scholarly journals Investigating the influence of spore maturation and sporulation conditions on MalS expression during Bacillus subtilis spore germination

2019 ◽  
Author(s):  
Bhagyashree Swarge ◽  
Chahida Nafid ◽  
Norbert Vischer ◽  
Stanley Brul
Keyword(s):  
Fusion Protein ◽  
Spore Germination ◽  
Time Lapse ◽  
Initial Increase ◽  
Bacillus Subtilis Spore ◽  
Gfp Fusion Protein ◽  
Food Borne ◽  
Germination Behaviour ◽  
Fluorescence Level ◽  
Spore Forming Bacteria

AbstractSpore forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and food-borne diseases. The spores remain in a dormant state for extended periods due to their highly resistant features. When environmental conditions become favourable, they can germinate as the germinant receptors located on the spore’s inner membrane get activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth. The present study focuses on the synthesis of protein MalS during B. subtilis spore germination by investigating the dynamics of the fluorescence level of a MalS-GFP fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase within the first 15 minutes of germination, followed by a drop and stabilization of the fluorescence throughout the spore ripening period. Western blot analyses, however, indicate no increase in the levels of the MalS-GFP fusion protein during the first 15 minutes after the addition of the germinants. Thus the instantaneous increase in fluorescence of MalS-GFP may likely due to a change in the physical environment as the spore germination is triggered. Our findings also show that the different sporulation conditions and the maturation time of spores affect the expression of MalS-GFP and the germination behaviour of the spores.

Inhibition of clathrin-coated pit assembly by an Eps15 mutant

1999 ◽  
Vol 112 (9) ◽  
pp. 1303-1311 ◽  
Author(s):  
A. Benmerah ◽  
M. Bayrou ◽  
N. Cerf-Bensussan ◽  
A. Dautry-Varsat
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Site ◽  
Specific Marker ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gfp Fusion Protein ◽  
Protein Encoding ◽  
Homology Domains

Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.

scholarly journals Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

1997 ◽  
Vol 139 (6) ◽  
pp. 1465-1476 ◽  
Author(s):  
Norio Sakai ◽  
Keiko Sasaki ◽  
Natsu Ikegaki ◽  
Yasuhito Shirai ◽  
Yoshitaka Ono ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Nmda Receptor ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Kinase C ◽  
Gfp Fusion Protein ◽  
Green Fluorescent

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

2010 ◽  
Vol 163 (1) ◽  
pp. 80-89 ◽  
Author(s):  
Julia Maria Naumann ◽  
Gabriele Küttner ◽  
Matthias Bureik
Keyword(s):  
Fusion Protein ◽  
Fission Yeast ◽  
Gfp Fusion Protein

scholarly journals Effect of Pasteurized Low Acidic Indigenous Beverages Produced in Northern Cameroon on Germination of Bacillus and Geobacillus Spores Species

2021 ◽  
Vol 8 (2) ◽  
pp. 52-62
Author(s):  
James Ronald Bayoï ◽  
François-Xavier Etoa
Keyword(s):  
Bacillus Subtilis ◽  
Incubation Time ◽  
Spore Germination ◽  
Inhibitory Effect ◽  
Geobacillus Stearothermophilus ◽  
Northern Cameroon ◽  
Natural Preservatives ◽  
Acidic Beverages ◽  
Good Hygiene ◽  
Spore Forming Bacteria

The present study aimed to investigate the influence of three commercially available traditional acidic beverages on spore germination. “Foléré”, red “té” and white “mpedli” sorghum beers have been produced at the laboratory scale assisted by experimented producers, and pH of samples were adjusted at 2.01, 2.63 and 2.8 respectively, then they were pasteurized. The samples produced were tested on four spore-forming bacteria (Bacillus cereus, Bacillus megapterium, Bacillus subtilis and Geobacillus stearothermophilus) and germination was assessed both on culture plate media and by loss of optical density (OD) methods. The results obtained showed that “foléré” at pH 2.01, and both indigenous sorghum red beer at pH 2.63 and white beer at pH 2.8 were effective on spore germination, and efficacy significantly increase (p < 0.05) with the incubation time. The presence of alcohol in the pasteurized white (2.43 %) and red (4.7 %) sorghum beers has significantly (p < 0.05) improved the anti-germinating activity compared to the non-alcoholic “foléré” beverage. The sensitivity of B. cereus and B. subtilis was positively and significantly correlated (r = 0.880; p < 0.01) likewise the sensitivity of B. megapterium and G. stearothermophilus (r = 0.725; p < 0.05), and the activity of traditional white and red sorghum beers was found to be very significant (p < 0.05) for each couple respectively. The loss of OD showed an inhibitory effect of indigenous beverages germination and exhibited a microcycle on all tested spore-forming bacteria. It was concluded that if the good hygiene and manufacturing practices were applied for production of indigenous beverages, they might easily be used as natural preservatives and for prevention of gastroenteritis induced by germination and outgrowth of spore-forming bacteria like B. cereus.

scholarly journals The Conserved Effector UvHrip1 Interacts with OsHGW and Infection of Ustilaginoidea virens Regulates Defense- and Heading Date-Related Signaling Pathway

2020 ◽  
Vol 21 (9) ◽  
pp. 3376
Author(s):  
Songhong Wei ◽  
Yingling Wang ◽  
Jianming Zhou ◽  
Shibo Xiang ◽  
Wenxian Sun ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Virulence Factors ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Heading Date ◽  
Ustilaginoidea Virens ◽  
Bifc Assay ◽  
Gfp Fusion Protein ◽  
False Smut ◽  
Insight Into

Ustilaginoidea virens, which causes rice false smut (RFS), is one of the most detrimental rice fungal diseases and poses a severe threat to rice production and quality. Effectors in U. virens often act as a group of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of individual effectors in U. virens need to be further explored. Here, we demonstrated a small secreted hypersensitive response-inducing protein (hrip), named UvHrip1, which was highly conserved in U. virens isolates. UvHrip1 was also proven to suppress necrosis-like defense symptoms in N. benthamiana induced by the oomycete elicitor INF1. The localization of UvHrip1 was mainly in the nuclei and cytoplasm via monitoring the UvHrip1-GFP fusion protein in rice cells. Furthermore, Y2H and BiFC assay demonstrated that UvHrip1 interacted with OsHGW, which is a critical regulator in heading date and grain weight signaling pathways in rice. Expression patterns of defense- and heading date-related genes, OsPR1#051 and OsMYB21, were down-regulated over U. virens infection in rice. Collectively, our data provide a theory for gaining an insight into the molecular mechanisms underlying the UvHrip1 virulence function.

scholarly journals Spinophilin and the immune synapse

2008 ◽  
Vol 181 (2) ◽  
pp. 181-183 ◽  
Author(s):  
Brian Seed ◽  
Ramnik Xavier
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Fusion Protein ◽  
Target Cells ◽  
Cellular Organization ◽  
Immune Synapse ◽  
Cellular Architecture ◽  
Gfp Fusion Protein ◽  
Cell Biol ◽  
In Cells

Extensive alterations in cellular organization are known to accompany the responses of sensitized T cells to target cells presenting an antigen of interest. Now, equally if not more dramatic changes are found to take place in cells presenting an antigen. With the help of a spinophilin-GFP fusion protein, Bloom et al. (Bloom, O., J.J. Unternaehrer, A. Jiang, J.-S. Shin, L. Delamarre, P. Allen, and I. Mellman. 2008. J. Cell Biol. 181:203–211) have captured a remarkable polarization of the cellular architecture of dendritic cells presenting an antigen to T cells.

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