scholarly journals Functional Regions of the Bacillus subtilis Spore Coat Morphogenetic Protein CotE

1999 ◽  
Vol 181 (22) ◽  
pp. 7043-7051 ◽  
Author(s):  
Tamara Bauer ◽  
Shawn Little ◽  
Axel G. Stöver ◽  
Adam Driks
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Coat Protein ◽  
Protein Assembly ◽  
Spore Coat ◽  
Coat Proteins ◽  
Outer Coat ◽  
Bacillus Subtilis Spore ◽  
Coat Protein Assembly

ABSTRACT The Bacillus subtilis spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat’s outer layer. We have used a series of mutant alleles of cotE to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.

scholarly journals Characterization of the Bacillus subtilis Spore Morphogenetic Coat Protein CotO

2005 ◽  
Vol 187 (24) ◽  
pp. 8278-8290 ◽  
Author(s):  
D. C. McPherson ◽  
H. Kim ◽  
M. Hahn ◽  
R. Wang ◽  
P. Grabowski ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Late Phase ◽  
Coat Proteins ◽  
Outer Coat ◽  
Bacillus Subtilis Spore ◽  
Fluorescence And Electron Microscopy ◽  
Complex Protein ◽  
Bacillus Spores ◽  
Critical Model

ABSTRACT Bacillus spores are protected by a structurally and biochemically complex protein shell composed of over 50 polypeptide species, called the coat. Coat assembly in Bacillus subtilis serves as a relatively tractable model for the study of the formation of more complex macromolecular structures and organelles. It is also a critical model for the discovery of strategies to decontaminate B. anthracis spores. In B. subtilis, a subset of coat proteins is known to have important roles in assembly. Here we show that the recently identified B. subtilis coat protein CotO (YjbX) has an especially important morphogenetic role. We used electron and atomic force microscopy to show that CotO controls assembly of the coat layers and coat surface topography as well as biochemical and cell-biological analyses to identify coat proteins whose assembly is CotO dependent. cotO spores are defective in germination and partially sensitive to lysozyme. As a whole, these phenotypes resemble those resulting from a mutation in the coat protein gene cotH. Nonetheless, the roles of CotH and CotO and the proteins whose assembly they direct are not identical. Based on fluorescence and electron microscopy, we suggest that CotO resides in the outer coat (although not on the coat surface). We propose that CotO and CotH participate in a late phase of coat assembly. We further speculate that an important role of these proteins is ensuring that polymerization of the outer coat layers occurs in such a manner that contiguous shells, and not unproductive aggregates, are formed.

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

2006 ◽  
Vol 188 (21) ◽  
pp. 7609-7616 ◽  
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.

scholarly journals Localization of Proteins to Different Layers and Regions of Bacillus subtilis Spore Coats

2009 ◽  
Vol 192 (2) ◽  
pp. 518-524 ◽  
Author(s):  
Daisuke Imamura ◽  
Ritsuko Kuwana ◽  
Hiromu Takamatsu ◽  
Kazuhito Watabe
Keyword(s):  
Bacillus Subtilis ◽  
Mother Cell ◽  
Fluorescent Proteins ◽  
Bacterial Spores ◽  
Spore Coat ◽  
Subtilis Spore ◽  
Proximal Pole ◽  
Outer Coat ◽  
Outermost Layer ◽  
Bacillus Subtilis Spore

ABSTRACT Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.

scholarly journals Bacillus subtilis Spore Coat

1999 ◽  
Vol 63 (1) ◽  
pp. 1-20 ◽  
Author(s):  
Adam Driks
Keyword(s):  
Bacillus Subtilis ◽  
Posttranslational Processing ◽  
Spore Coat ◽  
Coat Proteins ◽  
Spore Surface ◽  
Subtilis Spore ◽  
Cell Type ◽  
Dormant Cell ◽  
Bacillus Subtilis Spore ◽  
Specialized Program

SUMMARY In response to starvation, bacilli and clostridia undergo a specialized program of development that results in the production of a highly resistant dormant cell type known as the spore. A proteinacious shell, called the coat, encases the spore and plays a major role in spore survival. The coat is composed of over 25 polypeptide species, organized into several morphologically distinct layers. The mechanisms that guide coat assembly have been largely unknown until recently. We now know that proper formation of the coat relies on the genetic program that guides the synthesis of spore components during development as well as on morphogenetic proteins dedicated to coat assembly. Over 20 structural and morphogenetic genes have been cloned. In this review, we consider the contributions of the known coat and morphogenetic proteins to coat function and assembly. We present a model that describes how morphogenetic proteins direct coat assembly to the specific subcellular site of the nascent spore surface and how they establish the coat layers. We also discuss the importance of posttranslational processing of coat proteins in coat morphogenesis. Finally, we review some of the major outstanding questions in the field.

scholarly journals The Timing of cotE Expression Affects Bacillus subtilis Spore Coat Morphology but Not Lysozyme Resistance

2006 ◽  
Vol 189 (6) ◽  
pp. 2401-2410 ◽  
Author(s):  
Teresa Costa ◽  
Mónica Serrano ◽  
Leif Steil ◽  
Uwe Völker ◽  
Charles P. Moran ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Spore Coat ◽  
Outer Coat ◽  
Bacillus Subtilis Spore ◽  
Spore Resistance ◽  
Early Expression ◽  
Cell Gene Expression ◽  
Coat Layer

ABSTRACT The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σE factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme. cotE is expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σK control. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.

scholarly journals CotE Binds to CotC and CotU and Mediates Their Interaction during Spore Coat Formation in Bacillus subtilis

2009 ◽  
Vol 192 (4) ◽  
pp. 949-954 ◽  
Author(s):  
Rachele Isticato ◽  
Assunta Pelosi ◽  
Maurilio De Felice ◽  
Ezio Ricca
Keyword(s):  
Bacillus Subtilis ◽  
Direct Evidence ◽  
Spore Coat ◽  
Coat Proteins ◽  
Heterologous Host ◽  
Outer Coat ◽  
Genetic Experiment

ABSTRACT CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of Bacillus subtilis. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.

scholarly journals Morphogenetic Proteins SpoVID and SafA Form a Complex during Assembly of the Bacillus subtilis Spore Coat

2000 ◽  
Vol 182 (7) ◽  
pp. 1828-1833 ◽  
Author(s):  
Amanda J. Ozin ◽  
Adriano O. Henriques ◽  
Hong Yi ◽  
Charles P. Moran
Keyword(s):  
Bacillus Subtilis ◽  
Modular Design ◽  
Early Stage ◽  
Multilayered Structure ◽  
Terminal Region ◽  
Immunogold Electron Microscopy ◽  
Spore Coat ◽  
Coat Proteins ◽  
Bacillus Subtilis Spore ◽  
Cell Extracts

ABSTRACT During endospore formation in Bacillus subtilis, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination. In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore. A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat. Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly. This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as yrbA. We confirmed the results of a previous study that showed safA mutant spores have defective coats which are missing several proteins. We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation. Therefore, SafA may associate with SpoVID during the early stage of coat assembly. We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores. SafA appears to have a modular design. The C-terminal region of SafA is similar to those of several inner spore coat proteins. The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall. This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore.

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