scholarly journals The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1867-1875 ◽  
Author(s):  
Xue-Xian Zhang ◽  
Andrew George ◽  
Mark J. Bailey ◽  
Paul B. Rainey
Keyword(s):  
Sugar Beet ◽  
Pseudomonas Fluorescens ◽  
Bacterial Colonization ◽  
Temporal Distribution ◽  
Tetracycline Resistance ◽  
Dependent Manner ◽  
In The Wild ◽  
Pseudomonas Fluorescens Sbw25 ◽  
Functional Analyses

The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based in vivo expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium Pseudomonas fluorescens SBW25. Central to the project was a rhizosphere-inducible locus, rhi14, which functional analyses show is hutT, a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between hutT and a promoterless site-specific recombinase (tnpR mut168) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose- and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical hutT fusion to promoterless lacZ. To demonstrate the effectiveness of the strategy, the activity of hutT was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0.6 μg ml−1. The ecological significance of the hut locus was examined by competing a hutT deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

scholarly journals CsHscB as a novel TLR2 agonist from carcinogenic liver fluke Clonorchis sinensis modulates host immune response

2019 ◽  
Author(s):  
Chao Yan ◽  
Fang Fan ◽  
Yu-Zhao Zhang ◽  
Jing Wu ◽  
Xin Dong ◽  
...  
Keyword(s):  
Clonorchis Sinensis ◽  
Dependent Manner ◽  
Tlr2 Agonist ◽  
Therapeutic Implications ◽  
Intrahepatic Bile Ducts ◽  
Host Interaction ◽  
Tnf Α ◽  
Activated Macrophage ◽  
In The Wild

AbstractClonorchis sinensis-a fluke dwelling on the intrahepatic bile ducts causes clonorchiasis. During C. sinensis infection, worm-host interaction results in activation of PRRs and further triggers immune responses which determine the outcome of infection. However, the mechanisms by which pathogen-associated molecules patterns from C. sinensis interacted with TLRs were poorly understood. In the present study, we identified a ∼34 kDa lipoprotein CsHscB from C. sinensis which physically bound with TLR2. We also found that recombinant CsHscB (rCsHscB) potently activated macrophage to express various proteins including TLR2, CD80, MHCII, and cytokines like IL-6, TNF-α, and IL-10 in a TLR2-dependent manner but rCsHscB failed to induce IL-10 in macrophages from Tlr2-/- mice. Moreover, ERK1/2 activation was required for rCsHscB-induced IL-10 production in macrophages. In vivo study revealed that rCsHscB triggered a high induction of IL-10 in the wild-type (WT) but not in Tlr2-/- mice. Our data thus demonstrate that rCsHscB from C. sinensis is an unidentified TLR2 agonist with immune regulatory activities, and may have some therapeutic implications in future beyond parasitology.

scholarly journals Rhes, a striatal-enriched protein, promotes mitophagy via Nix

2019 ◽  
Vol 116 (47) ◽  
pp. 23760-23771 ◽  
Author(s):  
Manish Sharma ◽  
Uri Nimrod Ramírez-Jarquín ◽  
Oscar Rivera ◽  
Melissa Kazantzis ◽  
Mehdi Eshraghi ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Neuronal Cell ◽  
Dependent Manner ◽  
Systemic Injection ◽  
Neuronal Cell Line ◽  
In The Wild ◽  
Striatal Lesion ◽  
The Brain

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here, we find that striatal-enriched protein, Rhes, is a critical regulator of mitophagy and striatal vulnerability in brain. In vivo interactome and density fractionation reveal that Rhes coimmunoprecipitates and cosediments with mitochondrial and lysosomal proteins. Live-cell imaging of cultured striatal neuronal cell line shows Rhes surrounds globular mitochondria, recruits lysosomes, and ultimately degrades mitochondria. In the presence of 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, Rhes disrupts mitochondrial membrane potential (ΔΨm) and promotes excessive mitophagy and cell death. Ultrastructural analysis reveals that systemic injection of 3-NP in mice promotes globular mitochondria, accumulation of mitophagosomes, and striatal lesion only in the wild-type (WT), but not in the Rhes knockout (KO), striatum, suggesting that Rhes is critical for mitophagy and neuronal death in vivo. Mechanistically, Rhes requires Nix (BNIP3L), a known receptor of mitophagy, to disrupt ΔΨm and promote mitophagy and cell death. Rhes interacts with Nix via SUMO E3-ligase domain, and Nix depletion totally abrogates Rhes-mediated mitophagy and cell death in the cultured striatal neuronal cell line. Finally, we find that Rhes, which travels from cell to cell via tunneling nanotube (TNT)-like cellular protrusions, interacts with dysfunctional mitochondria in the neighboring cell in a Nix-dependent manner. Collectively, Rhes is a major regulator of mitophagy via Nix, which may determine striatal vulnerability in the brain.

scholarly journals OmpR phosphorylation regulates ompS1 expression by differentially controlling the use of promoters

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 733-741 ◽  
Author(s):  
Mario Alberto Flores-Valdez ◽  
Marcos Fernández-Mora ◽  
Miguel Ángel Ares ◽  
Jorge A. Girón ◽  
Edmundo Calva ◽  
...  
Keyword(s):  
Regulatory Sequence ◽  
Dependent Manner ◽  
In The Wild ◽  
P2 Promoter ◽  
Regulatory Effects ◽  
Encoding Genes ◽  
Effect Of Glucose

The Salmonella enterica ompS1 gene encodes a quiescent porin that belongs to the OmpC/OmpF family. In the present work we analysed the regulatory effects of OmpR phosphorylation on ompS1 expression. We found that in vivo, OmpR in its phosphorylated form (OmpR-P) was important in the regulation of the two ompS1 promoters: OmpR-P activated the P1 promoter and repressed the P2 promoter in an EnvZ-dependent manner; expression occurs from the P2 promoter in an ompR mutant. In vitro, OmpR-P had a higher DNA-binding-affinity to the ompS1 promoter region than OmpR and OmpRD55A, showing an affinity even higher than that of equivalent DNA regions in the 5′-upstream regulatory sequence of the major porin-encoding genes ompC and ompF. By analysing different environmental conditions, we found that glucose and glycerol enhanced ompS1 expression in the wild-type strain. Interestingly the stimulation by glycerol was OmpR-dependent while the effect of glucose was still observed in the absence of OmpR. Acetyl phosphate produced by the AckA-Pta pathway did not influence ompS1 regulation. These data indicate the important role of the phosphorylation in the activity of OmpR on the differential regulation of both ompS1 promoters and its impact on the pathogenesis.

scholarly journals Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology

2003 ◽  
Vol 71 (3) ◽  
pp. 1379-1388 ◽  
Author(s):  
Beatrice Saviola ◽  
Samuel C. Woolwine ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Low Ph ◽  
Dependent Manner ◽  
Promoter Regions ◽  
Gene Encoding ◽  
In Vivo Expression ◽  
Promoter Trap ◽  
In The Wild ◽  
Inducible Genes

ABSTRACT A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.

scholarly journals 3′,5′-Cyclic Diguanylic Acid Reduces the Virulence of Biofilm-Forming Staphylococcus aureus Strains in a Mouse Model of Mastitis Infection

2005 ◽  
Vol 49 (8) ◽  
pp. 3109-3113 ◽  
Author(s):  
Eric Brouillette ◽  
Mamoru Hyodo ◽  
Yoshihiro Hayakawa ◽  
David K. R. Karaolis ◽  
François Malouin
Keyword(s):  
Staphylococcus Aureus ◽  
Mouse Model ◽  
Bacterial Colonization ◽  
Dependent Manner ◽  
Signaling Systems ◽  
Cyclic Dinucleotides ◽  
Novel Drug ◽  
Dose Dependent

ABSTRACT The cyclic dinucleotide 3′,5′-cyclic diguanylic acid (c-di-GMP) is a naturally occurring small molecule that regulates important signaling systems in bacteria. We have recently shown that c-di-GMP inhibits Staphylococcus aureus biofilm formation in vitro and its adherence to HeLa cells. We now report that c-di-GMP treatment has an antimicrobial and antipathogenic activity in vivo and reduces, in a dose-dependent manner, bacterial colonization by biofilm-forming S. aureus strains in a mouse model of mastitis infection. Intramammary injections of 5 and 50 nmol of c-di-GMP decreased colonization (bacterial CFU per gram of gland) by 0.79 (P > 0.05) and 1.44 (P < 0.01) logs, respectively, whereas 200-nmol doses allowed clearance of the bacteria below the detection limit with a reduction of more than 4 logs (P < 0.001) compared to the untreated control groups. These results indicate that cyclic dinucleotides potentially represent an attractive and novel drug platform which could be used alone or in combination with other agents or drugs in the prevention, treatment, or control of infection.

Survival, Colonization and Dispersal of Genetically Modified Pseudomonas fluorescens SBW25 in the Phytosphere of Field Grown Sugar Beet

1995 ◽  
Vol 13 (12) ◽  
pp. 1493-1497 ◽  
Author(s):  
Ian P. Thompson ◽  
Andrew K. Lilley ◽  
Richard J. Ellis ◽  
Penny A. Bramwell ◽  
Mark J. Bailey
Keyword(s):  
Sugar Beet ◽  
Pseudomonas Fluorescens ◽  
Genetically Modified ◽  
Pseudomonas Fluorescens Sbw25

scholarly journals Collectin Kidney 1 Plays an Important Role in Innate Immunity against Streptococcus pneumoniae Infection

2017 ◽  
Vol 9 (2) ◽  
pp. 217-228 ◽  
Author(s):  
Insu Hwang ◽  
Kenichiro Mori ◽  
Katsuki Ohtani ◽  
Yasuyuki Matsuda ◽  
Nitai Roy ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Streptococcus Pneumoniae ◽  
Pulmonary Inflammation ◽  
Dependent Manner ◽  
Complement Pathway ◽  
Calcium Dependent ◽  
Host Protection ◽  
In The Wild

Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1-/-) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1-/- mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1-/- mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1-/- mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1-/- mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway.

scholarly journals Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Ulf Gerth ◽  
Eleonora Krieger ◽  
Daniela Zühlke ◽  
Alexander Reder ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Arginine Kinase ◽  
Adaptor Protein ◽  
Glycolytic Enzyme ◽  
Dependent Manner ◽  
Wild Type ◽  
Recognition Mechanism ◽  
Content Type ◽  
In The Wild

ABSTRACT Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type. IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

scholarly journals A neural m6A/YTHDF pathway is required for learning and memory in Drosophila

2020 ◽  
Author(s):  
Lijuan Kan ◽  
Stanislav Ott ◽  
Brian Joseph ◽  
Eun Sil Park ◽  
Crystal Dai ◽  
...  
Keyword(s):  
Short Term Memory ◽  
Rna Stability ◽  
Model Organism ◽  
Translational Efficiency ◽  
Neural Function ◽  
Dependent Manner ◽  
Genomic Analyses ◽  
Age Dependent ◽  
Functional Analyses

AbstractThe roles of epitranscriptomic modifications in mRNA regulation have recently received substantial attention, with appreciation growing for their phenotypically selective impacts within the animal. We adopted Drosophila melanogaster as a model system to study m6A, the most abundant internal modification of mRNA. Here, we report proteomic and functional analyses of fly m6A-binding proteins, confirming nuclear (YTHDC) and cytoplasmic (YTHDF) YTH domain proteins as the major m6A binders. Since all core m6A pathway mutants are viable, we assessed in vivo requirements of the m6A pathway in cognitive processes. Assays of short term memory revealed an age-dependent requirement of m6A writers working via YTHDF, but not YTHDC, comprising the first phenotypes assigned to Drosophila mutants of the cytoplasmic m6A reader. These factors promote memory via neural-autonomous activities, and are required in the mushroom body, the center for associative learning. To inform their basis, we mapped m6A from wild-type and mettl3 null mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites. In contrast to mammalian m6A, which is predominant in 3’ UTRs, Drosophila m6A is highly enriched in 5’ UTRs and occurs in an adenosine-rich context. Genomic analyses demonstrate that Drosophila m6A does not directionally affect RNA stability, but is preferentially deposited on genes with low translational efficiency. However, functional tests indicate a role for m6A in translational activation, since we observe reduced nascent protein synthesis in mettl3-KO cells. Finally, we show that ectopic YTHDF can increase m6A target reporter output in an m6A-binding dependent manner, and that this activity is required for in vivo neural function of YTHDF in memory. Altogether, we provide the first tissue-specific m6A maps in this model organism and reveal selective behavioral and translational defects for m6A/YTHDF mutants.

scholarly journals Global Regulatory Roles of the Histidine-Responsive Transcriptional Repressor HutC in Pseudomonas fluorescens SBW25

2020 ◽  
Vol 202 (13) ◽  
Author(s):  
Naran Naren ◽  
Xue-Xian Zhang
Keyword(s):  
Dna Binding ◽  
Pseudomonas Fluorescens ◽  
Bacterial Colonization ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Phenotypic Characterization ◽  
Growth Defect ◽  
Dependent Manner ◽  
Rna Seq ◽  
Content Type

ABSTRACT HutC is known as a transcriptional repressor specific for histidine utilization (hut) genes in Gram-negative bacteria, including Pseudomonas fluorescens SBW25. However, its precise mode of protein-DNA interactions hasn’t been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His6-tagged HutC and biotin-labeled probe of the hut promoter (PhutU). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a P value less than 10−4. HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic hutC deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, hutC deletion caused upregulation of 794 genes and downregulation of 525 genes at a P value of <0.05 with a fold change cutoff of 2.0. The hutC mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the hut locus. IMPORTANCE HutC in Pseudomonas is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization (hut) genes through binding to the PhutU promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its PhutU target site in a plant growth-promoting bacterium, P. fluorescens SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization in planta.

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