Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of <i>Bacillus subtilis</i> spore

Tang, Jilin; Krajcikova, Daniela; Zhu, Rong; Ebner, Andreas; Cutting, Simon M.; Gruber, Hermann J.; Barák, Imrich; Hinterdorfer, Peter

doi:10.1002/jmr.828

Cited by 30 publications

(32 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…YeeK fusions produced a smaller diameter than the other proteins. These results are consistent with previous reports that CotA and CotB are located on the outer surfaces of spores and that YeeK is located in the inner coat (15,44,45). Since the diameters measured with GFPfused proteins were typically slightly longer (up to 1 pixel) than those measured with the corresponding RFP-fused proteins, small differences in relative localizations are not very reliable.…”

Section: Resultssupporting

confidence: 90%

“…The locations of CotE, CotS, and SpoIVA in the spore coat were determined previously by immunoelectron microscopy (9, 43). CotA, CotB, CotC, and CotG were shown to be externally exposed on the surface of the spore by single-molecule recognition force spectroscopy or antibody accessibility (15,18,45,28). However, the positions of most of the spore coat proteins in the coat have not been determined experimentally, although provisional assignments were made based largely on the control of assembly into the coat by CotE (17).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

et al. 2010

View full text Add to dashboard Cite

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.Proper localization and assembly of proteins in cells and subcellular structures are essential features of living organisms. Complex protein assemblies, including ribosomes, flagella, and the cytokinetic machinery, play important roles in bacteria (26,27,40). Studying how these complex structures are formed is a fundamental theme in molecular biology. In this work, we developed a method to analyze one of the most complex bacterial protein assemblies: the spore coat of Bacillus subtilis.Sporulation of B. subtilis is initiated in response to nutrient limitation, and it involves a highly ordered program of gene expression and morphological change (33, 42). The first morphological change of sporulation is the appearance of an asymmetrically positioned septum that divides the cell into a larger mother cell and a smaller forespore. Next, the mother cell membrane migrates around the forespore membrane during a phagocytosislike process called engulfment. The completion of engulfment involves fusion of the mother cell membrane to pinch off the forespore within the mother cell. Compartment-specific gene expression brings about maturation of the spore and its release upon lysis of the mother cell (reviewed in reference 19). Mature spores remain viable during long periods of starvation and are resistant to heat, toxic chemicals, lytic enzymes, and other factors capable of damaging vegetative cells (30). Spores germinate and resume growth when nutrients become available (32).The outer portions of Bacillus spores consist of a cortex, a spore coat layer, and in some cases, an exosporium. The cortex, a thick layer of peptidoglycan, is deposited between the inner and the outer membranes of the forespore, and it is responsible for maintaining the highly dehydrated state of the core, thereby contri...

show abstract

Section: Resultssupporting

confidence: 90%

mentioning

confidence: 99%

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

et al. 2010

View full text Add to dashboard Cite

show abstract

“…3). These results suggest that some outer coat proteins are present in the spore crust in small amounts that are at least detectable by methods used in previous studies (e.g., each antibody exhibits different sensitivities) (6,8,15,25). However, our data demonstrated that the spore crust proteins CgeA, CotY, and CotZ were clearly more efficiently exposed externally on the spore surface than outer coat proteins (Fig.…”

Section: Discussionsupporting

confidence: 53%

“…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. 3).…”

Section: Discussionsupporting

confidence: 50%

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

et al. 2011

View full text Add to dashboard Cite

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.

show abstract

“…So the number of antibody-epitope interactions is an indirect measure of conformation. AFM tips can be functionalized with antibodies and measure antibody-epitope interaction on the biomaterial surface [112][113][114][115][116]. Figure 16 shows two examples of an antibody-modified AFM tip interacting with a fibronectin protein that has an RGD group exposed and one that has its RGD group buried.…”

Section: Phil Trans R Soc a (2012)mentioning

confidence: 99%

Designing nanostructured block copolymer surfaces to control protein adhesion

Schricker

Palacio

Bhushan

2012

Phil. Trans. R. Soc. A.

View full text Add to dashboard Cite

The profile and conformation of proteins that are adsorbed onto a polymeric biomaterial surface have a profound effect on its in vivo performance. Cells and tissue recognize the protein layer rather than directly interact with the surface. The chemistry and morphology of a polymer surface will govern the protein behaviour. So, by controlling the polymer surface, the biocompatibility can be regulated. Nanoscale surface features are known to affect the protein behaviour, and in this overview the nanostructure of self-assembled block copolymers will be harnessed to control protein behaviour. The nanostructure of a block copolymer can be controlled by manipulating the chemistry and arrangement of the blocks. Random, A-B and A-B-A block copolymers composed of methyl methacrylate copolymerized with either acrylic acid or 2-hydroxyethyl methacrylate will be explored. Using atomic force microscopy (AFM), the surface morphology of these block copolymers will be characterized. Further, AFM tips functionalized with proteins will measure the adhesion of that particular protein to polymer surfaces. In this manner, the influence of block copolymer morphology on protein adhesion can be measured. AFM tips functionalized with antibodies to fibronectin will determine how the surfaces will affect the conformation of fibronectin, an important parameter in evaluating surface biocompatibility.

show abstract

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Cited by 30 publications

References 35 publications

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

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

Designing nanostructured block copolymer surfaces to control protein adhesion

Contact Info

Product

Resources

About