scholarly journals Salicylic Acid Activates Sigma Factor B by rsbU-Dependent and -Independent Mechanisms

2006 ◽  
Vol 188 (16) ◽  
pp. 5896-5903 ◽  
Author(s):  
Marco Palma ◽  
Arnold Bayer ◽  
Leon I. Kupferwasser ◽  
Tammy Joska ◽  
Michael R. Yeaman ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Parental Strain ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Dependent Manner ◽  
Alpha Toxin ◽  
Reporter System ◽  
Fibronectin Binding ◽  
Green Fluorescent

ABSTRACT Salicylic acid (SAL) may impact Staphylococcus aureus virulence by activating the sigB operon (rsbU-V-W-sigB), thus leading to reductions in alpha-toxin production and decreased fibronectin binding (L. I. Kupferwasser et al., J. Clin. Investig. 112:222-233, 2003). As these prior studies were performed in strain RN6390 (an rsbU mutant) and its rsbU-repaired variant, SH1000, the current investigation was designed to determine if the SAL effect occurs via rsbU- and/or rsbV-dependent pathways in an rsbU-intact S. aureus strain (FDA486). We thus quantified the transcription from two sigB-dependent promoters (asp23 and sarA P3) in FDA486 in response to SAL exposure in vitro, using isogenic single-knockout constructs of rsbU, rsbV, or rsbW and a green fluorescent protein reporter system. SAL induced sarA P3 and asp23 promoter activities in a dose-dependent manner in the parental strain. In contrast, sigB activation by SAL was progressively more mitigated in the rsbU and rsbV mutants. As predicted, SAL caused significant reductions in both alpha-toxin production and fibrinogen and fibronectin binding in the parental strain. The extent of these reductions, compared with the parent, was reduced in the rsb mutants (rsbV > rsbU), especially at low SAL concentrations. Since generation of the free SigB protein usually requires a sequential rsbU-V-W-sigB activation cascade, the present phenotypic and genotypic data suggest key roles for both rsbU and rsbV in SAL-mediated activation of sigB in strains with a fully intact sigB operon.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1819-1828 ◽  
Author(s):  
James A. Carroll ◽  
Philip E. Stewart ◽  
Patricia Rosa ◽  
Abdallah F. Elias ◽  
Claude F. Garon
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Epifluorescence Microscopy ◽  
Reporter System ◽  
Environmental Signals ◽  
Gfp Reporter ◽  
Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

Activity of Cardiorespiratory Networks Revealed by Transsynaptic Virus Expressing GFP

2001 ◽  
Vol 85 (1) ◽  
pp. 435-438 ◽  
Author(s):  
Mustapha Irnaten ◽  
Robert A. Neff ◽  
Jijiang Wang ◽  
Arthur D. Loewy ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Nucleus Ambiguus ◽  
Dependent Manner ◽  
Neural Basis ◽  
Electrophysiological Properties ◽  
Functional Studies ◽  
Neuronal Systems ◽  
Green Fluorescent

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alphaherpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome

2001 ◽  
Vol 114 (11) ◽  
pp. 2095-2104
Author(s):  
Régis Giet ◽  
Claude Prigent
Keyword(s):  
Xenopus Laevis ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Egg Extracts ◽  
Green Fluorescent ◽  
Xenopus Egg Extracts

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.

scholarly journals Oncostatin M induces IL-33 expression in liver endothelial cells in mice and expands ST2+CD4+lymphocytes

2015 ◽  
Vol 309 (7) ◽  
pp. G542-G553 ◽  
Author(s):  
Muhammad Imran Arshad ◽  
Pierre Guihard ◽  
Yannic Danger ◽  
Gregory Noel ◽  
Jacques Le Seyec ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Oncostatin M ◽  
Dependent Manner ◽  
Liver Sinusoidal Endothelial Cells ◽  
Liver Endothelial Cells ◽  
Green Fluorescent

Interleukin (IL)-33 is crucially involved in liver pathology and drives hepatoprotective functions. However, the regulation of IL-33 by cytokines of the IL-6 family, including oncostatin M (OSM) and IL-6, is not well studied. The aim of the present study was to determine whether OSM mediates regulation of IL-33 expression in liver cells. Intramuscular administration in mice of an adenovirus encoding OSM (AdOSM) leads to increase in expression of OSM in muscles, liver, and serum of AdOSM-infected mice compared with control mice. The increase of circulating OSM markedly regulated mRNA of genes associated with blood vessel biology, chemotaxis, cellular death, induction of cell adhesion molecules, and the alarmin cytokine IL-33 in liver. Steady-state IL-33 mRNA was upregulated by OSM at an early phase (8 h) following AdOSM infection. At the protein level, the expression of IL-33 was significantly induced in liver endothelial cells [liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells] with a peak at 8 days post-AdOSM infection in mice. In addition, we found OSM-stimulated human microvascular endothelial HMEC-1 cells and human LSEC/TRP3 cells showed a significant increase in expression of IL-33 mRNA in a dose-dependent manner in cell culture. The OSM-mediated overexpression of IL-33 was associated with the activation/enrichment of CD4+ST2+cells in liver of AdOSM-infected mice compared with adenovirus encoding green fluorescent protein-treated control mice. In summary, these data suggest that the cytokine OSM regulates the IL-33 expression in liver endothelial cells in vivo and in HMEC-1/TRP3 cells in vitro and may specifically expand the target CD4+ST2+cells in liver.

scholarly journals Identification ofVibrio choleraeType III Secretion System Effector Proteins

2011 ◽  
Vol 79 (4) ◽  
pp. 1728-1740 ◽  
Author(s):  
Ashfaqul Alam ◽  
Kelly A. Miller ◽  
Mudit Chaand ◽  
J. Scott Butler ◽  
Michelle Dziejman
Keyword(s):  
Mapk Pathway ◽  
Toxin Production ◽  
Secretion System ◽  
Effector Proteins ◽  
Open Reading Frames ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Reporter System

ABSTRACTAM-19226 is a pathogenic O39 serogroupVibrio choleraestrain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a β-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins werebona fideeffectors translocated into HeLa cellsin vitroin a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only inV. choleraeandVibrio parahaemolyticusT3SS-positive strains and has not been previously studied. AvopXdeletion reduces the ability of strain AM-19226 to colonizein vivo, and the bile-induced expression of avopX-lacZtranscriptional fusionin vitrois regulated by the T3SS-encoded transcriptional regulators VttRAand VttRB. AnRLM1yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that theV. choleraeT3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.

scholarly journals Reassembling green fluorescent protein for in vitro evaluation of trans-translation

2020 ◽  
Vol 48 (4) ◽  
pp. e22-e22
Author(s):  
Charlotte Guyomar ◽  
Marion Thépaut ◽  
Sylvie Nonin-Lecomte ◽  
Agnès Méreau ◽  
Renan Goude ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Messenger Rna ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chemical Compounds ◽  
Reporter System ◽  
New Antibiotics ◽  
Green Fluorescent

Abstract In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

scholarly journals Liquid-liquid phase separation of florigen activation complex induces flowering

2021 ◽  
Author(s):  
Ken-ichiro Taoka ◽  
Hiroyuki Tsuji ◽  
Suai Anzawa ◽  
Mayu Enomoto ◽  
Yuka Koizumi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Gene Activation ◽  
Heading Date ◽  
Dependent Manner ◽  
Basic Leucine Zipper ◽  
Genes Encoding ◽  
Green Fluorescent

Floral transition, regulated by the systemic action of the mobile florigen protein FLOWERING LOCUS T (FT), is essential for successful plant reproduction1. How FT controls downstream gene expression remains incompletely understood, although it relies on the florigen activation complex (FAC), a core component of FT function2-4. The FAC is a nucleus-localized transcriptional activator of genes encoding MADS-box transcription factors critical to reproductive development and consists of florigen FT; a scaffold 14-3-3 protein that is a key component for complex assembly; and FD, a basic leucine-zipper protein that recruits the FAC to DNA. Here we report that the FAC exhibits phase separation. In rice shoot apical meristem cells, rice florigen Heading date 3a (Hd3a) fused to the green fluorescent protein formed speckles in the nucleus. The FAC speckle is formed in a FAC-dependent manner in tobacco cells. Recombinant Hd3a, but not OsFD1, phase-separated in vitro, and this effect was enhanced in the presence of 14-3-3 protein. Furthermore, mutations affecting functionally important residues in the pocket region or C-terminal disordered region of Hd3a affected FAC phase separation, providing a biochemical framework for the protein's effect on flowering. The ability to form condensates via phase transition represents a previously unknown mechanism for gene activation by the FAC.

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