Display of proteins on Bacillus subtilis endospores

Kim, Junehyung; Schumann, Wolfgang

doi:10.1007/s00018-009-0067-6

Cited by 75 publications

(43 citation statements)

References 107 publications

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“…The identification of spore surface proteins is becoming increasingly important for potential practical applications (9). It was proposed previously that CotA, CotB, CotC, and CotG are externally exposed on the surface of spores (6,8,15,25), whereas CotA-GFP, CotB-GFP, and CotC-GFP were not efficiently exposed on spores in this study (Fig.…”

Section: Discussionmentioning

confidence: 49%

“…Using a similar method, McKenney and coworkers also identified an outermost spore layer composed of CotG, CotW, and CotZ and demonstrated that this layer, which they termed the spore crust, is absent in cotXYZ mutant spores (16). The identification of spore surface proteins is also becoming increasingly important for potential practical applications (9). In this study, we demonstrate that the spore crust is the externally exposed, outermost component of the B. subtilis spore, forming a layer that covers the spore coat.…”

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confidence: 99%

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Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores

et al. 2011

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To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.

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Section: Discussionmentioning

confidence: 49%

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confidence: 99%

Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores

et al. 2011

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“…Successful display of functional enzymes to the microbial cell surface shows increasing promise in developing whole-cell biocatalysts, as our society progresses toward an industrialized and technologically dependent future (60). However, the major challenge of a microbial surface display at the moment is the development of a system that is sufficiently robust for industrial applications (4,32,37,48). Fragility of the outer membrane of Gram-negative bacteria (e.g., E. coli) can be a major concern for surface display.…”

Section: Characterization Of Yhcr By Oussenko and Coworkersmentioning

confidence: 99%

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

Liew¹,

Wang²,

Wong³

2011

Journal of Bacteriology

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Sortases catalyze the covalent anchoring of proteins to the cell surface on Gram-positive bacteria. Bioinformatic analysis suggests the presence of structural genes encoding sortases and their substrates in the Bacillus subtilis genome. In this study, a ␤-lactamase reporter was fused to the cell wall anchoring domain from a putative sortase substrate, YhcR. Covalent anchoring of this fusion protein to the cell wall was confirmed by using the eight-protease-deficient B. subtilis strain WB800 as the host. Inactivation of yhcS abolished the cell wall anchoring reaction. The amounts of fusion protein anchored to the cell wall were proportional to the levels of YhcS. These data demonstrate that YhcS and YhcR are the sortase and sortase substrate, respectively, in B. subtilis. Furthermore, yhcS is not essential for the survival of B. subtilis under the cultivation condition tested. YhcR fusions were distributed helically in the lateral cell wall. Interestingly, when viewed with an epifluorescence microscope, YhcS also appeared to form short helical arcs. This is the first report to illustrate such distribution of sortases in a rod-shaped bacterium. Models for the spatial distribution of both the sortase and its substrate are discussed. The amount of the reporters displayed on the surface was unambiguously quantified via a unique strategy. Under optimal conditions with the overproduction of YhcS, 47,300 YhcR fusions could be displayed per cell. Displayed reporters were biologically functional and surface accessible. Characterization of the sortase-substrate system allowed the successful development of a YhcR-based covalent surface display system. This system may have various biotechnological applications.

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“…Surface display, a powerful technique that uses different microbial components to express heterologous peptides or proteins, can effectively display the bioactive molecules on the surfaces of cells (16,32). Target proteins anchored on the outside of cells play a role in biocatalysis without the need for any purification or permeation procedure.…”

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Production of N -Acetyl- d -Neuraminic Acid by Use of an Efficient Spore Surface Display System

Gao

Zhang

et al. 2011

Appl Environ Microbiol

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Production of N-acetyl-D-neuraminic acid (Neu5Ac) via biocatalysis is traditionally conducted using isolated enzymes or whole cells. The use of isolated enzymes is restricted by the time-consuming purification process, whereas the application of whole cells is limited by the permeability barrier presented by the microbial cell membrane. In this study, a novel type of biocatalyst, Neu5Ac aldolase presented on the surface of Bacillus subtilis spores, was used for the production of Neu5Ac. Under optimal conditions, Neu5Ac at a high concentration (54.7 g liter ؊1 ) and a high yield (90.2%) was obtained under a 5-fold excess of pyruvate over N-acetyl-D-mannosamine. The novel biocatalyst system, which is able to express and immobilize the target enzyme simultaneously on the surface of B. subtilis spores, represents a suitable alternative for value-added chemical production.

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Display of proteins on Bacillus subtilis endospores

Cited by 75 publications

References 107 publications

Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores

Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

Production of N -Acetyl- d -Neuraminic Acid by Use of an Efficient Spore Surface Display System

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