Bacillus subtilis spore coat components, their assembly and use for surface display of heterologous antigens

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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Display of Bombyx mori Nucleopolyhedrovirus GP64 on the Bacillus subtilis Spore Coat

Current Microbiology ◽

10.1007/s00284-011-9867-7 ◽

2011 ◽

Vol 62 (5) ◽

pp. 1368-1373 ◽

Cited By ~ 11

Author(s):

Guohui Li ◽

Qi Tang ◽

Huiqing Chen ◽

Qin Yao ◽

Degang Ning ◽

...

Keyword(s):

Bacillus Subtilis ◽

Bombyx Mori ◽

Spore Coat ◽

Subtilis Spore ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Bacillus Subtilis Spore

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Interactions among CotB, CotG, and CotH during Assembly of the Bacillus subtilis Spore Coat

Journal of Bacteriology ◽

10.1128/jb.186.4.1110-1119.2004 ◽

2004 ◽

Vol 186 (4) ◽

pp. 1110-1119 ◽

Cited By ~ 53

Author(s):

Rita Zilhão ◽

Mónica Serrano ◽

Rachele Isticato ◽

Ezio Ricca ◽

Charles P. Moran ◽

...

Keyword(s):

Bacillus Subtilis ◽

Reverse Genetics ◽

Spore Coat ◽

Yeast Two Hybrid ◽

Bacillus Subtilis Spore ◽

Yeast Two Hybrid System ◽

Ordered Assembly ◽

Heterologous Antigens ◽

Two Hybrid ◽

Good Agreement

ABSTRACT Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component. cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.

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The spore coat is essential for Bacillus subtilis spore resistance to pulsed light, and pulsed light treatment eliminates some spore coat proteins

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2020.108592 ◽

2020 ◽

Vol 323 ◽

pp. 108592 ◽

Cited By ~ 1

Author(s):

Gérémy Clair ◽

Julia Esbelin ◽

Sabine Malléa ◽

Isabelle Bornard ◽

Frédéric Carlin

Keyword(s):

Bacillus Subtilis ◽

Light Treatment ◽

Spore Coat ◽

Coat Proteins ◽

Subtilis Spore ◽

Pulsed Light ◽

Bacillus Subtilis Spore ◽

Spore Resistance ◽

Spore Coat Proteins

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Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat

Journal of Bacteriology ◽

10.1128/jb.186.4.1129-1135.2004 ◽

2004 ◽

Vol 186 (4) ◽

pp. 1129-1135 ◽

Cited By ~ 56

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.

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Temporal and spatial regulation of protein cross-linking by the pre-assembled substrates of a Bacillus subtilis spore coat transglutaminase

PLoS Genetics ◽

10.1371/journal.pgen.1007912 ◽

2019 ◽

Vol 15 (4) ◽

pp. e1007912 ◽

Cited By ~ 3

Author(s):

Catarina G. Fernandes ◽

Diogo Martins ◽

Guillem Hernandez ◽

Ana L. Sousa ◽

Carolina Freitas ◽

...

Keyword(s):

Bacillus Subtilis ◽

Cross Linking ◽

Spore Coat ◽

Subtilis Spore ◽

Spatial Regulation ◽

Bacillus Subtilis Spore ◽

Temporal And Spatial ◽

Protein Cross Linking

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Removal of Ni2+ and Cd2+ by Surface Display of Polyhistidine on Bacillus subtilis Spore Using CotE Anchor Protein

Biotechnology and Bioprocess Engineering ◽

10.1007/s12257-018-0467-2 ◽

2019 ◽

Vol 24 (2) ◽

pp. 375-381 ◽

Cited By ~ 2

Author(s):

Wooil Kim ◽

Daeun Kim ◽

Sanggeun Back ◽

Yong-suk Lee ◽

Afrouzossadat Hosseini Abari ◽

...

Keyword(s):

Bacillus Subtilis ◽

Surface Display ◽

Subtilis Spore ◽

Anchor Protein ◽

Bacillus Subtilis Spore

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

Journal of Bacteriology ◽

10.1128/jb.01103-09 ◽

2009 ◽

Vol 192 (2) ◽

pp. 518-524 ◽

Cited By ~ 41

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.

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