Characterization of Glutamate-Gated Chloride Channels in the Pharynx of Wild-Type and Mutant Caenorhabditis elegansDelineates the Role of the Subunit GluCl-α2 in the Function of the Native Receptor

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

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Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions

eLife ◽

10.7554/elife.54740 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 14

Author(s):

Michael J Prigge ◽

Matthieu Platre ◽

Nikita Kadakia ◽

Yi Zhang ◽

Kathleen Greenham ◽

...

Keyword(s):

Genetic Analysis ◽

Early Embryo ◽

Wild Type ◽

The Family ◽

Dependent Inhibition ◽

Root Gravitropism ◽

Specialized Function ◽

Mutant Lines

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

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Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

Canadian Journal of Genetics and Cytology ◽

10.1139/g84-060 ◽

1984 ◽

Vol 26 (3) ◽

pp. 386-389 ◽

Cited By ~ 1

Author(s):

Linda J. Reha-Krantz ◽

Sükran Parmaksizoglu

Keyword(s):

Dna Polymerase ◽

Low Temperatures ◽

Bacteriophage T4 ◽

Effect Of Temperature ◽

In Vitro Mutagenesis ◽

Wild Type ◽

Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

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Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04694-14 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4669-4679 ◽

Cited By ~ 23

Author(s):

Nilmar Silvio Moretti ◽

Leonardo da Silva Augusto ◽

Tatiana Mordente Clemente ◽

Raysa Paes Pinto Antunes ◽

Nobuko Yoshida ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Drug Targets ◽

Wild Type ◽

Content Type ◽

Damage Repair ◽

Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

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Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

Eukaryotic Cell ◽

10.1128/ec.00181-13 ◽

2013 ◽

Vol 13 (4) ◽

pp. 438-451 ◽

Cited By ~ 20

Author(s):

Srisuda Pannanusorn ◽

Bernardo Ramírez-Zavala ◽

Heinrich Lünsdorf ◽

Birgitta Agerberth ◽

Joachim Morschhäuser ◽

...

Keyword(s):

Biofilm Formation ◽

Candida Parapsilosis ◽

Clinical Isolates ◽

Wild Type ◽

Content Type ◽

Initial Adhesion ◽

High Level ◽

Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

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Characterization of the Opp Peptide Transporter ofCorynebacterium pseudotuberculosisand Its Role in Virulence and Pathogenicity

BioMed Research International ◽

10.1155/2014/489782 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 18

Author(s):

Pablo M. R. O. Moraes ◽

Nubia Seyffert ◽

Wanderson M. Silva ◽

Thiago L. P. Castro ◽

Renata F. Silva ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Etiologic Agent ◽

Peptide Transporter ◽

Intercellular Signaling ◽

Wild Type ◽

Cell Nutrition ◽

Oligopeptide Permease ◽

Extracellular Environment

Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused byCorynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp inC. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of theoppDgene sequence. As occurred to the wild-type, the ΔoppDstrain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppDmutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.

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Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets

International Journal of Molecular Sciences ◽

10.3390/ijms21186563 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6563

Author(s):

Preeti Kumari Chaudhary ◽

Sanggu Kim ◽

Youngheun Jee ◽

Seung-Hun Lee ◽

Soochong Kim

Keyword(s):

Protein Kinase ◽

Kinase Inhibitor ◽

Wild Type ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Tyrosine Kinase 2 ◽

Platelet Spreading ◽

Protein Kinase Cδ

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

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Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N-Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

Journal of Bacteriology ◽

10.1128/jb.01546-09 ◽

2010 ◽

Vol 192 (9) ◽

pp. 2373-2384 ◽

Cited By ~ 20

Author(s):

Emilie Camiade ◽

Johann Peltier ◽

Ingrid Bourgeois ◽

Evelyne Couture-Tosi ◽

Pascal Courtin ◽

...

Keyword(s):

Mutant Strain ◽

Clostridium Perfringens ◽

Vegetative Growth ◽

Cell Separation ◽

Catalytic Domain ◽

Peptidoglycan Hydrolase ◽

Wild Type ◽

Induced Autolysis

ABSTRACT This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

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σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

10.1101/505099 ◽

2018 ◽

Cited By ~ 1

Author(s):

Rémi Hocq ◽

Maxime Bouilloux-Lafont ◽

Nicolas Lopes Ferreira ◽

François Wasels

Keyword(s):

Point Mutations ◽

Random Mutagenesis ◽

Value Added ◽

Clostridium Beijerinckii ◽

Phenotypic Traits ◽

Wild Type ◽

Genomic Comparison ◽

Alcohol Synthesis

Microbial production of butanol and isopropanol, two high value-added chemicals, is naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its ancient discovery, the precise mechanisms controlling alcohol synthesis in this microorganism are poorly understood. In this work, an allyl alcohol tolerant strain obtained by random mutagenesis was characterized. This strain, designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ54 sigma factor. Complementation with the wild-type sigL gene fully restored solvent production and sugar assimilation, demonstrating that σ54 plays a central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic comparison with other strains further revealed that these functions are probably conserved among the C. beijerinckii strains. The importance of σ54 in C. beijerinckii was further assessed by the characterization of a sigL deletion mutant of the model strain NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited phenotypic traits similar to the AA strain, and was subsequently complemented with the sigL gene from either the wild type or the AA strains. The results of this experiment confirmed the crucial role of σ54 in the regulation of both solventogenesis and sugar consumption pathways in C. beijerinckii.

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Genetic evidence for signal transduction within the Bacillus subtilis GerA germinant receptor

10.1101/2021.10.28.466341 ◽

2021 ◽

Author(s):

Jeremy D. Amon ◽

Lior Artzi ◽

David Z. Rudner

Keyword(s):

Signal Transduction ◽

Bacillus Subtilis ◽

Signal Transduction Pathway ◽

Dominant Negative ◽

Bacterial Spores ◽

Wild Type ◽

Enrichment Strategy ◽

Functional Receptor

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to L-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB-E105K) reduces the GerA complex's ability to respond to L-alanine, while another (gerAB-F259S) disrupts the germinant signal downstream of L-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway.

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