Analyse ultrastructurale et biochimique de mutants pléiotropes de Bacillus subtilis affectés dans le contrôle de la sporulation

1984 ◽  
Vol 30 (11) ◽  
pp. 1367-1376
Author(s):  
Joseph Zucca ◽  
Serge Renaudin
Keyword(s):  
Bacillus Subtilis ◽  
Pleiotropic Effect ◽  
Spore Coat ◽  
Coat Proteins ◽  
Extracellular Proteases ◽  
Transmission Electron ◽  
Mutant Strains ◽  
Spore Coat Proteins ◽  
Spore Ultrastructure ◽  
Spore Coat Protein

Derived from a Bacillus subtilis unstable mutant strains which hyperproduce extracellular proteases were found to contain mutations at loci scoC, scoD, or scoE; these were observed by transmission electron microscopy. In scoC and scoD strains, spores formed overproduced spore coat proteins and the spore ultrastructure was deeply altered. In scoE strains, stage III of the sporulation was blocked. The pleiotropic effect of scoC mutations was either positive or negative. In unstable strains, mutations at the scoC locus stimulated α-amylase and levane sucrase hyperproduction. In stable strains, the same mutations led to an absence of α-amylase synthesis but to a normal levane sucrase production. ScoD mutations only induced α-amylase hyperproduction, while scoE mutations allowed neither levane sucrase production nor spore coat protein synthesis.

scholarly journals Transglutaminase-Mediated Cross-Linking of GerQ in the Coats of Bacillus subtilis Spores

2004 ◽  
Vol 186 (17) ◽  
pp. 5567-5575 ◽  
Author(s):  
Katerina Ragkousi ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
Cross Linking ◽  
Spore Coat ◽  
Coat Proteins ◽  
Formation Pathway ◽  
Mutant Strains ◽  
Hydrolysis Of ◽  
Spore Coat Protein

ABSTRACT The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat. GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat. Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase. GerQ is the only substrate for this transglutaminase identified to date. In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis. These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway.

scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

scholarly journals The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins

2000 ◽  
Vol 192 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Hiromu Takamatsu ◽  
Atsuo Imamura ◽  
Takeko Kodama ◽  
Kei Asai ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Spore Coat ◽  
Coat Proteins ◽  
Specific Protease ◽  
Spore Coat Proteins

scholarly journals Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

2002 ◽  
Vol 184 (4) ◽  
pp. 1219-1224 ◽  
Author(s):  
Irina Bagyan ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
High Salt ◽  
Lytic Enzyme ◽  
Spore Coat ◽  
Coat Proteins ◽  
His Tag ◽  
Spore Coat Proteins ◽  
Triton X

ABSTRACT The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca2+-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.

Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 3011-3021 ◽  
Author(s):  
Ritsuko Kuwana ◽  
Satoko Yamamura ◽  
Hiromi Ikejiri ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Electron Microscopic Examination ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Systematic Screening ◽  
Consensus Sequences ◽  
Transmission Electron ◽  
Insertional Inactivation ◽  
Spore Coat Proteins

In systematic screening four sporulation-specific genes, yjcA, yjcB, yjcZ and yjcC, of unknown function were found in Bacillus subtilis. These genes are located just upstream of the cotVWXYZ gene cluster oriented in the opposite direction. Northern blot analysis showed that yjcA was transcribed by the SigE RNA polymerase beginning 2 h (t 2) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at t 4 of sporulation. The transcription of yjcZ was dependent on SigK and GerE. The consensus sequences of the appropriate sigma factors were found upstream of each gene. There were putative GerE-binding sites upstream of yjcZ. Insertional inactivation of the yjcC gene resulted in a reduction in resistance of the mutant spores to lysozyme and heat. Transmission electron microscopic examination of yjcC spores revealed a defect of sporulation at stage VI, resulting in loss of spore coats. These results suggest that YjcC is involved in assembly of spore coat proteins that have roles in lysozyme resistance. It is proposed that yjcC should be renamed as spoVIF.

scholarly journals The choice of anchoring protein influences interaction of recombinant Bacillus spores with the immune system

2017 ◽  
Vol 64 (2) ◽  
Author(s):  
Aurelia Piekarska ◽  
Paulina Pełka ◽  
Grażyna Peszyńska-Sularz ◽  
Alessandro Negri ◽  
Krzysztof Hinc ◽  
...  
Keyword(s):  
Immune System ◽  
Spore Coat ◽  
Coat Proteins ◽  
Heterologous Proteins ◽  
Protein Fusion ◽  
Mucosal Vaccination ◽  
Anchoring Protein ◽  
Bacillus Spores ◽  
Spore Coat Proteins ◽  
Spore Coat Protein

The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently there are no technical possibilities to predict, whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study we compared four variants of B. subtilis spores presenting a fragment of FliD protein of Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their phagocytosis and activate both, J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent on its abundance and localization in the spore coat structure.

scholarly journals Characterization of exosporium layer variability of Clostridioides difficile spores in the epidemically relevant strain R20291

2020 ◽  
Author(s):  
Marjorie Pizarro-Guajardo ◽  
Paulina Calderón ◽  
Alba Romero-Rodriguez ◽  
Daniel Paredes-Sabja
Keyword(s):  
Spore Coat ◽  
Coat Proteins ◽  
Anaerobic Conditions ◽  
Host Interactions ◽  
Clostridioides Difficile ◽  
Transmission Electron ◽  
Antibiotic Associated Diarrhea ◽  
Biological Aspects ◽  
Spore Coat Proteins

AbstractClostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea and spores are the transmission vehicle of the disease. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that the laminations of the spore-coat are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation.

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