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

2004 ◽  
Vol 186 (4) ◽  
pp. 1110-1119 ◽  
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.

scholarly journals Searching for Protein-Protein Interactions within the Bacillus subtilis Spore Coat

2009 ◽  
Vol 191 (10) ◽  
pp. 3212-3219 ◽  
Author(s):  
Daniela Krajčíková ◽  
Magda Lukáčová ◽  
Denisa Müllerová ◽  
Simon M. Cutting ◽  
Imrich Barák
Keyword(s):  
Bacillus Subtilis ◽  
Complex Structure ◽  
Insoluble Fraction ◽  
Size Exclusion ◽  
Strong Interactions ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid ◽  
Lytic Enzymes ◽  
Two Hybrid

ABSTRACT The capability of endospores of Bacillus subtilis to withstand extreme environmental conditions is secured by several attributes. One of them, the protein shell that encases the spore and is known as the coat, provides the spore with its characteristic resistance to toxic chemicals, lytic enzymes, and predation by unicellular and multicellular eukaryotes. Despite most of the components of the spore coat having been identified, we have only a vague understanding of how such a complex structure is assembled. Using the yeast two-hybrid system, we attempted to identify direct contacts among the proteins allocated to the insoluble fraction of the spore coat: CotV, CotW, CotX, CotY, and CotZ. We also examined whether they could interact with CotE, one of the most crucial morphogenetic proteins governing outer coat formation and also present in the insoluble fraction. Out of all 21 possible interactions we tested, 4 were found to be positive. Among these interactions, we confirmed the previous observation that CotE forms homo-oligomers. In addition, we observed homotypic interactions of CotY, strong interactions between CotZ and CotY, and relatively weak, yet significant, interactions between CotV and CotW. The results of this yeast two-hybrid analysis were confirmed by size exclusion chromatography of recombinant coat proteins and a pull-down assay.

Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner

Vaccine ◽  
2004 ◽  
Vol 22 (9-10) ◽  
pp. 1177-1187 ◽  
Author(s):  
Emilia M.F. Mauriello ◽  
Le H. Duc ◽  
Rachele Isticato ◽  
Giuseppina Cangiano ◽  
Huynh A. Hong ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Fusion Partner ◽  
Spore Coat ◽  
Subtilis Spore ◽  
Bacillus Subtilis Spore ◽  
Heterologous Antigens

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

2007 ◽  
Vol 131 (2) ◽  
pp. S63
Author(s):  
Ezio Ricca ◽  
Rachele Isticato ◽  
Loredana Baccigalupi ◽  
Maurilio De Felice
Keyword(s):  
Bacillus Subtilis ◽  
Surface Display ◽  
Spore Coat ◽  
Subtilis Spore ◽  
Bacillus Subtilis Spore ◽  
Heterologous Antigens

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

2021 ◽  
Vol 9 (2) ◽  
pp. 304
Author(s):  
Yao Chi ◽  
Li-Long Pan ◽  
Shu-Sheng Liu ◽  
Shahid Mansoor ◽  
Xiao-Wei Wang
Keyword(s):  
Coat Protein ◽  
Molecular Mechanisms ◽  
Cotton Leaf ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Vacuolar Protein ◽  
Leaf Curl Disease ◽  
Two Hybrid ◽  
Asia Ii 1 ◽  
Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

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

Catalysts ◽  
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.

scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

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