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The cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about thirty clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to B. anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain must be a representative of a novel bacterial species, for which the name Bacillus cytotoxis is proposed. This strain has a reduced genome size compared to other cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical psychrotrophe characteristic to unbiased soil communities. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530 kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrotrophic strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome in the cereus group to be 3,000?b200 genes and the total proteome to be 20-25,000 genes.

Food poisoning by Bacillus cereus group strains is becoming a notable problem, especially prominent in highly developed countries. An acquired ability of food contaminating strains, some of which can grow at low temperatures, to synthesize highly active toxins, can make them a threat to human health. The three main known toxins in question are hemolysin Hbl, non-hemolytic toxin Nhe and cytotoxin CytK. A gene homologous to CytK is also present in many strains in the form of a paralogue HlyII, but it is still unknown to be the cause of food poisoning. A representative psychrotrophic strain Bwe KBAB4, able to grow at 6OC, and a very distinct and rare strain NVH391-98, isolated from a severe food poisoning case, were chosen for complete genomic sequencing. Preliminary analysis of genomic data shows that the two strains differ in their genetic makeup. NVH391-98, already known as a severe food poisoner, produces in high amounts only one toxin CytK (Lund et al, 2000; Brillard and Lereclus, 2004). The strain does not encode any other of the above toxins and its genome represents a small 4 Mb chromosome, instead of 5.2-5.6 Mb usually reported in other strains of the B. cereus group. Furthermore, the strain has lost its entire trp operon and can only grow at temperatures higher than 20OC. Because the majority of chromosomal genes greatly diverge from those of all other representatives of the group, the strain can be considered as the only known representative of a new species, which we suggest to name Bacillus cytotoxis. The second strain sequenced, Bwe KBAB4, isolated from forest soil, is a typical representative of recently recognized species - B. weihenstephanensis (Lechner et al, 1998). It contains a total of 5.6 Mb of genetic material, which includes genes to encode Nhe and Hbl, but lacks genes for CytK and HlyII. Although closely related strains are frequently reported to be food contaminants, they are in almost all cases not poisonous. However, the presence of Nhe and Hbl encoding genes suggests that such strains can also become toxic. Details of genomic project (sequence, assembly, finishing) and data analysis will be presented.

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM. doi: 10.1128/JB.01315-09

The virulence of Bacillus cereus requires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalioproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with a lacZ reporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multiple inhA mutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lacking inhA1, inhA2, and inhA3 shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases of B. cereus are important virulence factors that may allow the bacteria to counteract the host immune system. doi: 10.1128/JB.00264-09

The novel protein MclX (mother cell lysis X) in Bacillus thuringiensis subsp. kurstaki strain HD73 (B. thuringiensis HD73) was characterized in this work. MclX has no known domain and its gene deletion in HD73 resulted in Cry1Ac encapsulation in the mother cell and did not influence Cry1Ac protein production or insecticidal activity. In vitro cell wall hydrolysis experiments showed that MclX cannot hydrolyze the cell wall. In mclX deletion mutants, the expression of cwlC (which encodes a key cell wall hydrolase) was significantly decreased, as shown by the β-galactosidase activity assay. MclX cannot directly bind to the cwlC promoter, based on the electrophoretic mobility shift assay (EMSA). The cwlC was reported to be regulated by σK and GerE. However, the transcriptional activities of sigK and gerE showed no difference between HD73 and the mclX deletion mutant. It is indicated that MclX influenced cwlC expression independently of σK or GerE, through a new pathway to regulate cwlC expression. mclX deletion could be a new approach for insecticidal protein encapsulation in Bacillus thuringiensis. [ABSTRACT FROM AUTHOR]

Several approaches are used to characterize Bacillus thuringiensis FlhA mutant and elucidate an eventual relationship between motility, secretion of virulence factors and pathogenesis. The results have indicated that the transcription of many PlcR-regulated virulence factors is coordinated with the flagellar apparatus and hence, FlhA appears to be an essential virulence factor with a pleiotropic role.

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.

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 identification and characterization of two genes, yqgB and yqfZ, of unknown function are reported. The findings suggest that the yqgB and ygfZ genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host.

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.

[beta]-Exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfX-ecfY, that is essential for [beta]-exotoxin I production in B. thuringiensis subsp, thuringiensis strain 407-1. In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants. sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively. EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1([Cry.sup.-])([Pig.sup.+]), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1 ([Cry.sup.-])([Pig.sup.+]) mutant. This suggests that in the wild type-407([Cry.sup.+]) strain, [beta]-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce [beta]-exotoxin I production in B. thuringiensis in the absence of cry gene-bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that [beta]-exotoxin I production in B. thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.