Adsorption of β-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis
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The 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.
We 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.
Our 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.
Display systems to present biologically active molecules on the surface of microorganisms have become an increasingly used strategy to address biotechnological issues [1, 2]. For biomedical applications surface display systems have been mostly used for the identification of neutralizing epitopes, the development of whole cell diagnostic tools, or vaccine delivery [3, 4]. More recent is a strategy to engineer bacterial endospores (spores) to display heterologous proteins on their surface . Endospore-forming bacteria are Gram-positive microorganisms belonging to different genera and including more than 1,000 species . The common feature of these organisms is the ability to form a quiescent cellular type (the spore) in response to harsh environments. The spore can survive in this dormant state for long periods, resisting to a vast range of stresses such as high temperature, dehydration, absence of nutrients, presence of toxic chemicals. When the environmental conditions ameliorate, the spore germinates originating a vegetative cell able to grow and sporulate . The ability of the spore to survive non-physiological conditions is, in part, due to the presence of the spore coat, a proteinaceous structure surrounding the spore. At least seventy different proteins (Cot proteins) form the multilayered coat structure, composed of an inner part, an outer part  and the crust, the latter being a recently discovered outermost layer of the spore [8, 9].
Spore-based display systems provide several advantages with respect to systems based on the use of phages and bacterial cells . The remarkable and well documented resistance of spores to various environmental and toxic effects  ensures high stability of the display system. Proteins to be displayed on the spore are produced in the mother cell compartment of the sporangium and are assembled around the forming spore without the need to be translocated across a membrane, thus eliminating the size constrains of cell-based display systems [5, 10]. The safety record of several endospore-forming species , makes spores of those species ideal candidates as vehicles to deliver molecules to mucosal surfaces .
The strategy to obtain the spore surface display of heterologous proteins is based on the construction of gene fusions between the gene coding for a selected spore surface protein (carrier) and the heterologous DNA coding for the protein to be displayed . By this approach a variety of heterologous proteins have been displayed and recombinant spores proposed as vaccine vehicles (see ref. 6 for a review), as biocatalysts (see ref. 10 for a review), or as a bioremediation tool . To optimize and rationalize this display strategy an inner (OxdD ) and various outer (CotB , CotC [14, 15], CotG ) coat components have been tested as carriers.
The spore-based display system, like other cell- or phage-based systems, relies on the genetic engineering of the host to display immunogenic peptides or proteins and obtain a recombinant organism to be used as a live biotechnological tool [5, 6, 10]. This is a major drawback since it causes the release of live recombinant organisms into nature, raising concerns over the use and clearance of genetically modified microorganisms . To overcome this obstacle, a non-recombinant approach to use spores as a display system has been recently proposed and model proteins efficiently exposed. In the first study suggesting that heterologous proteins can be adsorbed on the spore surface, the mammalian NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, was over-expressed in sporulating B. subtilis cells and released into the culture medium after sporulation by autolysis of the mother cell. However, part of the CPR activity was found associated to spores and the displayed enzyme shown to be accessible to anti-CPR antibodies . In a different study a collections of purified antigens (TTFC of Clostridium tetani, PA of Bacillus anthracis, Cpa of Clostridium perfringens and glutathione S transferase of Shistosomas japonica) were adsorbed to B. subtilis spores and shown to be able to induce specific and protective immune responses in mucosally immunised mice . Spore adsorption resulted to be more efficient when the pH of the binding buffer was acidic (pH 4) and less efficient or totally inhibited at pH values of 7 or 10 . A combination of electrostatic and hydrophobic interactions between spores and antigens were suggested to drive the adsorption, that was shown to be not dependent on specific spore coat components but rather on the negatively charged and hydrophobic surface of the spore . Hydrophobic and electrostatic interactions were suggested as the main forces involved also in the interaction between the E. coli phytase and spores of B. polyfermenticus.
We used a well-characterized and biotechnologically important enzyme, a β-galactosidase of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius, as a model to study enzyme adsorption on B. subtilis spores. This enzyme belongs to the glycoside hydrolase family 42 (GH42) and is characterized by an optimal activity and stability at 65°C . By using this system we tested whether adsorbed β-galactosidase molecules retained their activity and whether and how spore-adsorption affected the properties of the enzyme. With the dual aim of identifying spore surface structures involved in β-galactosidase adsorption and to improve the efficiency of the process we also screened for enzyme binding a collection of mutant strains of B. subtilis lacking a single or several spore coat proteins. A better understanding of the spore surface structure is likely to lead to a rationalization of the adsorption system, such that wild type or mutant spores will be utilized, depending upon the specific application or the heterologous enzyme to display.
β-Galactosidase of A. acidocaldarius adsorbs to B. subtilis spores and retains its enzymatic activity
pH-dependent adsorption efficiency with 1 × 1010spores
pH values of the adsorption reaction
Total units (%)
262.55 ± 5.15 (100)
308.92 ± 11.13 (100)
353.64 ± 23.80 (100)
Spore-bound units (%)
139.64 ± 5.65 (53.18)
158.98 ± 2.41 (51.46)
26.89 ± 4.68 (7.60)
92.46 ± 8.24 (35.21)
127.77 ± 12.38 (41.36)
306.72 ± 26.96 (86.73)
Adsorption to spores stabilizes β-Gal
Spores with altered surface show increased efficiency of adsorption
Next, we decided to test the adsorption efficiency of mutants lacking also other coat layers. In particular, we used spores of strains lacking cotH, gerE or cotE. cotH spores lack at least 9 outer coat components , gerE codes for a transcriptional regulator and gerE mutant spores lack the latest class of coat components , and cotE spores totally lack the outer coat . As shown in Figure5C, with spores of all three mutants 100% of the β-Gal activity was adsorbed, indicating that the spores lacking the crust and at least part of the outer coat are extremely efficient in adsorbing β-Gal.
Adsorption to mutant spores lacking cotE or cotH
Effect of wild type and mutant spores on pH
n° of sporesa
5.98 ± 0.08
Water + WT spores
2.0 × 109
7.23 ± 0.07
1.0 × 1010
7.14 ± 0.24
Water + cotE spores
2.0 × 109
6.06 ± 0.07
1.0 × 1010
6.18 ± 0.04
Water + cotH spores
2.0 × 109
5.88 ± 0.07
1.0 × 1010
5.85 ± 0.07
The adsorption of antigens and enzymes to bacterial spores has been reported previously and the involvement of a combination of physicochemical forces has been suggested [18, 19, 20]. In this frame, our work was aimed at gaining a better understanding of the spore-enzyme interaction. We used spores of a laboratory strain of B. subtilis, for which a collection of isogenic mutants altered in spore surface proteins was available, and as a model enzyme the well characterized β-Gal of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius.
Our results indicate that spores bind β-Gal without affecting its properties but, instead, stabilizing it at acidic pH and high temperatures. Spore adsorption is a very efficient process with each wild type spore able to adsorb about 7.75×103 molecules of β-Gal. Gene fusion-based display of heterologous antigens was slightly less efficient than adsorption, with 1.5×103 molecules of TTFC (5) and 4.8×103 molecules of LTB (15) exposed on each spore. Spores lacking the outermost structures, the crust and the outer coat layer, have an adsorption efficiency even higher than wild type spores, with 1.55×104 molecules of β-Gal adsorbed on each spore. This indicates that those structures, mainly formed by proteins and glycoproteins , have an inhibitory effect on the adhesion of the enzyme. While the efficiency of adhesion is improved, the stabilization at low pH is reduced in the mutants after 24 hours of incubation at pH 4.0. In the case of β-Gal, the loss of activity (30%) at low pH is compensated by the high amount of enzyme adsorbed. Although other enzymes have not been tested yet, our data suggest that the use of wild type or mutants spores can be planned according to the specific application and the heterologous enzyme to be displayed.
The carboxyl groups were identified as the major ionizable groups in the spore and proton diffusion was found much lower in the spore core than within the coats and cortex, suggesting the inner membrane, separating core from the external layers of the spore, as a major permeability barrier for protons . Then, the carboxyl groups in the coat and in the cortex have been suggested as responsible of the negative charge of spores . The different effects of wild type and mutant spores on the pH of an aqueous solution (Table2), indicate that wild type spores attract more protons than cotE or cotH mutant spores and therefore, suggest that thay have a net negative charge higher than that of the mutant spores. Since spores of those mutants lack either the entire outer coat (cotE) or a large part of it (cotH), they have a reduced number of proteins, and as a consequence of carboxyl groups, in their cortex and coat. Based on this we hypothesize that the reduced number of carboxyl groups present in the regions of the spore more permeable to proton diffusion  is responsible of the reduced number of protons attracted by mutant spores. However, the different efficiency of β-Gal adsorption observed between wild type and mutant spores is not due to a different effect on the pH, since the effect of spores on the pH is buffered at the adsorption conditions (citrate buffer pH 4.0).
Our results, indicating that spores with altered surface structures have altered adsorption efficiency, point to the physicochemical properties of the spore surface as responsible of the interaction with the model enzyme. β-Gal, having a deduced isoelectric point of 5.77, at the adsorption conditions (pH 4.0) is expected to have a net positive charge and to be attracted by negatively charged spores. In spite of this, β-Gal binds more efficiently to mutant (lower negative charge) than wild type (higher negative charge) spores. We conclude that, at least in the case of β-Gal, the electrostatic force does not seem to be the predominant force involved in the interaction with the spore.
The hypothesis that the different negative charge of wild type and mutant spores is somehow responsible of the different stabilization effect observed at pH 4.0 is an intriguing possibility that needs to be addressed. Answering the questions of what is the basis of spore adsorption and of enzyme stabilization are challenging future goals that will necessarily require further experiments and the use of sophisticated physicochemical tools.
Although the molecular details of adsorption and enzyme stabilization have not been totally elucidated, spores have shown clear potentials as a novel, surface display system that, being non-recombinant, able to protect the heterologous enzyme from acidic pH and based on a host with a remarkable safety record [10, 11], appear particularly well suited for the delivery of biotherapeutic molecules to animal and human mucosal surfaces.
Bacterial strains and transformation
B. subtilis strains
Sequence (5’- 3’)a
Position of annealingb
Genetic and molecular procedures
For the construction of insertional null mutations DNA fragments internal to genes cotS, cotZ and cgeA were amplified by PCR using the B. subtilis chromosome as a substrate and synthetic oligonucleotides listed in Table2 to prime the reactions. The PCR products were visualized on ethidium bromide-stained agarose gels and gel purified by the QIAquick gel extraction kit (Qiagen) as specified by the manufacturer. Amplified fragments were ligated into plasmids pBEST501  (cotZ mutant) or pER19  (cotS mutant), carrying an antibiotic resistance cassette selectable in B. subtilis. The mutant in cgeAB was obtained by cloning amplified internal fragment of cgeA gene in a pGEM-T (Promega) vector in which was previously cloned a spectinomycin cassette (from pAH256 ) in a Pst I restriction site. Those recombinant plasmids were used to transform competent cells of strain PY79. Transformants were the result of a single (Campbell-like) recombination event between homologous DNA present on the plasmid and on the chromosome. Transformants were selected by antibiotic resistance and confirmed by PCR analysis of chromosomal DNA. Mutants in the cotX and cotXYZ genes were already available but carried in a B. subtilis strain with a different genetic background (strain MB24) . To obtain cotX and cotXYZ mutants isogenic with the wild type and other mutants used in this study, chromosomal DNA of the existing strains was extracted and used to transform competent cells of PY79 competent cells.
Purification of spores and β-galactosidase
Sporulation of wild type and recombinant strains was induced by the exhaustion method. After 30 h of growth in Difco Sporulation medium (DSM) at 37°C with vigorous shaking , spores were collected, washed three times with distilled water and purified by gastrografin gradient as described before . Spore counts were determined by serial dilution and plate-counting.
A recombinant plasmid containing the lacB gene of Alyciclobacillus acidocaldarius into the expression vector pET29a has been previously described . Expression of lacB was induced by 0.1 mM isopropyl-β D-thiogalactoside (IPTG) in E. coli BL21RB791 cells and β-Gal purified using the GST-tag and the thrombin cleavage on the matrix as described by the manufacturer (Amersham Biotech).
Binding assay and enzyme detection
Purified β-Gal was added to a suspension of 1×1010 spores in sodium citrate 50 mM at pH 4.0 at 25°C in a final volume of 200 μl. After 1 hour of incubation, an aliquot (70 μl) of the binding mixture was stored at 4°C while the remaining part of the binding mixture was centrifuged (10 min at 13,000 rpm) to fractionate pellet and supernatant. All fractions were then used for β-Gal assays: 20 μl of each fraction were added to the reaction buffer (50 mM sodium citrate buffer at pH 5.5, 2NP-β-D-Gal 10 mM) and mixtures incubated at 65°C for 5 minutes; the reaction was then blocked by addition of 800 μl of 1 M Na2CO3. When the assay was performed on samples containing spores, the samples were centrifuged prior to measurement of optical density at 420 nm. We expressed results of enzymatic assays in total units, where 1 unit is defined as an amount of β-Gal able to hydrolyze 1 μmol of substrate in 1 min at standard conditions .
We thank G. Pesce, C. Lapegna and M. Trifuoggi for valuable suggestions and help with the pH measures. This work was supported by a grant (KBBE-476 2007–207948) from the EU 7th Framework to E.R. and by a grant MoMa n. 1/014/06/0 of the Agenzia Spaziale Italiana to M.M.
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