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 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 Maintaining the Transcription Factor SpoIIID Level Late during Sporulation Causes Spore Defects in Bacillus subtilis

2007 ◽  
Vol 189 (20) ◽  
pp. 7302-7309 ◽  
Author(s):  
Lijuan Wang ◽  
John Perpich ◽  
Adam Driks ◽  
Lee Kroos
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Mother Cell ◽  
Structural Defects ◽  
Spore Coat ◽  
Multicopy Plasmid ◽  
Altered Expression ◽  
Cell Gene Expression ◽  
Cell Gene

ABSTRACT During sporulation of Bacillus subtilis, four regulatory proteins act in the order σE, SpoIIID, σK, and GerE to temporally control gene expression in the mother cell. σE and σK work sequentially with core RNA polymerase to transcribe different sets of genes. SpoIIID and GerE are small, sequence-specific DNA-binding proteins that activate or repress transcription of many genes. Previous studies showed that transcriptionally active σK RNA polymerase inhibits early mother cell gene expression, reducing accumulation of SpoIIID late in sporulation. Here, the effects of perturbing the mother cell gene regulatory network by maintaining the SpoIIID level late during sporulation are reported. Persistent expression was obtained by fusing spoIIID to the σK-controlled gerE promoter on a multicopy plasmid. Fewer heat- and lysozyme-resistant spores were produced by the strain with persistent spoIIID expression, but the number of spores resistant to organic solvents was unchanged, as was their germination ability. Transmission electron microscopy showed structural defects in the spore coat. Reporter fusions to σK-dependent promoters showed lower expression of gerE and cotC and higher expression of cotD. Altered expression of cot genes, which encode spore coat proteins, may account for the spore structural defects. These results suggest that one role of negative feedback by σK RNA polymerase on early mother cell gene expression is to lower the level of SpoIIID late during sporulation in order to allow normal expression of genes in the σK regulon.

scholarly journals Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat

2004 ◽  
Vol 186 (4) ◽  
pp. 1129-1135 ◽  
Author(s):  
Rachele Isticato ◽  
Giovanni Esposito ◽  
Rita Zilhão ◽  
Sofia Nolasco ◽  
Giuseppina Cangiano ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Posttranslational Modifications ◽  
Mother Cell ◽  
Spore Coat ◽  
Spore Surface ◽  
Subtilis Spore ◽  
Cell Compartment ◽  
Bacillus Subtilis Spore ◽  
Molecular Masses

ABSTRACT We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

scholarly journals Erratum. Bacillus subtilis spore coat assembly requires cotH gene expression.

1996 ◽  
Vol 178 (21) ◽  
pp. 6407-6407 ◽  
Author(s):  
G Naclerio ◽  
L Baccigalupi ◽  
R Zilhao ◽  
M De Felice ◽  
E Ricca
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Spore Coat ◽  
Subtilis Spore ◽  
Bacillus Subtilis Spore

scholarly journals Protozoal Digestion of Coat-Defective Bacillus subtilis Spores Produces “Rinds” Composed of Insoluble Coat Protein

2008 ◽  
Vol 74 (19) ◽  
pp. 5875-5881 ◽  
Author(s):  
Alicia Monroe Carroll ◽  
Marco Plomp ◽  
Alexander J. Malkin ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Amorphous Layer ◽  
Spore Coat ◽  
Outer Coat ◽  
Force Microscopy ◽  
Atomic Force ◽  
Spore Resistance ◽  
Contrast Microscope ◽  
Coat Material

ABSTRACT The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon.

scholarly journals Assembly of an Oxalate Decarboxylase Produced under σK Control into the Bacillus subtilis Spore Coat

2004 ◽  
Vol 186 (5) ◽  
pp. 1462-1474 ◽  
Author(s):  
Teresa Costa ◽  
Leif Steil ◽  
Lígia O. Martins ◽  
Uwe Völker ◽  
Adriano O. Henriques
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Decarboxylase Activity ◽  
Spore Coat ◽  
Oxalate Decarboxylase ◽  
Bacillus Subtilis Spore ◽  
Morphogenetic Protein ◽  
Interior Layers ◽  
Coat Layer ◽  
Green Fluorescent

ABSTRACT Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of σK and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.

scholarly journals Involvement of Superoxide Dismutase in Spore Coat Assembly in Bacillus subtilis

1998 ◽  
Vol 180 (9) ◽  
pp. 2285-2291 ◽  
Author(s):  
Adriano O. Henriques ◽  
Lawrence R. Melsen ◽  
Charles P. Moran
Keyword(s):  
Superoxide Dismutase ◽  
Bacillus Subtilis ◽  
Spore Coat ◽  
Coat Proteins ◽  
Wild Type ◽  
Outer Coat ◽  
Sod Activity ◽  
Cell Extracts ◽  
Spore Coat Proteins ◽  
Coat Layer

ABSTRACT Endospores of Bacillus subtilis are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the sodA locus.sodA is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from B. subtilis. Disruption of the sodA locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The sodA mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of sodA mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the cotG gene in sporulating cells of the sodA mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a sodA mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in acotG mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer.

scholarly journals Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation

2000 ◽  
Vol 66 (2) ◽  
pp. 620-626 ◽  
Author(s):  
Paul J. Riesenman ◽  
Wayne L. Nicholson
Keyword(s):  
Hydrogen Peroxide ◽  
Bacillus Subtilis ◽  
Uv Radiation ◽  
Spore Coat ◽  
Wild Type ◽  
Spore Resistance ◽  
Solar Uv ◽  
Coat Layer ◽  
Artificial Uv ◽  
Uv C

ABSTRACT Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing thegerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H2O2) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H2O2, as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing thegerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing thecotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 andcotE::cat mutations behaved likegerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.

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