Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus

ABSTRACT The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation.

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

10.1101/2020.04.05.024893 ◽

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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Functional Characterization of Clostridium difficile Spore Coat Proteins

Journal of Bacteriology ◽

10.1128/jb.02104-12 ◽

2013 ◽

Vol 195 (7) ◽

pp. 1492-1503 ◽

Cited By ~ 58

Author(s):

P. Permpoonpattana ◽

J. Phetcharaburanin ◽

A. Mikelsone ◽

M. Dembek ◽

S. Tan ◽

...

Keyword(s):

Clostridium Difficile ◽

Functional Characterization ◽

Spore Coat ◽

Coat Proteins ◽

Spore Coat Proteins

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SpoVID Guides SafA to the Spore Coat inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.183.10.3041-3049.2001 ◽

2001 ◽

Vol 183 (10) ◽

pp. 3041-3049 ◽

Cited By ~ 56

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.

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Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2273-2278.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2273-2278

Author(s):

B C Dowds ◽

W F Loomis

Keyword(s):

Dictyostelium Discoideum ◽

Cdna Clone ◽

Single Gene ◽

Polyclonal Antiserum ◽

Cloning And Expression ◽

Spore Coat ◽

Coat Proteins ◽

Developmentally Regulated ◽

Gene Coding ◽

Spore Coat Proteins

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

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