Shin Okazaki ◽  
Masayuki Sugawara ◽  
Kiwamu Minamisawa
Keyword(s):  
Glycine Max ◽  
Type Strain ◽  
Wild Type Strain ◽  
Null Mutant ◽  
Wild Type ◽  
In Planta ◽  
Distinct Effect ◽  
Nodulation Competitiveness ◽  
Macroptilium Atropurpureum ◽  
Bradyrhizobium Elkanii

ABSTRACT We disrupted the rtxC gene on the chromosome of Bradyrhizobium elkanii USDA94 by insertion of a nonpolar aph cartridge. The rtxC mutant, designated ΔrtxC, produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta. The introduction of cosmids harboring the rtxC gene into the ΔrtxC mutant complemented rhizobitoxine production, suggesting that rtxC is involved in the final step of rhizobitoxine biosynthesis in B. elkanii USDA94. Glycine max cv. Lee inoculated with ΔrtxC or with a null mutant, Δrtx::Ω1, showed no foliar chlorosis, whereas the wild-type strain USDA94 caused severe foliar chlorosis. The two mutants showed significantly less nodulation competitiveness than the wild-type strain on Macroptilium atropurpureum. These results indicate that dihydrorhizobitoxine, the immediate precursor of the oxidative form of rhizobitoxine, has no distinct effect on nodulation phenotype in these legumes. Thus, desaturation of dihydrorhizobitoxine by rtxC-encoded protein is essential for the bacterium to show rhizobitoxine phenotypes in planta. In addition, complementation analysis of rtxC by cosmids differing in rtxC transcription levels suggested that rhizobitoxine production correlates with the amount of rtxC transcript.

scholarly journals Mutants Suppressing Novobiocin Hypersensitivity of a mukB Null Mutation

2003 ◽  
Vol 185 (13) ◽  
pp. 3690-3695 ◽  
Author(s):  
Shun Adachi ◽  
Sota Hiraga
Keyword(s):  
Chromosome Segregation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Beta Subunit ◽  
Null Mutation ◽  
Null Mutant ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Sister Chromosome

ABSTRACT The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.

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 EspC Is Involved in Controlling the Timing of Development in Myxococcus xanthus

2005 ◽  
Vol 187 (14) ◽  
pp. 5029-5031 ◽  
Author(s):  
Bongsoo Lee ◽  
Penelope I. Higgs ◽  
David R. Zusman ◽  
Kyungyun Cho
Keyword(s):  
Fruiting Body ◽  
Type Strain ◽  
Wild Type Strain ◽  
Myxococcus Xanthus ◽  
Null Mutation ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Body Development ◽  
Fruiting Body Development

ABSTRACT The espC null mutation caused accelerated aggregation and formation of tiny fruiting bodies surrounded by spores, which were also observed in the espA mutant and in CsgA-overproducing cells in Myxococcus xanthus. In addition, the espC mutant appeared to produce larger amounts of the complementary C-signal than the wild-type strain. These findings suggest that EspC is involved in controlling the timing of fruiting body development in M. xanthus.

scholarly journals The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

2015 ◽  
Vol 82 (1) ◽  
pp. 244-254 ◽  
Author(s):  
Manuel S. Godoy ◽  
Pablo I. Nikel ◽  
José G. Cabrera Gomez ◽  
M. Julia Pettinari
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Oxygen Availability ◽  
Biochemical Network ◽  
Metabolic Effects ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type ◽  
E Coli

ABSTRACTThe CreBC (carbon source-responsive) two-component regulation system ofEscherichia coliaffects a number of functions, including intermediary carbon catabolism. The impacts of differentcreCmutations (a ΔcreCmutant and a mutant carrying the constitutivecreC510allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carryingcreC510and shown to be affected by oxygen availability. The ΔcreCstrain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P)+ratios showed that thecreC510strain had a more reduced intracellular redox state, while the opposite was observed for the ΔcreCmutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase fromE. coliand a NADH-forming formate dehydrogenase fromCandida boidiniiin the ΔcreCmutant further increased the yield of succinate on glucose. Interestingly, the elimination ofackAandadhEdid not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network ofE. colimake it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.

scholarly journals Epsilon Toxin Is Essential for the Virulence of Clostridium perfringens Type D Infection in Sheep, Goats, and Mice

2013 ◽  
Vol 81 (7) ◽  
pp. 2405-2414 ◽  
Author(s):  
J. P. Garcia ◽  
V. Adams ◽  
J. Beingesser ◽  
M. L. Hughes ◽  
R. Poon ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Null Mutant ◽  
Wild Type ◽  
Histological Changes ◽  
Content Type ◽  
Type D ◽  
Necrotizing Colitis ◽  
Epsilon Toxin

ABSTRACTClostridium perfringenstype D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX toC. perfringenstype D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulentC. perfringenstype D wild-type strain (WT), an isogenic ETX null mutant (etxmutant), and a strain where theetxmutation has been reversed (etxcomplemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenicetxmutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation ofetxknockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.

scholarly journals A Negative Regulator of Carotenogenesis in Blakeslea trispora

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Wei Luo ◽  
Zunyang Gong ◽  
Na Li ◽  
Yuzheng Zhao ◽  
Huili Zhang ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Carotenoid Biosynthesis ◽  
Negative Regulator ◽  
Blakeslea Trispora ◽  
Regulation Mechanism ◽  
Null Mutant ◽  
Structural Genes ◽  
Wild Type ◽  
Β Carotene

ABSTRACT As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora. In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (−) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora. In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids. IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

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.

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