Comparative Genomic and Phylogenomic Analyses Clarify Relationships Within and Between Bacillus cereus and Bacillus thuringiensis: Proposal for the Recognition of Two Bacillus thuringiensis Genomovars

Abstract

The present study was designed to clarify the taxonomic status of two species classified as Bacillus cereus sensu lato, namely B. cereus sensu stricto and Bacillus thuringiensis. To this end, nearly 900 whole genome sequences of strains assigned to these taxa were the subject of comparative genomic and phylogenomic analyses. A phylogenomic tree based on core gene sequences showed that the type strains of B. cereus and B. thuringiensis formed a well-supported monophyletic clade that was clearly separated from corresponding clades composed of the remaining validly published species classified as B. cereus sensu lato. However, since average nucleotide identity and digital DNA-DNA hybridization similarities between the two types of Bacillus were slightly higher than the thresholds used to distinguish between closely related species we conclude that B. cereus and B. thuringiensis should continue to be recognized as validly published species. The B. thuringiensis strains were assigned to two genomically distinct groups, we propose that these taxa be recognized as genomovars, that is, as B. thuringiensis gv. thuringiensis and B. thuringiensis gv. cytolyticus. The extensive comparative genomic data clearly show that the distribution of pesticidal genes is irregular as strains identified as B. thuringiensis were assigned to several polyphyletic groups/subclades within the B. cereus-B. thuringiensis clade. Consequently, we recommend that genomic or equivalent molecular systematic features should be used to identify B. thuringiensis strains as the presence of pesticidal genes cannot be used as a diagnostic marker for this species. Comparative taxonomic studies are needed to find phenotypic properties that can be used to distinguish between the B. thuringiensis genomovars and between them and B. cereus.

Keywords: Bacillus cereus; Bacillus thuringiensis; Bt toxin; Cry toxin; phylogenomic analysis.

FIGURE 1

Maximum-likelihood tree based on 92 single copy core gene sequences showing relationships between the type trains of B. cereus and B. thuringiensis and between them and corresponding strains of other B. cereus sensu lato species. The numbers at the nodes are bootstrap values based on 1000 replicates. The bar stands for the number of substitutions per nucleotide.

FIGURE 2

(A) Collapsed OrthoANIu dendrogram and (B) collapsed phylogenomic tree based on 92 universal bacterial genes showing relationships between the three subclades of the B. cereus–B. thuringiensis clade. Bacillus toyonensis BCT-7112^T was used as the outgroup (not shown). The scale bar on the OrthoANIu dendrogram stands for OrthoANIu values (%). Bar on the phylogenomic tree indicates the number of substitutions per nucleotide.

FIGURE 3

The bacterial core gene-based phylogenomic tree of Bacillus cereus and Bacillus thuringiensis strains together with labeled enterotoxin genes. The numbers at each branching point are bootstrap values. Bar indicates the nucleotide substitution rate per site. Bacillus toyonensis BCT-7112^T was used as the outgroup. Taxonomic groups are indicated in different colors: red, B. cereus; yellow, B. thuringiensis gv. thuringiensis; and green, B. thuringiensis gv. cytolyticus. Genes are displayed on the outer circles: EntA, enterotoxin A; EntFM, enterotoxin FM; CytK, cytotoxin K; Hbl, hemolysin BL; Nhe, non-hemolytic enterotoxin.

FIGURE 4

The bacterial core gene-based phylogenomic tree of Bacillus cereus and Bacillus thuringiensis strains together with labeled pesticidal genes. The numbers at each branching points are bootstrap values. Bar indicates the nucleotide substitution rate per site. Bacillus toyonensis BCT-7112^T was used as the outgroup. Taxonomic groups are indicated in different colors: red, B. cereus; yellow, B. thuringiensis gv. thuringiensis; and green, B. thuringiensis gv. cytolyticus. Genes are displayed on the outer circles: Cry 3D, insecticidal crystal protein with three protein domains; Cry Mtx, insecticidal crystal protein that sequentially resembles the toxin of Clostridium perfringens; Cry Bin, a conjugated insecticidal crystal protein that sequentially resembles the toxin of Lysinibacillus sphaericus; Cry6 like, Cry toxins with shorter sequence and distinct structures; Cyt, insecticidal cytotoxic protein; Vip, insecticidal vegetative protein.