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This work details a science experiment on garlic's antibiotic properties designed to acquaint high school and introductory-level undergraduate students with concepts such as natural antibiotics, bioactive substances, and biosafety. This activity is optimized to be implemented by teachers with limited experience in laboratory activities and/or in poorly equipped schools. A list of materials is provided, along with safety and procedural instructions, discussion topics, and assessment suggestions. Key Words: Allicin; Allium sativum L.; science experiment; natural antibiotics., Garlic (Allium sativum L.) is widely used for its culinary and pharmacological properties, which include antimicrobial traits. Garlic extracts are known to inhibit the growth of fungi, protozoa, viruses, and [...]

Bacillus cereus 569 spores germinate either with inosine as a sole germinant or with a combination of nucleosides and L-alanine. Whereas the inosine-only germination pathway requires the presence of two different germination receptors (Gerl and GerQ) to be activated, the nucleoside/alanine germination pathway only needs one of the two receptors. To differentiate how nucleoside recognition varies between the inosine-only germination pathway and the nucleoside/ alanine germination pathway, we tested 61 purine analogues as agonists and antagonists of the two pathways in wild-type, [DELTA]gerl and [DELTA]gerQ spores. The structure-activity relationships of germination agonists and antagonists suggest that the inosine-only germination pathway is restricted to recognize a single germinant (inosine), but can be inhibited in predictable patterns by structurally distinct purine nucleosides. B. cereus spores encoding Gerl as the only nucleoside receptor ([DELTA]gerQ mutant) showed a germination inhibition profile similar to wild-type spores treated with inosine only. Thus, Gerl seems to have a well-organized binding site that recognizes inosine and inhibitors through specific substrate-protein interactions. Structure-activity analysis also showed that the nucleoside/alanine germination pathway is more promiscuous toward purine nucleoside agonists, and is only inhibited by hydrophobic analogues. B. cereus spores encoding GerQ as the only nucleoside receptor ([DELTA]gerl mutant) behaved like wild-type spores treated with inosine and L-alanine. Thus, the GerQ receptor seems to recognize substrates in a more flexible binding site through non-specific interactions. We propose that the Gerl receptor is responsible for germinant detection in the inosine-only germination pathway. On the other hand, supplementing inosine with L-alanine allows bypassing of the Gerl receptor to activate the more flexible GerQ receptor. DOI 10.1099/mic.0.030270-0

Bacillus cereus is a food-berne pathogen and a frequent contaminant of food production plants. The persistence of this pathogen in various environments results from the formation of spores and of biofilms. To investigate the role of the B. cereus flagellar apparatus in biofilm formation, we constructed a non-flagellated mutant and a flagellated but non-motile mutant. Unexpectedly, we found that the presence of flagella decreased the adhesion of the bacterium to g ass surfaces. We hypothesize that this decrease is a consequence of the flagella hindering a direct interaction between the bacterial cell wall and the surface. In contrast, in specific conditions, motility promotes biofilm formation. Our results suggest that motility could influence biofilm formation by three mechanisms. Motility is necessary for the bacteria to reach surfaces suitable for biofilm formation, In static conditions, reaching the air--liquid interface, where the biofilm forms, is a strong requirement, whereas in flow cells bacteria can have access to the bosom glass slide by sedimentation. Therefore, motility is important for biofilm formation in glass tubes and in microtitre plates, but not in flew cells. Motility also promotes recruitment of planktonic cells within the biofilm by allowing motile bacteria to invade the whole biofilm, Finally, motility is involved in the spreading of the biofilm on glass surfaces. DOI 10.1099/mic.0.034827-0

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in grampositive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The [[DELTA]dlt.sub.Bc] mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodopterafrugiperda. [[DELTA]dlt.sub.Bc] was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. meUonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in [[DELTA]dlt.sub.Bc]). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans. doi: 10.1128/JB.00892-09

Bacterial extracellular proteases play an important role in cell survival and cell-cell communication. A high-molecular-mass minor extracellular protease (Vpr) from a feather-degrading bacterium, Bacillus cereus DCUW, has been reported by our laboratory. In the present study, we cloned and expressed Vpr in Escherichia coli. Complete nucleotide sequencing of this gene predicted that the protease is a member of the serine protease family, and SMART domain analysis revealed that the protease consists of an N-terminal signal sequence for secretion, a subtilisin_N sequence that is a signature for N-terminal processing, a catalytic S_8 peptidase domain, and finally a long C-terminal protease-associated (PA) region containing nine intrinsically disordered subdomains. Four truncated constructs of the Vpr protease were cloned and expressed in E. coli. We found that the catalytic domain (amino acid residues 172-583) is sufficient for protease activity. Maturation of the Vpr protease needed both N-terminal and C-terminal processing. We have demonstrated that the oligomerization property is associated with the C-terminal protease-associated domain and also shown that the substrate-binding specificity to raw feather resides in this domain.

Several Baciflus strains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strain Bacillus [cereus.sub.CH] displayed the best adhesion. Different biochemical treatments revealed that surface proteins of B. [cereus.sub.CH] were involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores of B. [cereus.sub.CH] were extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probiotic Bacillus strain within the human gastrointestinal tract.

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PIcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PIcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The [[sigma].sup.A]-promoted transcription of spoOA was found to be crucial for cereulide production, while the [[sigma].sup.H]-driven transcription of spoOA did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a [Na.sup.+]/[H.sup.+]-[K.sup.+] antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or [Na.sup.+] stress.

Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged ([Fnr.sub.His]) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) ([sub.strep]Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, [apoFnr.sub.His] was mainly monomeric, while [apo.sub.Strep]Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

Flagellar arrangement is a highly conserved feature within bacterial species. However, only a few genes regulating cell flagellation have been described in polar flagellate bacteria. This report demonstrates that the arrangement of flagella in the peritrichous flagellate Bacillus cereus is controlled by flhF. Disruption of flhF in B. cereus led to a reduction in the number of flagella from 10-12 to 1-3 filaments per cell in the insertion mutant MP06. Moreover, compared to the parental strain, MP06 exhibited: (i) shorter smooth swimming phases, causing reduced swimming motility but not affecting chemotaxis; (ii) complete inhibition of swarming motility, as differentiated swarm cells were never detected; (iii) an increased amount of extracellular proteins; and (iv) differential export of virulence determinants, such as haemolysin BL (HBL), phosphatidylcholinepreferring phospholipase C (PC-PLC) and non-haemolytic enterotoxin (NHE). Introduction of a plasmid harbouring flhF (pDGflhF) into MP06 completely restored the wild-type phenotype in the trans-complemented strain MP07. B. cereus flhF was found to constitute a monocistronic transcriptional unit and its overexpression did not produce abnormal features in the wild-type background. Characterization of a B. cereus mutant (MP05) carrying a partial flhF deletion indicated that the last C-terminal domain of FlhF is involved in protein export while not required for flagellar arrangement and motility behaviour. Taken together, these data suggest that B. cereus FlhF is a promising candidate for connecting diverse cellular functions, such as flagellar arrangement, motility behaviour, pattern of protein secretion and virulence phenotype.

Tolerance to bile salts was investigated in forty Bacillus cereus strains, including 17 environmental isolates, 11 dairy isolates, 3 isolates from food poisoning outbreaks, and 9 other clinical isolates. Growth of all strains was observed at low bile salt concentrations, but no growth was observed on LB agar plates containing more than 0.005% bile salts. Preincubation of the B. cereus type strain, ATCC 14579, in low levels of bile salts did not increase tolerance levels. B. cereus ATCC 14579 was grown to mid-exponential growth phase and shifted to medium containing bile salts (0.005%). Global expression patterns were determined by hybridization of total eDNA to a 70-mer oligonueleotide microarray. A general stress response and a specific response to bile salts were observed. The general response was similar to that observed in cultures grown in the absence of bile salts but at a higher (twofold) cell density. Up-regulation of several putative multidrug exporters and transcriptional regulators and down-regulation of most motility genes were observed as part of the specific response. Motility experiments in soft agar showed that motility decreased following bile salts exposure, in accordance with the transcriptional data. Genes encoding putative virulence factors were either unaffected or down-regulated.

The alternative sigma factor [[sigma].sup.B] has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of [[sigma].sup.B]-regulated genes in B. cereus by DNA microarray analysis of the transcriptome upon a mild heat shock. Twenty-four genes could be identified as being [[sigma].sup.B] dependent as witnessed by (i) significantly lower expression levels of these genes in mutants with a deletion of sigB and rsbY (which encode the alternative sigma factor [[sigma].sup.B] and a crucial positive regulator of [[sigma].sup.B] activity, respectively) than in the parental strain B. cereus ATCC 14579 and (ii) increased expression of these genes upon a heat shock. Newly identified [[sigma].sup.B]-dependent genes in B. cereus include a histidine kinase and two genes that have predicted functions in spore germination. This study shows that the [[sigma].sup.B] regulon of B. cereus is considerably smaller than that of other gram-positive bacteria. This appears to be in line with phylogenetic analyses where [[sigma].sup.B] of the B. cereus group was placed close to the ancestral form of [[sigma].sup.B] in gram-positive bacteria. The data described in this study and previous studies in which the complete [[sigma].sup.B] regulon of the gram-positive bacteria Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus were determined enabled a comparison of the sets of [[sigma].sup.B]-regulated genes in the different gram-positive bacteria. This showed that only three genes (rsbV, rsbW, and sigB) are conserved in their [[sigma].sup.B] dependency in all four bacteria, suggesting that the [[sigma].sup.B] regulon of the different gram-positive bacteria has evolved to perform niche-specific functions.

The motility behavior of and the secretion of hymolysin BL (HBL) is assessed by Bacillus cereus strains isolated from different sources. It is found that swarming differentiation by Bacillus cereus contributes to the virulence potential of this opportunistic human pathogen.

Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of [O.sub.2] and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PIcR to regulate enterotoxin gene expression.

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 [mm.sup.2]. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 [+ or -] 31 (n = 11), 2742 [+ or -] 38 (n = 3), 3053 [+ or -] 19 (n = 2), 2777 [+ or -] 26 (n = 2), 2953 [+ or -] 24 (n = 2), and 3105 [+ or -] 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 [min.sup.-1]. A hindrance factor (6) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x [10.sup.5] BT + BC spores/mL, suggesting that [alpha] is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ~272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ~270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXOl-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Piasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

The occurrence of scanning electron microscopy (SEM) and atomic force microscopy (AFM) side-by-side is becoming increasingly common in analytical research. This article shows microscopy techniques to image Bacillus spores, to measure spore dimensions, and to demonstrate how these methods provide supplementary information to study plasma torch effects. This paper demonstrates that observed morphologies of spores before and after exposure to a plasma torch are remarkably different. The use of SEM and AFM as a tool complex enables examination of spore morphology and dimensions as well as their alterations during decontamination using plasma torch. Index Terms--Atomic force microscopy (AFM), Bacillus cereus, decontamination, plasma torch, scanning electron microscopy (SEM).

An objective is presented of investigating the occurrence of the Bacillus cereus group, to characterize the cereulide producers with regard to psychrotolerance and compare them to popular cereulide producers at the pheno- and genotypic levels. Two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118 producing cereulide are reported.

A chemically defined medium in combination with an airlift fermentor system is used to study the growth and sporulation of Bacillus cereus ATCC 14579. Findings reveal that Bacillus cereus is able to form spores on a chemically defined medium without glucose but with lactate as a main carbon source.

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants or B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glyeoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.

Crystal structures of the An Arg121Cys mutant of the Bacillus cereus were solved at pH 7.0, 5.0, and 4.5, in presence of 20 (micro)M and 1 mM Zn(super 2+) to generate the mono- and bizinc forms. Experimental evidence is provided for an earlier proposed mechanism in which protonation of Asp120 and the Zn1-bound hydroxide are the two events that lead to the loss of activity at low pH.

A total of 333 Bacillus strains from various sources related to foods are examined for cytotoxicity and production of putative emetic toxins using a cytotoxicity assay of Vero cells, the boar spermatozoa motility assay and a liquid chromatography-mass spectrometry method. The analysis shows that the toxin-producing Bacillus spp. seems to be rare among isolates from water, food and food environments and none of the toxins detected are similar to the B. cereus enterotoxins or to cereulide.

The expression of extracellular virulence factors in various species of the Bacillus cereus group is controlled by the plcR and papR genes, which encode a transcriptional regulator and a cell-cell signaling peptide, respectively. A processed form of PapR, presumably a pentapeptide, specifically interacts with plcR to facilitate its binding to its DNA targets. This activating mechanism is strain specific, with this specificity being determined by the first residue of the pentapeptide. We carried out in vivo complementation assays and compared the PlcR-PapR sequences of 29 strains from the B. cereus group. Our findings suggested that the fifth amino acid of the pentapeptide is also involved in the specificity of activation. We identified four classes of PlcR-PapR pairs, defining four distinct pherotypes in the B. cereus group. We used these findings to look at the evolution of the PlcR-PapR quorum-sensing system with regard to the phylogeny of the species forming the B. cereus group.

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a [beta]-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The [DELTA]fur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene. Abbreviations: FTIR, Fourier transform Infrared; Hbl, haemolysin BL; MLST, multilocus sequence typing; Nhe, non-haemolytic enterotoxin; RAPD, random amplification of polymorphic DNA.

The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus. Nhe is composed of NheA, NheB and NheC. The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site. The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coil Separate expression showed that all three components were required for biological activity. In addition, NheA and NheB were purified from B. cereus culture supernatants. As NheC seems to be expressed in only small amounts by B. cereus, NheC was expressed and purified as a histidine-tagged fusion protein. The maximum cytotoxic activity was obtained when the molar ratio between NheA:NheB : [His.sub.6]-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

Representative strains of the Bacillus cereus group of bacteria, including Bacillus anthracis (11 isolates), B. cereus (38 isolates), Bacillus mycoides (1 isolate), Bacillus thuringiensis (53 isolates from 17 serovars), and Bacillus weihenstephanensis (2 isolates) were assigned to 59 sequence types (STs) derived from the nueleotide sequences of seven alleles, glpF, gmk, ilvD, pta, pur, pycA, and tpi. Comparisons of the maximum likelihood (ML) tree of the concatenated sequences with individual gene trees showed more congruence than expected by chance, indicating a generally clonal structure to the population. The STs followed two major lines of descent. Clade 1 comprised B. anthracis strains, numerous B. cereus strains, and rare B. thuringiensis strains, while clade 2 included the majority of the B. thuringiensis strains together with some B. cereus strains. Other species were allocated to a third, heterogeneous clade. The ML trees and split decomposition analysis were used to assign STs to eight lineages within clades 1 and 2. These lineages were defined by bootstrap analysis and by a preponderance of fixed differences over shared polymorphisms among the STs. Lineages were named with reference to existing designations: Anthracis, Cereus I, Cereus II, Cereus III, Kurstaki, Sotto, Thuringiensis, and Tolworthi. Strains from some B. thuringiensis serovars were wholly or largely assigned to a single ST, for example, serovar aizawai isolates were assigned to ST-15, serovar kenyae isolates were assigned to ST-13, and serovar tolworthi isolates were assigned to ST-23, while other serovars, such as serovar canadensis, were genetically heterogeneous. We suggest a revision of the nomenclature in which the lineage and clone are recognized through name and ST designations in accordance with the clonal structure of the population.

The binding of benzylpencillin to mono-zinc metallo-beta-lactamase from bacillus cereus is investigated in a docking procedure applying a combined quantum mechanical/molecular mechanical method. It is demonstrated that the substitute could bind with the carbonyl oxygen of the lactum ring coordinating to the zinc ion and with the zinc-bound hydroxide ion in position for a nucleophilic attack on the carbonyl carbon of the lactum ring.

A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.

Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor a ~155-hp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcrl repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TITAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.

CytK is a pore-forming toxin of Bacillus cereus that has been linked to a case of necrotic enteritis. PCR products of the expected size were generated with cytK primers in 13 of 29 strains. Six strains were PCR-positive for the related gene hly-II, which encodes haemolysin II, a protein that is 37 % identical to the original CytK. Five of the strains were positive for both genes. The DNA sequences of putative cytK genes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK. The authors have designated this new cytK variant cytK-2, and refer to the original cytK as cytK-1. The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing. BLAST analysis using the cytK-2 gene sequences revealed very high homology with two cytK-2 sequences in the genomes of B. cereus strains ATCC 14579 and ATCC 10987. The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins. The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1. Both native and recombinant CytK-2 proteins from B. cereus 1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1. It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains of B. cereus, although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.

The cytotoxin CytK produced by Bacillus cereus is believed to be involved in food-borne diseases. The transcriptional activity of the cytK promoter region in a food-poisoning strain was studied using a reporter gene and compared with that in the reference B. cereus strain ATCC 14579. In the food-poisoning strain, cytK is more strongly transcribed, possibly explaining the pathogenicity. The global regulator PlcR in B. cereus controls several putative virulence factors. It was found that PlcR regulates cytK in this clinical strain despite a mismatch in the PlcR recognition site, as currently defined. This suggests that the PlcR box consensus should be reconsidered and that the PlcR regulon might be larger than suspected. It is also shown that the high level of cytK transcription is not caused by a modification in the PlcR recognition site.

The alternative sigma factor [[sigma].sup.B] of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells. This study describes the identification of [[sigma].sup.B]-dependent genes in B. cereus. Two-dimensional gel electrophoresis was performed with protein extracts from a [[sigma].sup.B]-overproducing B. cereus strain. Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing. The [[sigma].sup.B]-dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B. cereus ATCC 14579 and its sigB null mutant. Northern blot analysis also revealed that six other genes were part of [[sigma].sup.B]-dependent operons. The proteins that are predicted to be encoded by the [[sigma].sup.B]-dependent genes include an intracellular protease, a [Mg.sup.2+] transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all [[sigma].sup.B]-dependent genes, with the exception of thiG. By searching the B. cereus genome for this conserved promoter sequence, five more candidate [[sigma].sup.B]-dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus [[sigma].sup.B]-RNA polymerase holoenzyme confirmed the [[sigma].sup.B] dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also [[sigma].sup.B] dependent. In conclusion, [[sigma].sup.B] of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.

Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis are closely related species belonging to the Bacillus cereus group. B. thuringiensis and B. cereus generally produce extracellular proteins, including phospholipases and hemolysins. Transcription of the genes encoding these factors is controlled by the pleiotropic regulator PlcR. Disruption of plcR in B. cereus and B. thuringiensis drastically reduces the hemolytic, lecithinase, and cytotoxic properties of these organisms. B. anthracis does not produce these proteins due to a nonsense mutation in the plcR gene. We screened 400 B. thuringiensis and B. cereus strains for their hemolytic and lecithinase properties. Eight [Hly.sup.-] [Lec.sup.-] strains were selected and analyzed to determine whether this unusual phenotype was due to a mutation similar to that found in B. anthracis. Sequence analysis of the DNA region including the plcR and papR genes of these strains and genetic complementation of the strains with functional copies of plcR and papR indicated that different types of mutations were responsible for these phenotypes. We also found that the plcR genes of three B. anthracis strains belonging to different phylogenetic groups contained the same nonsense mutation, suggesting that this mutation is a distinctive trait of this species.

The phenotypic adaptation of membrane lipids in seven strains of the food-poisoning bacterium Bacillus cereus, isolated from Bangladeshi rice, is reported in relation to their ability to grow under conditions of low water activity ([a.sub.w]), reduced temperature and the presence of soluble rice starch. The strains have different membrane phospholipid head-group and fatty acyl compositions, and they display individual differences in their responses to both low aw and reduced temperature. The extent of the increase in anionic membrane lipids in response to low [a.sub.w] varies from strain to strain, is solute specific and in one strain does not occur. Growth is Stimulated by the presence of soluble rice starch and results in a large rise in the proportion of diphosphatidylglycerol (DPG) at the expense of phosphatidylglycerol (PG), without any change in the proportion of total anionic phospholipids. Growth at 15 [degrees]C compared with 37 [degrees]C increases the proportions of DPG and phosphatidylethanolamine at the expense of PG. At the lower temperature there are changes in phospholipid fatty acyl composition characteristic of those expected to maintain membrane fluidity, including increases in the amount of total branched fatty acids and the anteiso-/iso-branched ratio, and a decrease in the equivalent chain-length, but there are strain differences in how those changes were achieved. In contrast to some other bacilli, there are persistent large increases in the proportions of unsaturated fatty acyl chains in phospholipids during growth at 15 [degrees]C.

The conditions for growth and sporulation of Bacillus cereus ATCC 14579, which has been characterized at genome level, in a chemically defined medium with an airlift fermentor system are described. The growth and sporulation can be monitored and manipulated so that a synchronous growth and sporulation occurs.

The Bacillus cereus group includes insecticidal bacteria (B. thuringiensis), food-borne pathogens (B. cereus and B. weihenstephanensis) and B. anthracis, the causative agent of anthrax. The precise number of rRNA operons in 12 strains of the B. cereus group was determined. Most of the tested strains possess 13 operons and the tested psychrotolerant strains contain 14 operons, the highest number ever found in bacteria. The separate clustering of the tested psychrotolerant strains was confirmed by partial sequencing of several genes distributed over the chromosomes. Analysis of regions downstream of the 23S rRNA genes in the type strain B. cereus ATCC 14579 indicates that the rRNA operons can be divided into two classes, I and II, consisting respectively of eight and five operons. Class II operons exhibit multiple tRNA genes downstream of the 5S rRNA gene and a putative promoter sequence in the 23S-5S intergenic region, suggesting that 5S rRNA and the downstream tRNA genes can be transcribed independently of the 16S and 23S genes. Similar observations were made in the recently sequenced genome of B. anthracis strain Ames. The existence of these distinct types of rRNA operons suggests an unknown mechanism for regulation of rRNA and tRNA synthesis potentially related to the pool of amino acids available for protein synthesis.

Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis are three closely related species of the B.cereus group with each of them being economically important and their DNA isolates were analyzed using fluorescent amplified fragment length polymorphism (AFLP). Phytogenetic analysis of AFLP data showed diversity with B.thuringinesis and B.cereus compared to the monomorphic nature of B.anthracis.

Researchers have developed a chemical assay that can measure the amount of cereulide produced by Bacillus cereus. Cereulide is a toxin that can cause serious illness in humans. However, the exact amount of toxin that causes symptoms is not known. Consequently, food safety standards for cereulide cannot be created.

Results from MD simulations of the Michaelis complex formed between the B. cereus zinc-beta-lactamase enzyme and benzylpenicillin is presented. The experimental rate data for the B. cereus enzyme is compatible with a one-step mechanism for the hydrolysis of beta-lactam substrates in which His210 acts as a proton donor is proposed.

Germinating spores of Bacillus cereus are sensitive to pulsed-electric-field (PEF) treatment or nisin. Carvacrol increased the sensitivity of the germinating spores to nisin but not to PEF treatment or nisin combined with PEF treatment.

Research has been conducted on the members of Bacillus cereus group of bacteria. Results indicate that B. anthracis is closely related to B. cereus and B. thuringiensis strains.

Two monoclonal antibodies (MAbs), 8D3 and 9B7, against vegetative cells of Bacillus cereus have been produced and characterized. The MAbs were selected by enzyme-linked immunosorbent assay for their reactivity with the cells. They did not recognize the spores of the organism. Molecular masses were about 58 and 62 kDa for the two antigens MAb 9B7 recognized. Together the two MAbs can perhaps be used to develop an immunological method to detect B. cereus vegetative cells in foods.

Research has been conducted on the Bacillus cereus phospholipase C. The X-ray structure of this phospholipase has been studied.

Results demonstrate that cortical-fragment-lytic enzyme of Bacillus cereus functions as N-acetylglucosaminidase coded by the gene sleL. Data indicate that the enzyme plays a role in the spore germination by hydrolyzing muramic acid-delta-lactam on peptidoglycan.

Perturbed angular correlation of gamma-ray spectroscopy has been used to study Cd-substituted forms of the Bacillus cereus matallo-beta-lactamases. The differences between Zn(II) and Cd(II) have been compared.

The procedure for making 'home-style' French fries usually involves blanching prior to finish frying to cut preparation time, but it is possible for pathogens to contaminate blanched potato strips during the process. A new study investigates the growth potential of the pathogen Bacillus cereus on blanched 'home-style' French fries and determines whether the final frying process inactivates the microorganism. It is shown that oil-blanched French fry strips can support growth of B. cereus at temperatures of 21 deg C or 26.7 deg C, but growth is slowed at below 21 deg C. French fries should be fried within 3-4 hours or else discarded.

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

A study was conducted to analyze the rate-determining step in the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(BC))-catalyzed hydrolysis of phospholipids. Assays in the presence of increasing concentrations of sucrose were carried out on recombinant and commercially available PLC(BC). Results indicated that the hydrolysis of monomeric phospholipids by PLC(BC) is negatively influenced by a chemical process.

A study was conducted to analyze the biological significance and chemical basis for the inhibition of Bacillus cereus UW85 by alfalfa seed exudate. The colonization and biological control capabililties of mutants resistant to canavanine were also examined. Experimental results showed that canavanine inhibited the growth of B. cereus UW85. Findings also indicated that alfalfa seeds in a cultivar differed in the inhibitory nature of their exudates.