Proteomic analysis of Bacillus cereus growing in liquid soil organic matter

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Degradation of N-acyl-L-homoserine lactones by Bacillus cereus in culture media and pork extract

Degradation of the quorum-sensing signal molecule N-acyl-L-homoserine lactone (AHL) in cocultures was verified with Bacillus cereus and Yersinia enterocolitica in culture medium and in pork extract. Results showed evidence of microbial interaction when the AHL-degrading bacterium and AHL-producing bacterium were cocultured in a food-simulating condition.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

Detection of Enterotoxic Bacillus cereus and Bacillus thuringiensis Strains by PCR Analysis

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

From soil to gut: Bacillus cereus and its food poisoning toxins

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.     

Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil

Bacillus weihenstephanensis strains are psychrotolerant and grow from below 7°C to 38°C. Closely related mesophilic Bacillus cereus strains can grow from above 7°C to 46°C. We classified 1060 B. cereus group isolates from different soil samples with respect to their psychrotolerant and mesophilic genotypes by polymerase chain reaction (PCR) targeting of specific 16S rDNA and cold shock protein A gene signatures. In parallel, growth tests at 7°C were carried out to determine the thermal phenotype. The geographic distribution of psychrotolerant and mesophilic isolates was found to depend significantly on the prevalent annual average temperature. In one tropical, one temperate and two alpine habitats, the proportion of psychrotolerant cspA genotypes was found to be 0%, 45% and 86% and 98%, respectively, with the corresponding annual average temperatures being 28°C, 7°C, 4°C and 1°C. In the tropical habitat, only the mesophilic B. cereus was found, characterized by correspondence of thermal genotype and phenotype. In the alpine habitat, almost only the psychrotolerant B. weihenstephanensis was isolated. In the temperate habitat, mesophilic B. cereus and psychrotolerant B. weihenstephanensis as well as 'intermediate thermal types' occurred, the latter having opposite thermal genotypes and phenotypes or opposing sets of thermal DNA signatures, characterized by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single isolate. Both sugar utilization and DNA fingerprinting patterns revealed a high, probably non-clonal microsite diversity within the population of the temperate habitat. We interpret our observations in terms of a temperature-dependent selection regime, acting on recombining B. cereus / B. weihenstephanensis populations in soil.     

Protoplast fusion in Bacillus cereus

Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.     

Enumeration of Bacillus cereus in Foods

For the enumeration of vegetative cells and spores of Bacillus cereus in foods, a mannitol-egg yolk-phenol red-agar has been developed which exploits the failure of B. cereus to dissimilate mannitol, and the ability of most strains to produce phospholipase C. When a high degree of selectivity was required, polymyxin B sulfate in a concentration of 10 ppm appeared to be the most effective selective additive. Useful characteristics for the identification of presumptive isolates of B. cereus were found to be: morphology, dissimilation of glucose mostly to acetyl methyl carbinol under anaerobic conditions, hydrolysis of starch and gelatin, reduction of nitrate, and growth on 0.25% chloral hydrate agar.