Teja Sirec

Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

BackgroundThe Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process.ResultsWe report that purified β- galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed β- galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust) were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the β- galactosidase molecules present in the adsorption reaction.ConclusionOur results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the β- galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-β- galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure. Although the molecular details of spore adsorption have not been fully elucidated, the efficiency of the process and the pH-stability of the adsorbed molecules, together with the well documented robustness and safety of spores of B. subtilis, propose the spore as a novel, non-recombinant system for enzyme display.

Non-recombinant display of the B subunit of the heat labile toxin of Escherichia coli on wild type and mutant spores of Bacillus subtilis

BackgroundMucosal infections are a major global health problem and it is generally accepted that mucosal vaccination strategies, able to block infection at their entry site, would be preferable with respect to other prevention approaches. However, there are still relatively few mucosal vaccines available, mainly because of the lack of efficient delivery systems and of mucosal adjuvants. Recombinant bacterial spores displaying a heterologous antigen have been shown to induce protective immune responses and, therefore, proposed as a mucosal delivery system. A non-recombinant approach has been recently developed and tested to display antigens and enzymes.ResultsWe report that the binding subunit of the heat-labile toxin (LTB) of Escherichia coli efficiently adsorbed on the surface of Bacillus subtilis spores. When nasally administered to groups of mice, spore-adsorbed LTB was able to induce a specific immune response with the production of serum IgG, fecal sIgA and of IFN-γ in spleen and mesenteric lymph nodes (MLN) of the immunized animals. Dot blotting experiments showed that the non-recombinant approach was more efficient than the recombinant system in displaying LTB and that the efficiency of display could be further increased by using mutant spores with an altered surface. In addition, immunofluorescence microscopy experiments showed that only when displayed on the spore surface by the non-recombinant approach LTB was found in its native, pentameric form.ConclusionOur results indicate that non-recombinant spores displaying LTB pentamers can be administered by the nasal route to induce a Th1-biased, specific immune response. Mutant spores with an altered coat are more efficient than wild type spores in adsorbing the antigen, allowing the use of a reduced number of spores in immunization procedures. Efficiency of display, ability to display the native form of the antigen and to induce a specific immune response propose this non-recombinant delivery system as a powerful mucosal vaccine delivery approach.

Flexibility of the Prograamme of Spore Coat Formation in Bacillus subtilis: Bypass of CotE Requirement by Over-Production of CotH

Bacterial spores are surrounded by the coat, a multilayered shell that contributes in protecting the genome during stress conditions. In Bacillus subtilis, the model organism for spore formers, the coat is composed by about seventy different proteins, organized into four layers by the action of several regulatory proteins. A major component of this regulatory network, CotE, is needed to assemble the outer coat and develop spores fully resistant to lysozyme and able to germinate efficiently. Another regulator, CotH, is controlled by CotE and is present in low amounts both during sporulation and in mature spores. In spite of this CotH controls the assembly of at least nine outer coat proteins and cooperates with CotE in producing fully resistant and efficiently germinating spores. In order to improve our understanding of CotH role in spore formation, we over-produced CotH by placing its coding region under the control of a promoter stronger than its own promoter but with a similar timing of activity during sporulation. Over-production of CotH in an otherwise wild type strain did not cause any major effect, whereas in a cotE null background a partial recovery of the phenotypes associated to the cotE null mutation was observed. Western blot, fluorescence microscopy and Surface-Enhanced Raman Scattering spectroscopy data indicate that, in the absence of CotE, over-production of CotH allowed the formation of spores overall resembling wild type spores and carrying in their coat some CotE−/CotH-dependant proteins. Our results suggest that the B. subtilis spore differentiation programme is flexible, and that an increase in the amount of a regulatory protein can replace a missing partner and partially substitute its function in the assembly of the spore coat.

Surface charge and hydrodynamic coefficient measurements of Bacillus subtilis spore by optical tweezers.

In this work we report on the simultaneous measurement of the hydrodynamic coefficient and the electric charge of single Bacillus subtilis spores. The latter has great importance in protein binding to spores and in the adhesion of spores onto surfaces. The charge and the hydrodynamic coefficient were measured by an accurate procedure based on the analysis of the motion of single spores confined by an optical trap. The technique has been validated using charged spherical polystyrene beads. The excellent agreement of our results with the expected values demonstrates the quality of our procedure. We measured the charge of spores of B. subtilis purified from a wild type strain and from two isogenic mutants characterized by an altered spore surface. Our technique is able to discriminate the three spore types used, by their charge and by their hydrodynamic coefficient which is related to the hydrophobic properties of the spore surface.

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

ABSTRACT The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform.

Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis

BackgroundBacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet.ResultsWe used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface.ConclusionsOur results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively.

Antagonistic role of CotG and CotH on spore germination and coat formation in Bacillus subtilis.

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.

The Direct Interaction between Two Morphogenetic Proteins Is Essential for Spore Coat Formation in Bacillus subtilis.

In Bacillus subtilis the protective layers that surround the mature spore are formed by over seventy different proteins. Some of those proteins have a regulatory role on the assembly of other coat proteins and are referred to as morphogenetic factors. CotE is a major morphogenetic factor, known to form a ring around the forming spore and organize the deposition of the outer surface layers. CotH is a CotE-dependent protein known to control the assembly of at least nine other coat proteins. We report that CotH also controls the assembly of CotE and that this mutual dependency is due to a direct interaction between the two proteins. The C-terminal end of CotE is essential for this direct interaction and CotH cannot bind to mutant CotE deleted of six or nine C-terminal amino acids. However, addition of a negatively charged amino acid to those deleted versions of CotE rescues the interaction.

Electrical-charge accumulation enables integrative quality control during B. subtilis sporulation

Quality control of offspring is important for the survival of cells. However, the mechanism by which quality of offspring cells may be monitored while running genetic programs of cellular differentiation remains largely unclear. Here we investigated a quality control system during Bacillus subtilis spore formation by combining single-cell time-lapse microscopy, molecular biology and mathematical modelling. Our results revealed that the quality-control system via premature germination is coupled with the accumulation of cations on the surface of developing forespores. Specifically, the forespores accumulating less cations on their surface are more likely to be aborted. This charge accumulation system enables the projection of multidimensional information about the external environment and morphological development of the forespore onto a one-dimensional information of cation accumulation. Based on the insight we gain, we propose a novel use of Nernstian chemicals for reducing the yield and quality of Bacillus endospores.