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

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.

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

Journal of Bacteriology ◽

10.1128/jb.01353-06 ◽

2006 ◽

Vol 189 (6) ◽

pp. 2401-2410 ◽

Cited By ~ 21

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.

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Dynamic Patterns of Subcellular Protein Localization during Spore Coat Morphogenesis in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.186.14.4441-4448.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4441-4448 ◽

Cited By ~ 44

Author(s):

Christiaan van Ooij ◽

Patrick Eichenberger ◽

Richard Losick

Keyword(s):

Bacillus Subtilis ◽

Subcellular Localization ◽

Fluorescent Protein ◽

Protein Localization ◽

Distinct Pattern ◽

Spore Coat ◽

Short Axis ◽

Subcellular Protein Localization ◽

Associated Proteins ◽

Green Fluorescent

ABSTRACT Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.

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Heterochronic Phosphorelay Gene Expression as a Source of Heterogeneity in Bacillus subtilis Spore Formation

Journal of Bacteriology ◽

10.1128/jb.01484-09 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2053-2067 ◽

Cited By ~ 61

Author(s):

Imke G. de Jong ◽

Jan-Willem Veening ◽

Oscar P. Kuipers

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Fluorescent Protein ◽

Large Cell ◽

Time Lapse ◽

Nutrient Sources ◽

Bacillus Subtilis Spore ◽

Artificial Induction ◽

Limiting Nutrient ◽

Green Fluorescent

ABSTRACT In response to limiting nutrient sources and cell density signals, Bacillus subtilis can differentiate and form highly resistant endospores. Initiation of spore development is governed by the master regulator Spo0A, which is activated by phosphorylation via a multicomponent phosphorelay. Interestingly, only part of a clonal population will enter this developmental pathway, a phenomenon known as sporulation bistability or sporulation heterogeneity. How sporulation heterogeneity is established is largely unknown. To investigate the origins of sporulation heterogeneity, we constructed promoter-green fluorescent protein (GFP) fusions to the main phosphorelay genes and perturbed their expression levels. Using time-lapse fluorescence microscopy and flow cytometry, we showed that expression of the phosphorelay genes is distributed in a unimodal manner. However, single-cell trajectories revealed that phosphorelay gene expression is highly dynamic or “heterochronic” between individual cells and that stochasticity of phosphorelay gene transcription might be an important regulatory mechanism for sporulation heterogeneity. Furthermore, we showed that artificial induction or depletion of the phosphorelay phosphate flow results in loss of sporulation heterogeneity. Our data suggest that sporulation heterogeneity originates from highly dynamic and variable gene activity of the phosphorelay components, resulting in large cell-to-cell variability with regard to phosphate input into the system. These transcriptional and posttranslational differences in phosphorelay activity appear to be sufficient to generate a heterogeneous sporulation signal without the need of the positive-feedback loop established by the sigma factor SigH.

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A Four-Dimensional View of Assembly of a Morphogenetic Protein during Sporulation in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.181.3.781-790.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 781-790 ◽

Cited By ~ 54

Author(s):

Kirsten D. Price ◽

Richard Losick

Keyword(s):

Bacillus Subtilis ◽

Outer Surface ◽

Mother Cell ◽

Fluorescent Protein ◽

Mature Spore ◽

Time Lapse ◽

Homologous Protein ◽

C Terminus ◽

Morphogenetic Protein ◽

Green Fluorescent

ABSTRACT We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacteriumBacillus subtilis. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, likespoIVA, are under the control of the mother cell transcription factor ςE. One such gene wasspoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.

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Expression of yeeK during Bacillus subtilis Sporulation and Localization of YeeK to the Inner Spore Coat using Fluorescence Microscopy

Journal of Bacteriology ◽

10.1128/jb.01269-08 ◽

2008 ◽

Vol 191 (4) ◽

pp. 1220-1229 ◽

Cited By ~ 12

Author(s):

Hiromu Takamatsu ◽

Daisuke Imamura ◽

Ritsuko Kuwana ◽

Kazuhito Watabe

Keyword(s):

Bacillus Subtilis ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Microscopy ◽

Molecular Size ◽

Northern Hybridization ◽

Minimal Amount ◽

Spore Coat ◽

Wild Type ◽

Green Fluorescent

ABSTRACT The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.

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Laccase and Its Mutant Displayed on the Bacillus subtilis Spore Coat for Oxidation of Phenolic Compounds in Organic Solvents

Catalysts ◽

10.3390/catal11050606 ◽

2021 ◽

Vol 11 (5) ◽

pp. 606

Author(s):

Silu Sheng ◽

Edgardo T. Farinas

Keyword(s):

Bacillus Subtilis ◽

Organic Solvents ◽

Environmental Changes ◽

Sinapic Acid ◽

Product Yield ◽

Spore Coat ◽

Subtilis Spore ◽

Bacillus Subtilis Spore ◽

Cota Laccase ◽

Inert Surface

Enzymes displayed on the Bacillus subtilis spore coat have several features that are useful for biocatalysis. The enzyme is preimmobilized on an inert surface of the spore coat, which is due to the natural sporulation process. As a result, protein stability can be increased, and they are resistant to environmental changes. Next, they would not lyse under extreme conditions, such as in organic solvents. Furthermore, they can be easily removed from the reaction solution and reused. The laboratory evolved CotA laccase variant T480A-CotA was used to oxidize the following phenolic substrates: (+)-catechin, (−)-epicatechin, and sinapic acid. The kinetic parameters were determined and T480A-CotA had a greater Vmax/Km than wt-CotA for all substrates. The Vmax/Km for T480A-CotA was 4.1, 5.6, and 1.4-fold greater than wt-CotA for (+)-catechin, (−)-epicatechin, and sinapic acid, respectively. The activity of wt-CotA and T480A-CotA was measured at different concentrations from 0–70% in organic solvents (dimethyl sulfoxide, ethanol, methanol, and acetonitrile). The Vmax for T480A-CotA was observed to be greater than the wt-CotA in all organic solvents. Finally, the T480A-CotA was recycled 7 times over a 23-h period and up to 60% activity for (+)-catechin remained. The product yield was up to 3.1-fold greater than the wild-type.

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Classic Spotlight: Green Fluorescent Protein in Bacillus subtilis and the Birth of Bacterial Cell Biology

Journal of Bacteriology ◽

10.1128/jb.00380-16 ◽

2016 ◽

Vol 198 (16) ◽

pp. 2141-2141

Author(s):

Conrad W. Mullineaux

Keyword(s):

Bacillus Subtilis ◽

Green Fluorescent Protein ◽

Bacterial Cell ◽

Cell Biology ◽

Fluorescent Protein ◽

Green Fluorescent

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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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Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

Journal of Bacteriology ◽

10.1128/jb.184.7.1998-2004.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1998-2004 ◽

Cited By ~ 47

Author(s):

Takako Murakami ◽

Koki Haga ◽

Michio Takeuchi ◽

Tsutomu Sato

Keyword(s):

Bacillus Subtilis ◽

Membrane Proteins ◽

Vegetative Growth ◽

Fluorescent Protein ◽

Specific Expression ◽

Rich Media ◽

Green Fluorescent ◽

High Level ◽

Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

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