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A total of thirty-two aerobic spore former strains were isolated from intestinal samples of healthy children and analyzed for their hemolytic and antibiotic-resistant activities. Four strains selected as non-hemolytic and sensitive to all antibiotics recommended as relevant by regulatory agencies were short-listed and evaluated for their in silico and in vitro probiotic potentials. The four selected strains were assigned to the Bacillus velezensis (MV4 and MV11), B. subtilis (MV24), and Priestia megaterium (formerly Bacillus megaterium) (MV30) species. A genomic analysis indicated that MV4, MV11, and MV24 contained a homolog of the gene coding for the fibrinolytic enzyme nattokinase while only MV30 encoded a glutamic acid decarboxylase essential to synthesize the neurotransmitter GABA. All four strains contained gene clusters potentially coding for new antimicrobials, showed strong antioxidant activity, formed biofilm, and produced/secreted quorum-sensing peptides able to induce a cytoprotective stress response in a model of human intestinal (HT-29) cells. Altogether, genomic and physiological data indicate that the analyzed strains do not pose safety concerns and have in vitro probiotic potentials allowing us to propose their use as an alternative to antibiotics. [ABSTRACT FROM AUTHOR]

The development of efficient mucosal vaccines is strongly dependent on the use of appropriate vectors. Various biological systems or synthetic nanoparticles have been proposed to display and deliver antigens to mucosal surfaces. The Bacillus spore, a metabolically quiescent and extremely resistant cell, has also been proposed as a mucosal vaccine delivery system and shown able to conjugate the advantages of live and synthetic systems. Several antigens have been displayed on the spore by either recombinant or non-recombinant approaches, and antigen-specific immune responses have been observed in animals immunized by the oral or nasal route. Here we review the use of the bacterial spore as a mucosal vaccine vehicle focusing on the advantages and drawbacks of using the spore and of the recombinant vs. non-recombinant approach to display antigens on the spore surface. An overview of the immune responses induced by antigen-displaying spores so far tested in animals is presented and discussed. [ABSTRACT FROM AUTHOR]

The fraction of low-abundance microbiota in the marine environment is a promising target for discovering new bioactive molecules with pharmaceutical applications. Phenomena in the ocean such as diel vertical migration (DVM) and seasonal dynamic events influence the pattern of diversity of marine bacteria, conditioning the probability of isolation of uncultured bacteria. In this study, we report a new marine bacterium belonging to the rare biosphere, Leeuwenhoekiella parthenopeia sp. nov. Mr9T, which was isolated employing seasonal and diel sampling approaches. Its complete characterization, ecology, biosynthetic gene profiling of the whole genus Leeuwenhoekiella, and bioactivity of its extract on human cells are reported. The phylogenomic and microbial diversity studies demonstrated that this bacterium is a new and rare species, barely representing 0.0029% of the bacterial community in Mediterranean Sea metagenomes. The biosynthetic profiling of species of the genus Leeuwenhoekiella showed nine functionally related gene cluster families (GCF), none were associated with pathways responsible to produce known compounds or registered patents, therefore revealing its potential to synthesize novel bioactive compounds. In vitro screenings of L. parthenopeia Mr9T showed that the total lipid content (lipidome) of the cell membrane reduces the prostatic and brain tumor cell viability with a lower effect on normal cells. The lipidome consisted of sulfobacin A, WB 3559A, WB 3559B, docosenamide, topostin B-567, and unknown compounds. Therefore, the bioactivity could be attributed to any of these individual compounds or due to their synergistic effect. Beyond the rarity and biosynthetic potential of this bacterium, the importance and novelty of this study is the employment of sampling strategies based on ecological factors to reach the hidden microbiota, as well as the use of bacterial membrane constituents as potential novel therapeutics. Our findings open new perspectives on cultivation and the relationship between bacterial biological membrane components and their bioactivity in eukaryotic cells, encouraging similar studies in other members of the rare biosphere. [ABSTRACT FROM AUTHOR]

Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities. [ABSTRACT FROM AUTHOR]

GerR is a sporulation-specific transcriptional factor of Bacillus subtilis that has been identified as a negative regulator of genes transcribed by [[sigma].sup.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].sup.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. doi: 10.1128/JB.00329-10

Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.

Summary: Bacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation‐specific and heat‐labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo‐regulator CotH contributes to the switch between the coat of 25°C and that of 42°C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components. [ABSTRACT FROM AUTHOR]

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 [[sigma].sup.l] and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to forma 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.

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains [epsilon]-([gamma]-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under [[sigma].sup.K] control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.

Citrobacter rodentium is a murine pathogen that is now widely used as an in vivo model for gastrointestinal infections due to its similarities with human enteropathogens, such as the possession of a locus for enterocyte effacement (the LEE island). We studied the lrp gene of C. rodentium and found that it encodes a product highly similar to members of the Lrp (leucine-responsive regulatory protein) family of transcriptional regulators, able to recognize leucine as an effector and to repress the expression of its own structural gene. In enterobacteria, Lrp is a global regulator of gene expression, as it controls a large variety of genes, including those coding for cell appendages and other potential virulence factors. Based on the well-established role of Lrp on the expression of pilus genes in Escherichia coli, we also studied the role of Lrp in controlling the formation of the type I pilus in C. rodentium. Type I pili, produced by the fim system, are virulence factors of uropathogens, involved in mediating bacterial adhesion to bladder epithelial cells. Yeast agglutination assays showed that Lrp is needed for type I pilus formation and real-time PCR experiments indicated that Lrp has a strong leucine-mediated effect on the expression of the fim AICDFGH operon. Mutant studies indicated that this positive action is exerted mainly through a positive control of Lrp on the phase variation mechanism that regulates fimAICDFGH expression. A quantitative analysis of its expression suggested that this operon may also be negatively regulated at the level of transcription.

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 GorE. 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.

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 CotB46 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.

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.

Domestication is an intriguing evolutionary process. Many domestic populations are subjected to strong human‐mediated selection, and when some individuals return to the wild, they are again subjected to selective forces associated with new environments. Generally, these feral populations evolve into something different from their wild predecessors and their members typically possess a combination of both wild and human selected traits. Feralisation can manifest in different forms on a spectrum from a wild to a domestic phenotype. This depends on how the rewilded domesticated populations can readapt to natural environments based on how much potential and flexibility the ancestral genome retains after its domestication signature. Whether feralisation leads to the evolution of new traits that do not exist in the wild or to convergence with wild forms, however, remains unclear. To address this question, we performed population genomic, olfactory, dietary, and gut microbiota analyses on different populations of Sus scrofa (wild boar, hybrid, feral and several domestic pig breeds). Porcine single nucleotide polymorphisms (SNPs) analysis shows that the feral population represents a cluster distinctly separate from all others. Its members display signatures of past artificial selection, as demonstrated by values of FST in specific regions of the genome and bottleneck signature, such as the number and length of runs of homozygosity. Generalised FST values, reacquired olfactory abilities, diet, and gut microbiota variation show current responses to natural selection. Our results suggest that feral pigs are an independent evolutionary unit which can persist so long as levels of human intervention remain unchanged. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract Background Spore-forming Bacilli are Gram-positive bacteria commonly found in a variety of natural habitats, including soil, water and the gastro-intestinal (GI)-tract of animals. Isolates of various Bacillus species produce pigments, mostly carotenoids, with a putative protective role against UV irradiation and oxygen-reactive forms. Results We report the annotation of carbohydrate active enzymes (CAZymes) of two pigmented Bacilli isolated from the human GI-tract and belonging to the Bacillus indicus and B. firmus species. A high number of glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) were found in both isolates. A detailed analysis of CAZyme families, was performed and supported by growth data. Carbohydrates able to support growth as the sole carbon source negatively effected carotenoid formation in rich medium, suggesting that a catabolite repression-like mechanism controls carotenoid biosynthesis in both Bacilli. Experimental results on biofilm formation confirmed genomic data on the potentials of B. indicus HU36 to produce a levan-based biofilm, while mucin-binding and -degradation experiments supported genomic data suggesting the ability of both Bacilli to degrade mammalian glycans. Conclusions CAZy analyses of the genomes of the two pigmented Bacilli, compared to other Bacillus species and validated by experimental data on carbohydrate utilization, biofilm formation and mucin degradation, suggests that the two pigmented Bacilli are adapted to the intestinal environment and are suited to grow in and colonize the human gut.

Abstract 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 × 103 recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 × 103 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.

Abstract Background RecA is a highly conserved prokaryotic protein that not only plays several important roles connected to DNA metabolism but also affects the cell response to various stress conditions. While RecA is highly conserved, the mechanism of transcriptional regulation of its structural gene is less conserved. In Escherichia coli the LexA protein acts as a recA repressor and is able, in response to DNA damage, of RecA-promoted self-cleavage, thus allowing recA transcription. The LexA paradigm, although confirmed in a wide number of cases, is not universally valid. In some cases LexA does not control recA transcription while in other RecA-containing bacteria a LexA homologue is not present. Results We have studied the recA transcriptional regulation in S. thermophilus, a bacterium that does not contain a LexA homologue. We have characterized the promoter region of the gene and observed that its expression is strongly induced by DNA damage. The analysis of deletion mutants and of translational gene fusions showed that a DNA region of 83 base pairs, containg the recA promoter and the transcriptional start site, is sufficient to ensure normal expression of the gene. Unlike LexA of E. coli, the factor controlling recA expression in S. thermophilus acts in a RecA-independent way since recA induction was observed in a strain carrying a recA null mutation. Conclusion In S. thermophilus, as in many other bacteria,recA expression is strongly induced by DNA damage, however, in this organism expression of the gene is controlled by a factor different from those well characterized in other bacteria. A small DNA region extending from 62 base pairs upstream of the recA transcriptional start site to 21 base pairs downstream of it carries all the information needed for normal regulation of the S. thermophilus recA gene.

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self‐organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C‐terminal moiety of CotB, SKRB, formed by serine‐rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine‐repeat region, SKRG, interacts with the C‐terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKRB region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers. [ABSTRACT FROM AUTHOR]

Abstract Bacterial spores are robust and dormant life forms with formidable resistance properties, in part, attributable to the multiple layers of protein that encase the spore in a protective and flexible shield. The coat has a number of features pertinent to the emerging field of nanobiotechnology including self-assembling protomers and the capacity for engineering and delivery of foreign molecules. This review gives an account of recent progress describing the use of the spore, and specifically, the spore coat as a vehicle for heterologous antigen presentation and protective immunization (vaccination). As interest in the spore coat increases it seems likely that they will be exploited further for drug and enzyme delivery as well as a source of novel self-assembling proteins.