Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1

Abstract

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

FIG. 1.

Similarity of newly identified plasmids to B. anthracis pXO1 and other pXO1-like plasmids. For panel A, a BLAST score ratio analysis was performed using the proteome of B. anthracis pXO1 as reference. Panels B and C use pPER272 and pCER270, respectively, as the reference. Each block represents an individual reference plasmid CDS compared to the most similar CDS in the query plasmid. The names of the query plasmids are indicated at the top of each panel. A green block indicates a highly conserved CDS (BSR > 0.8) on the reference and query plasmids. A red box indicates a CDS that is not conserved between the reference and query plasmids (BSR < 0.4). Black boxes indicate that the two CDS share some similarity but are divergent (0.4 < BSR < 0.8). “Rep” in panels A to C indicates the location of the putative replication machinery. Regions with similar or distinct features are highlighted to the left of each panel.

FIG. 2.

Conserved replication protein and initiation of replication region. (A) Peptide alignment of RepX (pXO1-45) from pXO1-like plasmids. Black boxes and asterisks indicate the locations of amino acid substitutions. None of the differences are within the active sites predicted by Tinsley and Khan (42). Panel B, The putative origin of replication of the pXO1-like plasmids as identified by Tinsley et al. (42). The alignment was constructed by using CLUSTAL W (40) and displayed with Boxshade (http://bioweb.pasteur.fr/docs/softgen.html#BOXSHADE). The gray boxes and arrows represent the palindromic sequences putatively thought to facilitate the initiation of replication. The nucleotides in the black boxes indicate differences among the compared putative plasmid origins. Notably, the palindrome is exact in only the B. anthracis Sterne pXO1 plasmid, whereas all other sequenced plasmids have differences in the palindromic region.

FIG. 3.

Phylogenetic analysis based on the conserved core region of the pXO1-like plasmids. A neighbor-joining tree was constructed by using the conserved core nucleotide sequences of 40 conserved genes. The tree shows that, based on this region, pBC10987 is more similar to pXO1 than either of the pathogenesis related plasmids, pCER270 or pPER272.

FIG. 4.

BLAST score ratio-based synteny plots of the novel plasmids with B. anthracis pXO1 demonstrate a lack of conservation of the pXO1 pathogenicity island. (A) Comparison of pXO1 and pPER272. (B) Comparison of pXO1 and pCER270. The gray box in each panel indicates the location of the pXO1-pathogenicity island, which is absent in each of the plasmids analyzed. Each point on the figure represents an individual peptide in pXO1 compared to the proteome of the novel plasmid. The associated color of each point indicates the level of similarity between the compared peptides.

FIG. 5.

BSR-based synteny plots of the novel plasmids with other pXO1-like plasmids identifying regions of similarity and dissimilarity. (A to C) pPER272. Gray boxes indicate regions unique to pPER272. The red box in panel B identifies a region in the pPER272 plasmids that is shared with pBC10987, both in gene content and synteny. (D to F) All comparisons are to pCER270. The red box in all three panels illustrates the location of the cereulide biosynthetic gene cluster. The gray boxes indicate unique regions to pCER270.

FIG. 6.

Unique regions from each of the B. cereus group plasmids analyzed. Genes are colored according to function: red, toxin or virulence related; green, sporulation and germination; blue, transposon or mobile element related; orange, regulation; pink, membrane associated; brown, proteases; black with annotation, genes for which no functional class could be assigned; and black with no annotation, hypothetical and conserved hypothetical proteins.