Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.

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Phosphorylation of spore coat proteins during development of Dictyostelium discoideum

Biochemical Journal ◽

10.1042/bj2120699 ◽

1983 ◽

Vol 212 (3) ◽

pp. 699-703 ◽

Cited By ~ 3

Author(s):

S J Delaney ◽

D G Wilkinson ◽

B D Hames

Keyword(s):

Coat Protein ◽

Dictyostelium Discoideum ◽

Spore Coat ◽

Coat Proteins ◽

Cellular Slime Mould ◽

Slime Mould ◽

Cellular Slime ◽

Spore Coat Proteins ◽

Spore Coat Protein

Immunological evidence is presented which confirms that pp95, one of the major phosphoproteins accumulated in development of the cellular slime mould Dictyostelium discoideum, is identical with spore coat protein SP13. The site of phosphorylation is identified as phosphoserine. The second major phosphorylated component, pp74, corresponds to two co-migrating spore coat proteins known collectively as SP74.

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

Acta Biochimica Polonica ◽

10.18388/abp.2016_1315 ◽

2017 ◽

Vol 64 (2) ◽

Cited By ~ 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.

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Outside-In Signaling of Cellulose Synthesis by a Spore Coat Protein in Dictyostelium

Eukaryotic Cell ◽

10.1128/ec.1.2.281-292.2002 ◽

2002 ◽

Vol 1 (2) ◽

pp. 281-292 ◽

Cited By ~ 10

Author(s):

Christopher M. West ◽

Ping Zhang ◽

Aiko C. McGlynn ◽

Lee Kaplan

Keyword(s):

Coat Protein ◽

Cell Surface ◽

De Novo ◽

Cellulose Synthesis ◽

Spore Coat ◽

Coat Proteins ◽

C2 Domains ◽

Partial Length ◽

C1 Domain ◽

Spore Coat Protein

ABSTRACT The spore coat of Dictyostelium is formed de novo from proteins secreted from vesicles and cellulose synthesized across the plasma membrane as differentiating spores rise up the stalk. The mechanism by which these events are coordinated is not understood. In the course of experiments designed to test the function of the inner layer coat protein SP85 (PsB), expression of a specific partial length fragment was found to interrupt coat assembly after protein secretion and prior to cellulose synthesis in 85% of the cells. This fragment consisted of SP85's N-terminal domain, containing prespore vesicle targeting information, and its Cys-rich C1 domain. The effect of the NC1 fusion was not cell autonomous in interstrain chimeras, suggesting that it acted at the cell surface. SP85-null spores presented an opposite phenotype in which spores differentiated prematurely before reaching the top of the stalk, and cellulose was slightly overproduced in a disorganized fashion. A similar though less severe phenotype occurred when a fusion of the N and C2 domains was expressed. In a double mutant, absence of SP85 was epistatic to NC1 expression, suggesting that NC1 inhibited SP85 function. Together, these results suggest the existence of an outside-in signaling pathway that constitutes a checkpoint to ensure that cellulose synthesis does not occur until coat proteins are properly organized at the cell surface and stalk formation is complete. Checkpoint execution is proposed to be regulated by SP85, which is in turn under the influence of other coat proteins that interact with SP85 via its C1 and C2 domains.

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

Canadian Journal of Microbiology ◽

10.1139/m84-219 ◽

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.

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Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37974-7 ◽

1978 ◽

Vol 253 (19) ◽

pp. 6694-6701

Author(s):

L. Munoz ◽

Y. Sadaie ◽

R.H. Doi

Keyword(s):

Bacillus Subtilis ◽

Coat Protein ◽

Spore Coat ◽

Precursor Synthesis ◽

Spore Coat Protein

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Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

Journal of Bacteriology ◽

10.1128/jb.01116-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7609-7616 ◽

Cited By ~ 36

Author(s):

Alicia Monroe ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Coat Protein ◽

Cross Linking ◽

Spore Coat ◽

Bacillus Subtilis Spore ◽

Binding Partners ◽

Lysine Residues ◽

Cross Links ◽

Hydrolysis Of ◽

Spore Coat Protein

ABSTRACT The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an ε-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

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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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