The Bacillus cereus Group comprises organisms that are widely distributed in the environment and are of health and economic interest. We demonstrate an 'ecotypic' structure of populations in the B. cereus Group using (i) molecular data from Fluorescent Amplified Fragment Length Polymorphism patterns, ribosomal gene sequences, partial panC gene sequences, 'psychrotolerant' DNA sequence signatures and (ii) phenotypic and descriptive data from range of growth temperature, psychrotolerance and thermal niches. Seven major phylogenetic groups (I to VII) were thus identified, with ecological differences that provide evidence for a multiemergence of psychrotolerance in the B. cereus Group. A moderate thermotolerant group (VII) was basal to the mesophilic group I, from which in turn distinct thermal lineages have emerged, comprising two mesophilic groups (III, IV), an intermediate group (V) and two psychrotolerant groups (VI, II). This stepwise evolutionary transition toward psychrotolerance was particularly well illustrated by the relative abundance of the 'psychrotolerant' rrs signature (as defined by Pruss et al.) copies accumulated in strains that varied according to the phylogenetic group. The 'psychrotolerant' cspA signature (as defined by Francis et al.) was specific to group VI and provided a useful way to differentiate it from the psychrotolerant group II. This study illustrates how adaptation to novel environments by the modification of temperature tolerance limits has shaped historical patterns of global ecological diversification in the B. cereus Group. The implications for the taxonomy of this Group and for the human health risk are discussed.

A transcriptional analysis of the phosphatidylinositol-specific phospholipase C (plcA) gene of Bacillus thuringiensis indicated that its transcription was activated at the onset of the stationary phase in B. thuringiensis but was not activated in B. subtilis. The B. thuringiensis gene encoding a transcriptional activator required for plcA expression was cloned by using a B. subtilis strain carrying a chromosomal plcA-lacZ fusion as a heterologous host for selection. This trans activator (designated PlcR) is a protein of a calculated molecular weight of 33,762 which appears to be distantly related to PreL and NprA, regulator proteins enhancing transcription of neutral protease genes during the stationary phase of a Lactobacillus sp. and B. stearothermophilus, respectively. plcR gene transcription was analyzed in B. thuringiensis and in B. subtilis. PlcR positively regulated its own transcription at the onset of the stationary phase. There is a highly conserved DNA sequence (17 bp) 34 nucleotides upstream from the plcR transcriptional start site and 49 nucleotides upstream from the plcA transcriptional start site. As PlcR positively regulates its own transcription and plcA transcription, this conserved DNA sequence may be the specific recognition target for PlcR activation.Phospholipases C that specifically cleave phosphatidylinositol (PI-PLCs) have been isolated from several gram-positive bacteria (for a review, see reference 48). The plcA genes encoding PI-PLCs from Bacillus cereus, B. thuringiensis, Staphylococcus aureus, and Listeria monocytogenes have been cloned and sequenced (6,15,21,22,32), and the deduced amino acid sequences show extensive similarity (about 50%). The PI-PLC of L. monocytogenes contributes to the growth of bacteria in infected cells and is therefore considered to be a virulence factor (45). Expression of plcA in L. monocytogenes is positively regulated during the growth phase by the pleiotropic transcriptional activator PrfA (33). The S. aureus PI-PLC is also a potential virulence factor. Its production is positively regulated by Agr, a pleiotropic regulator which activates expression of several staphylococcal exoproteins at the end of the exponential growth (6, 41).B. thuringiensis is known for its entomopathogenic properties which are partly due to the production of a variety of larvicidal crystal proteins designated Cry (for reviews, see references 17 and 26). When ingested by susceptible insect larvae, these crystal proteins are dissolved in the insect gut and activated. They bind to specific receptors on the surface of the midgut epithelial cells, forming transmembrane pores and causing cell lysis. This toxic effect either kills the susceptible insects or weakens them, creating favorable conditions for the germination of spores in the gut environment. Thereafter, the bacteria can invade the hemocoel from the gut and cause septicemia. The contribution of the B. thuringiensis spores to overall virulence has been demonstrated: insecticidal activity against some lepidopteran species requ...

Many virulence factors are secreted by the gram-positive, spore forming bacterium Bacillus cereus. Most of them are regulated by the transcriptional activator, PlcR, which is maximally expressed at the beginning of the stationary phase. We used a proteomic approach to study the impact of the PlcR regulon on the secreted proteins of B. cereus, by comparing the extracellular proteomes of strains ATCC 14579 and ATCC 14579 Delta plcR, in which plcR has been disrupted. Our study indicated that, quantitatively, most of the proteins secreted at the onset of the stationary phase are putative virulence factors, all of which are regulated, directly or indirectly, by PlcR. The inactivation of plcR abolished the secretion of some of these virulence factors, and strongly decreased that of others. The genes encoding proteins that are not secreted in the DeltaplcR mutant possessed a regulatory sequence, the PlcR box, upstream from their coding sequence. These proteins include collagenase, phospholipases, haemolysins, proteases and enterotoxins. Proteins for which the secretion was strongly decreased, but not abolished, in the DeltaplcR mutant did not display the PlcR box upstream from their genes. These proteins include flagellins and InhA2. InhA2 is a homologue of InhA, a Bacillus thuringiensis metalloprotease that specifically degrades antibacterial peptides. The mechanism by which PlcR affects the production of flagellins and InhA2 is not known.

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The Bacillus cereus sensu lato group contains diverse Gram-positive spore-forming bacteria that can cause gastrointestinal diseases and severe eye infections in humans. They have also been incriminated in a multitude of other severe, and frequently fatal, clinical infections, such as osteomyelitis, septicaemia, pneumonia, liver abscess and meningitis, particularly in immuno-compromised patients and preterm neonates. The pathogenic properties of this organism are mediated by the synergistic effects of a number of virulence products that promote intestinal cell destruction and/or resistance to the host immune system. This review focuses on the pore-forming haemolysins produced by B. cereus: haemolysin I (cereolysin O), haemolysin II, haemolysin III and haemolysin IV (CytK). Haemolysin I belongs to the cholesterol-dependent cytolysin (CDC) family whose best known members are listeriolysin O and perfringolysin O, produced by L. monocytogenes and C. perfringens respectively. HlyII and CytK are oligomeric ß-barrel pore-forming toxins related to the α-toxin of S. aureus or the ß-toxin of C. perfringens. The structure of haemolysin III, the least characterized haemolytic toxin from the B. cereus, group has not yet been determined.

-Bacillus thuringiensis, known as Bt, is a spore-forming bacterium that occurs naturally in soil and that produces highly specific insecticidal proteins called Cry proteins. These proteins are stomach poisons that specifically affect insects. Today, Bt preparations are considered as the most effective, specific and environmentally-friendly bioinsecticides; they have been used as biological pesticides in agriculture, forestry and in human health for the elimination of vectors of diseases for more than 60 years and their implementation far exceeds other microbial agents such as fungi, protozoa or viruses. This review on the use of this entomopathogenic bacterium in crop protection is not intended to be a compilation of the results of all the investigations made in this field. Instead, it is an attempt to provide an overview of the major trends and developments of Bt for the control of agricultural insect pests and to describe the main approaches that have been used to improve this natural bioinsecticide. Bt-based insecticides are considered safe for mammals and birds, and are safer for non-target insects than conventional insecticides; they have become the most widely used microbial insecticides. However, Bt products have several limitations, such as a narrow activity spectrum, instability in rain and sunlight, and inefficiency against pest feeding on internal tissues of the plants. The first step towards improving Bt has involved the isolation of new strains with higher and broader insecticidal activity against targeted insect pests and the cloning of cry genes encoding new insecticidal crystal proteins. A second strategy was to increase the persistence of its toxins in the field by encapsulation in recombinant asporogenic Bt strains or other heterologous recombinant microbial hosts; this protected the toxins against UV degradation and had the advantage that the transgenic microorganisms released into the environment were non-viable. Bt has also become a key source of genes for transgenic expression to provide pest resistance in plants and in so-called genetically modified plants. The engineering of plants to express Bt cry genes has been especially helpful against pests that attack parts of the plant that are usually not well protected by conventional insecticide application. The potential effects on human health and the environment of the large-scale use of these Bt crops are also in the scope of this review.

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are closely related species that share a similar genetic background but occupy different ecological niches. Virulence plasmids bearing genes coding for toxins, may explain, at least partly, this specialization. We have compared by 2-DE in the early stationary phase of growth the extracellular proteomes of three strains of these species that have lost their virulence plasmids. Proteins expected to be secreted or to belong to the cell wall or to the cytosol were found in the three proteomes. For the cell wall and cytosolic proteins located in the extracellular space, the three proteomes were similar. Cytosolic proteins included enolase, GroEL, PdhB, PdhD, SodA and others. Cell surface proteins were mainly autolysins, proteases, nucleotidases and OppAs. In contrast, the secreted proteins profiles of B. cereus and B. thuringiensis were quite different from that of B. anthracis. B. cereus and B. thuringiensis extracellular proteomes both contained large amounts of secreted degradative enzymes and toxins, including nine proteases, three phospholipases, two haemolysins and several enterotoxins. Most of the genes encoding these enzymes and toxins are controlled by the transcriptional activator PlcR. The extracellular proteome of the pXO1-, pXO2- B. anthracis 9131 strain contained only one secreted protein: the metalloprotease InhA1, also found in the proteomes of the two other strains and possibly involved in antibacterial peptide degradation.

We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters-C, T, and W-based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6°C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.