We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescentactivated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 ؋ 10 3 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

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؇ 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.Endospores of the gram-positive bacterium Bacillus subtilis are encased in a thick protein shell known as the coat (2). The coat is composed of 15 or more polypeptides arranged in an electron-dense outer layer and a lamellar inner layer. These layers protect the spore from bactericidal enzymes and chemicals, such as lysozyme and chloroform. So far, the genes for 13 of these coat proteins have been identified. These are located at diverse positions on the chromosome and code for polypeptides of 65 (CotA), 59 (CotB), 10 (CotC), 9 (CotD), 24 (CotE), 19 (CotF), 24 (CotG and CotJ), 41 (CotS), 10 (CotT), 19 (CotX), 26 (CotY), and 18 (CotZ) kDa (1,3,6,7,10,18,22,24). Certain proteins, such as CotD, are located in the inner coat, and others, such as CotA, CotB, CotC, and CotG, are located in the outer coat (18, 24). The coat is produced at a relatively late stage in the process of sporulation, when the developing spore (or forespore) is present as a free protoplast within the mother cell compartment of the sporangium (14). Coat proteins are organized on the outer surface of the membrane surrounding the forespore by the sporulation protein SpoIVA (8,17,21). SpoIVA controls the assembly of a ring of CotE proteins around the forespore (8). The CotE ring is thought to regulate the assembly of the proteins of the outer coat and is separated from the outer surface of the forespore membrane by a small gap, which is believed to be the site at which the inner coat will be assembled (8). The production of coat proteins is governed by a regulatory cascade of four transcription factors acting in the mother cell compartment of the sporangium in the sequence E , SpoIIID, K , and GerE (26). E and K are RNA polymerase sigma factors, whereas SpoIIID and GerE are DNA-binding proteins that act in conjunction with E -and K -containing forms of RNA polymerase, respectively (4,9,13,23,25).We report the identification of a gene, herein called cotH, located at 300Њ on the B. subtilis chromosomal map, where it is clustered with two previously described cot genes, cotB and cotG (7,18). cotH codes for a 42.8-kDa protein, apparently located in the inner laye...

Bacillus subtilis spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since B. subtilis is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as Lactobacillus spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.Probiotics, or "friendly bacteria," are becoming increasingly available to the public as beneficial functional foods that purport to promote specific health benefits to consumers (2,14,18). In some countries probiotics are available for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in humans. Often these disorders, many of which lead to diarrhea, are a direct result of antibiotic use, which produces an imbalance in the composition of the normal intestinal microbial flora. In the livestock industry the use of probiotics has potential as an alternative to antibiotics by competitive exclusion of pathogenic microorganisms (19), with some commercial products, such as Paciflor, already available. Bacteria most commonly used as probiotics include the lactic acid bacteria (e.g., lactobacilli, enterococci, streptococci, and bifidobacteria). Experimental evidence now suggests that the ingestion of substantial numbers of harmless bacteria does indeed provide a beneficial effect to the enteric flora (18). Precisely how this is achieved and whether the commercial claims are justified remains a contentious issue, though (14).In addition to the lactic acid bacteria, Bacillus species are also sold as probiotics. These consist of preparations of bacterial spores, with the potential advantage that the spore can survive transit through the stomach intact. Bacillus species are substantially different from other probiotic bacteria, though, being primarily aerobic saprophytes found in the soil. If indeed they have any health benefit, then one obviously important question is how? Do spores germinate and colonize the gut, do they competively exclude colonization by potential pathogens, or does the dormant spore provide some unique stimulus to the gut microbiota, such as enhanced local immunity?In an earlier study we have shown that one major Bacillus probiotic marketed in Europe contained spores of a taxonomically and phylogenetically unrelated Bacillus species (4). This was surprising, considering that in Europe probiotics must be licensed to be used as a functional or novel food.In this work we have examined and characterized five commercial Bacillus spore probiotics as a first step in understanding the nature of spore probiotics. MATERIALS AND METHODSBacterial strains. Bacteria were recovered by suspension of dried probiotic preparations in dist...

We describe the identification and characterization of a gene, herein designated cotG, encoding an abundant coat protein from the spores of Bacillus subtilis. The cotG open reading frame is 195 codons in length and is capable of encoding a polypeptide of 24 kDa that contains nine tandem copies of the 13-amino-acid long, approximately repeated sequence H/Y-K-K-S-Y-R/C-S/T-H/Y-K-K-S-R-S.cotG is located at 300؇ on the genetic map close to another coat protein gene, cotB. The cotG and cotB genes are in divergent orientation and are separated by 1.3 kb. Like the promoter for cotB, the cotG promoter is induced at a late stage of sporulation under the control of the RNA polymerase sigma factor K and the DNA-binding protein GerE. The ؊10 and ؊35 nucleotide sequences of the cotG promoter resemble those of other promoters recognized by K -containing RNA polymerase, and centered 70 bp upstream of the apparent start site is a sequence that matches the consensus binding site for GerE. Spore coat proteins from a newly constructed cotG null mutant lack not only CotG but also CotB, a finding that suggests that CotG may be a morphogenetic protein that is required for the incorporation of CotB into the coat.Spores of the gram-positive bacterium Bacillus subtilis are encased in a complex protein shell known as the coat consisting of 15 or more different proteins. So far, genes for 10 of these coat proteins have been identified. These are located at diverse positions on the chromosome and encode polypeptides of 65 (CotA), 59 (CotB), 10 (CotC), 9 (CotD), 24 (CotE), 19 (CotF), 10 (CotT), 19 (CotX), 26 (CotY), and 18 (CotZ) kDa (2,4,7,29,31). In the cases of CotF and CotT, little of the full-length gene products is found in the coat. Rather, CotF is present as two proteolytic fragments of 8 and 5 kDa, and CotT is present as a proteolytic fragment of 8 kDa (2, 4). Coat proteins are organized in two principal layers, a lamellar inner coat and an electron-dense outer coat (1), with certain proteins, such as CotD, being present in the inner coat, and other proteins, such as CotA, CotB, and CotC, being present in the outer coat (31). The coat is assembled at intermediate to late stages of sporulation when the nascent spore (known as the forespore) is present as a free protoplast, wholly engulfed within the mother cell compartment of the developing sporangium. Coat proteins are recruited to the outer surface of the membrane surrounding the forespore by the sporulation protein SpoIVA (which has not been detected in mature spores and hence is not referred to as a coat protein) (8,19,21). Morphogenetic events under the control of SpoIVA lead to the assembly of a ring of CotE protein around the forespore (8). The CotE ring is separated from the outer surface of the forespore (where SpoIVA is located) by a small gap, which is believed to be the site at which the inner coat will be assembled. Meanwhile, CotE dictates the assembly of the proteins of the outer coat, where it is itself located (8, 31). The production of coat proteins is governed by ...