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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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Mucosal 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., Results: We 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., Conclusion: Our 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.

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.

Background: 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., Results: 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., Conclusion: 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.

Citrobacter rodentium is a mouse pathogen that, because of its similarities with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) strains of Escherichia coli is widely used as a model system for in vivo and in vitro studies. Similarly to EPEC and EHEC, C. rodentium carries the LEE (locus of enterocyte effacement) pathogenicity island, encoding virulence factors essential for causing transmissible colonic hyperplasia in mice by attaching and effacing (A/E) lesions. Expression of the genes carried by the LEE pathogenicity island is controlled by complex networks of transcriptional factors, including the global regulators H-NS, IHF, and Fis. In this study, we analyzed the role of Lrp, another global regulator of gene expression in enteric bacteria, on the expression of LEE genes of C. rodentium. To this aim, a real-time PCR approach was used and revealed a negative role of Lrp on the expression of all analyzed LEE genes. Mobility-shift experiments indicated that Lrp action is direct on LEE1 and indirect on all other analyzed LEE genes., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The cotG and cotH genes of Bacillus subtilis encode two previously characterized spore coat proteins. The two genes are adjacent on the chromosome and divergently transcribed by σ(K), a sporulation-specific σ factor of the RNA polymerase. We report evidence that the cotH promoter maps 812 bp upstream of the beginning of its coding region and that the divergent cotG gene is entirely contained between the promoter and the coding part of cotH. A bioinformatic analysis of all entirely sequenced prokaryotic genomes showed that such chromosomal organization is not common in spore-forming bacilli. Indeed, CotG is present only in B. subtilis, B. amyloliquefaciens, and B. atrophaeus and in two Geobacillus strains. When present, cotG always encodes a modular protein composed of tandem repeats and is always close to but divergently transcribed with respect to cotH. Bioinformatic and phylogenic data suggest that such genomic organizations have a common evolutionary origin and that the modular structure of the extant cotG genes is the outcome of multiple rounds of gene elongation events of an ancestral minigene.

GerR is a sporulation-specific transcriptional factor of Bacillus subtilis that has been identified as a negative regulator of genes transcribed by sigma(E)-containing RNA polymerase and as a positive effector of the expression of three late sporulation genes. Here we confirmed that gerR transcription is dependent on sigma(E)-containing RNA polymerase but also observed that it requires the transcriptional regulator SpoIIID. The study of the role of GerR in regulating the expression of several late sporulation genes allowed us to observe that its effect is strongly positive on spoVIF, cotC, and cotG, weakly positive on cotB, and negative on cotU. The results of chromatin immunoprecipitation (ChIP) experiments indicated that GerR binds to the promoter regions of some, but not all, of the GerR-controlled genes, leading us to propose that GerR controls late sporulation genes in two ways: (i) directly, by acting on the transcription of cotB, cotU and spoVIF; and (ii) indirectly, through the activation of SpoVIF, which stabilizes the transcriptional activator GerE and consequently induces the expression of the GerE-dependent genes cotC and cotG.

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of Bacillus subtilis. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.

Background: The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections., Results: We expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 x 10(3) recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 x 10(3) recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed., Conclusion: UreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of sigmaK and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to form a coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis. Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE. In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH. These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.

Evidence is presented that expression of the cotH gene, whose product is required for the correct assembly of the Bacillus subtilis spore coat, is negatively controlled by the transcriptional regulator GerE. Mutations in the GerE-box, present in the cotH promoter region, increased expression of this gene, which also remained elevated during late stages of sporulation, when in wild-type cells cotH is normally turned off. Such alterations of cotH expression did not significantly affect spore coat structure or function but caused the accumulation of CotC molecules in the mother cell compartment, most likely as a consequence of CotH-mediated protection of CotC.

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

We report the characterization of the ccpA gene of Lactobacillus plantarum, coding for catabolite control protein A. The gene is linked to the pepQ gene, encoding a proline peptidase, in the order ccpA-pepQ, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two ccpA transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (cre) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a ccpA mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, beta-glucosidase and beta-galactosidase, was relieved from carbon catabolite repression in the ccpA mutant strain. In vivo footprinting analysis of the catabolite-controlled bglH gene regulatory region in the ccpA mutant strain showed loss of protection of the cre under repressing conditions.

A newly-identified bglGPT operon of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed three open reading frames encoding (i) a 237-amino acid protein (BglG), (ii) a 577-amino acid protein (BglP) and (iii) a 486-amino acid protein (BglT). BglG, BglP and BglT were shown to be homologous to the BglG family of transcriptional antiterminators, to permeases of the phosphoenolpyruvate-dependent phosphotransferase system and to beta-glucosidases, respectively. Complementation of E. coli mutant strains showed that BglP and BglT are a permease and a beta-glucosidase active on the beta-glucosides, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-glucoside, respectively. BglG was also shown to promote expression of a bglG-lacZ gene fusion in an E. coli bglG(-) background. A ribonucleic antiterminator sequence, the antiterminator-responsive cis-element and a 'catabolite responsive element', were found downstream of the transcriptional start point. Transcription of the operon was repressed 10-fold in L. plantarum cells grown on glucose as compared to ribose.

Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.

Transcription of the ilvIH operon was reduced during amino acid starvation of wild-type Escherichia coli. The effect was abolished by a relA mutation and was enhanced by a spoT mutation, thus suggesting a possible negative role of ppGpp accumulation on ilvIH transcription. No effect of amino acid downshift was observed on the synthesis of lrp mRNA, encoding the positive regulator (Lrp) of ilvIH transcription.

An in vivo footprinting analysis of the ilvIH regulatory region of Escherichia coli showed that the transcription activator Lrp binds to six sites, scattered over 250 bp upstream of the transcriptional start point. When Lrp-mediated activation was impaired by the presence of exogenous leucine, only one promoter-distal site (site 2) was partially protected by Lrp binding. Equilibrium dialysis experiments showed the formation of an Lrp-leucine complex in vitro. These results suggest that leucine negatively affects ilvIH transcription because its interaction with Lrp reduces the efficiency of binding of the regulatory protein to the promoter region.

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvIH. Lrp binds to six sites upstream of ilvIH, and Lrp binding is required for ilvIH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvIH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvIH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.