Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains.

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 degrees 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.

Two distinct types of rRNA operons in the Bacillus cereus group.

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.

Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis.

Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndromes. It is closely related to the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the former being used as a biological weapon and the latter as a pesticide. B. anthracis and B. thuringiensis are readily distinguished from B. cereus by the presence of plasmid-borne specific toxins (B. anthracis and B. thuringiensis) and capsule (B. anthracis). But phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus, B. anthracis and B. thuringiensis are varieties of the same species or different species. Here we report the sequencing and analysis of the type strain B. cereus ATCC 14579. The complete genome sequence of B. cereus ATCC 14579 together with the gapped genome of B. anthracis A2012 enables us to perform comparative analysis, and hence to identify the genes that are conserved between B. cereus and B. anthracis, and the genes that are unique for each species. We use the former to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers.