The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

Lanzilli, Mariamichela; Donadio, Giuliana; Addevico, Roberta; Saggese, Anella; Cangiano, Giuseppina; Baccigalupi, Loredana; Christie, Graham; Ricca, Ezio; Isticato, Rachele

doi:10.3389/fmicb.2016.01752

Cited by 17 publications

(20 citation statements)

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“…Two thermophilic enzymes, GH10-XA of A. acidocaldarius and GH3-XT of T. thermarum , were independently reacted with 1.0 × 10 10 purified spores of B. subtilis strain PY79 [ 24 ]. The adsorption reaction was performed by using 50 g of either one of the two purified proteins in 50 mM sodium citrate at pH 4.0, as previously described for other proteins [ 7 , 20 – 22 ]. After the adsorption, spores were washed and collected by centrifugation.…”

Section: Resultsmentioning

confidence: 99%

“…To evaluate the efficiency of adsorption, we followed a well-established procedure [ 20 – 22 ] and analyzed the amount of each enzyme left unbound, i.e., post-adsorbed spores were collected by centrifugation and the supernatant serially diluted and analyzed by dot blotting (Fig. 1 c, d).…”

Section: Resultsmentioning

confidence: 99%

“…Both recombinant and non-recombinant systems have been initially developed using spores of Bacillus subtilis , the model organism for spore formers. However, other species have been also proposed and a recent study has shown that spores of B. megaterium , also for their large size, are particularly efficient in displaying high amounts of heterologous proteins [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

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Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

et al. 2017

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BackgroundThe Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini.ResultsWe report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation.ConclusionOur results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

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“…2 ), which might suggest that FliD protein penetrates deeper into the spore coat structure. As recently reported, such phenomenon might result in lowered binding of antibodies directed against adsorbed protein and hence altered staining pattern [ 21 ].…”

Section: Discussionmentioning

confidence: 97%

The combination of recombinant and non-recombinant Bacillus subtilis spore display technology for presentation of antigen and adjuvant on single spore

et al. 2017

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Background: Bacillus subtilis spores can be used for presentation of heterologous proteins. Two main approaches have been developed, the recombinant one, requiring modification of bacterial genome to express a protein of interest as a fusion with spore-coat protein, and non-recombinant, based on the adsorption of a heterologous protein onto the spore. So far only single proteins have been displayed on the spore surface. Results:We have used a combined approach to adsorb and display FliD protein of Clostridium difficile on the surface of recombinant IL-2-presenting spores. Such spores presented FliD protein with efficiency comparable to FliDadsorbed spores produced by wild-type 168 strain and elicited FliD-specific immune response in intranasally immunized mice. Conclusions:Our results indicate that such dual display technology may be useful in creation of spores simultaneously presenting adjuvant and antigen molecules. Regarding the characteristics of elicited immune response it seems plausible that such recombinant IL-2-presenting spores with adsorbed FliD protein might be an interesting candidate for vaccine against infections with Clostridium difficile.

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“…Spores of several strains of Bacillus megaterium have also been the focus of studies on exosporium morphology and composition (11)(12)(13)(14)(15)(16). The species is comprised of many strains, which form a clade within the Bacillus genus that is quite distinct from both the B. subtilis and B. cereus clades.…”

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Orthologues of Bacillus subtilis Spore Crust Proteins Have a Structural Role in the Bacillus megaterium QM B1551 Spore Exosporium

Manetsberger

Ghosh

Hall

et al. 2018

Appl Environ Microbiol

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The exosporium ofBacillus megateriumQM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of theBacillus megateriumexosporium. Here, we provide evidence that pBM600-encoded orthologues of theBacillus subtilisCotW and CotX proteins, which form the crust layer in spores of that species, are structural components of theBacillus megateriumQM B1551 spore exosporium. The introduction of plasmid-bornecotWand orthologouscotXgenes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in theBacillus megateriumQM B1551 exosporium.IMPORTANCEWhen starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, inBacillus megateriumQM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenicBacillus cereusfamily. In this work, we provide evidence that structurally important protein constituents of theBacillus megateriumexosporium are different from those in theBacillus cereusfamily. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.

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The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

Cited by 17 publications

References 29 publications

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

The combination of recombinant and non-recombinant Bacillus subtilis spore display technology for presentation of antigen and adjuvant on single spore

Orthologues of Bacillus subtilis Spore Crust Proteins Have a Structural Role in the Bacillus megaterium QM B1551 Spore Exosporium

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