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

Abstract

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.

FIG. 1.

UPGMA-based dendrogram and BURST-based clonal complexes of the allelic profiles of the 134 strains used in this study. Each strain name is followed by the strain origin marker (consisting of the letters Bt, Bc, Ba, or Bw [phenotypically defined B. thuringiensis, B. cereus, B. anthracis, or B. weihenstephanensis, respectively], or BtF or BtS [strains from the Versailles Collection originating from deep in the forest or the forest edge], and a number indicating the soil sample [33]), the sequence-based strain cluster designation, the cold-growth phenotype (M, P, I, or N [see Materials and Methods for an explanation]), the ST, and the corresponding allelic profile. The allelic profile, in parentheses, contains arbitrary allelic numbers for the clpC, dinB, gdpD, panC, purF, and yhfL loci, respectively. Most of the strains fall into clonal complexes (or groups, designated G1, G2, etc.) of closely related STs, defined as groups of STs in which every ST shares at least four of six identical alleles with at least one other ST in the group. Singletons are STs not belonging to the clonal complexes. Groups marked with asterisks have ancestral allelic profiles identified by START. Relationships between single-locus variants (red), double-locus variants (blue), and the ancestor are indicated by concentric circles or connecting lines of the corresponding color, generated by START (18). (A, B, and C) Allelic profile trees for the T, W, and C clusters, respectively. Green vertical bars indicate clonal complexes of psychrotrophic strains.

FIG. 1.

UPGMA-based dendrogram and BURST-based clonal complexes of the allelic profiles of the 134 strains used in this study. Each strain name is followed by the strain origin marker (consisting of the letters Bt, Bc, Ba, or Bw [phenotypically defined B. thuringiensis, B. cereus, B. anthracis, or B. weihenstephanensis, respectively], or BtF or BtS [strains from the Versailles Collection originating from deep in the forest or the forest edge], and a number indicating the soil sample [33]), the sequence-based strain cluster designation, the cold-growth phenotype (M, P, I, or N [see Materials and Methods for an explanation]), the ST, and the corresponding allelic profile. The allelic profile, in parentheses, contains arbitrary allelic numbers for the clpC, dinB, gdpD, panC, purF, and yhfL loci, respectively. Most of the strains fall into clonal complexes (or groups, designated G1, G2, etc.) of closely related STs, defined as groups of STs in which every ST shares at least four of six identical alleles with at least one other ST in the group. Singletons are STs not belonging to the clonal complexes. Groups marked with asterisks have ancestral allelic profiles identified by START. Relationships between single-locus variants (red), double-locus variants (blue), and the ancestor are indicated by concentric circles or connecting lines of the corresponding color, generated by START (18). (A, B, and C) Allelic profile trees for the T, W, and C clusters, respectively. Green vertical bars indicate clonal complexes of psychrotrophic strains.

FIG. 2.

Concatenated sequence-based phylogenetic tree. Three clusters containing most of the strains are designated by the letters C, T, and W. Each strain name is followed by the ST number and the number of identical strains corresponding to this ST. Approximate locations of clonal clusters (or groups) are indicated, including G1, G2, and G8, corresponding to the lineages Tolworthi, Sotto, and Kurstaki identified by Priest et al. (26). The small cluster X, containing strains KBAE4, KBBC4, KBCF5, and KBCC8, is not labeled. Bc10987, B. cereus 10987; BAames, B. anthracis Ames.