Prior to the present study, detailed sequence analysis of large B. cereus
group plasmids was mostly limited to those of B. anthracis
and of a few environmental isolates of B. cereus
or B. thuringiensis
). The present study identifies and compares the primary sequence of pXO1-like plasmids harbored by two periodontal and an emetic B. cereus
with the goal of determining the role of plasmid-encoded genes in virulence.
The pXO1-like plasmids range in size from ca. 181 to 272 kb and, interestingly, pXO1, is the smallest. This suggests that pXO1 has undergone or is undergoing gene loss, retaining factors that mainly are required for pathogenesis and plasmid maintenance. The pPER272 and pCER270 plasmids are the largest pXO1-like plasmids, ~272 and ~270 kb, respectively, potentially reflecting adaptation to multiple ecological niches in addition to encoding potential virulence factors. We sequenced two plasmids from periodontal isolates and demonstrated that these ~272-kb plasmids were identical except for a single nucleotide (see Fig. S1 in the supplemental material). These two isolates were from obtained from distinct geographic and temporal locations, and yet they harbor essentially the same plasmid. The size of the pCER270 was previously predicted to be ~208 kb by using pulsed-field gel electrophoresis and hybridization methods (12
), whereas our sequencing effort revealed a larger plasmid of 270 kb. Both of these novel plasmids exhibit a mosaic structure and contain sets of genes previously found on the chromosomes of B. cereus
group members (16
). This observation suggests that these large plasmids of the B. cereus
group, like other bacterial plasmids, can act as the vehicle for the exchange of genetic information.
Analysis of the copy number of the pXO1-like plasmids indicates that they are maintained at one to three copies per chromosome (Table ). This value is significantly different from a report by Coker et al., who estimated that the copy number of B. anthracis
pXO1 ranged from 13 to 40 copies per chromosome and that a higher copy number leads to increased virulence (9
). It must be noted that different methodologies were used to calculate the plasmid copy number; we used the depth of clone coverage from random shotgun sequencing, whereas Coker et al. used a single-locus quantitative PCR method. A large number of factors could have resulted in such disparate results, including growth time, media, and growth conditions, all of which were variable between the present study and the study by Coker et al. (9
). In each case, the plasmid copy number calculation, no matter how it is measured, is a narrow snapshot of the dynamic process of plasmid replication and maintenance and as such can vary from isolate to isolate.
It has been demonstrated that the chromosomes of members of B. cereus
group are very similar, both in sequence similarity and synteny (19
), and that plasmids can play a significant adaptive role in the pathogen host range, virulence, and ecology of the isolate (16
). The complete sequence of multiple pXO1-like plasmids enabled comparative studies to identify regions of similarity and divergence potentially responsible for the observed phenotypes (virulence and nutritional adaptation). Analysis of the pXO1-like plasmids resulted in the identification of unique virulence-associated regions in pCER270. Based on the nucleotide similarity of the conserved core region and the BLAST score ratio analysis, pCER270 is the most distantly related plasmid to pXO1 and contains the greatest proportion of unique DNA (see Table S6 in the supplemental material). The unique regions of pCER270 contain the cereulide synthesis gene cluster previously described (12
), in addition to a region shared with pPER272 and the chromosomes of spore-forming bacilli. These conserved genes include sporulation and germination genes, as well as a formaldehyde detoxification locus (17
). The presence of these genes on pCER270 and pPER272 suggests that there is genetic exchange between the chromosomes and plasmids of the B. cereus
group. When the complete chromosome sequences of the strains containing pCER270 or pPER272 become available, it will be interesting to look for the presence or absence of chromosomal homologs for these genes. One can speculate that if these genes are only present on the plasmids, they might represent a mechanism to guarantee plasmid maintenance. However, it is still unclear whether these plasmid-encoded chromosomal genes provide a competitive advantage in virulence, metabolic, or environmental adaptation for these isolates.
Examination of pPER272 did not result in the conclusive identification of classical virulence factors. It is still debatable as to what role the plasmids play in periodontal disease, but a large pXO1-like plasmid is more often associated with periodontal B. cereus
isolates than environmental isolates, suggesting a possible connection (Table ) (18
). Plasmid-encoded genes were identified that may be associated with the periodontal phenotype by comparing pPER272 with pXO1-like plasmids from clinical and environmental B. cereus
isolates. For example, pPER272 contains a region previously described in pBC10987 that replaced pXO1-PI (33
). This region encodes an MIP channel homolog and other membrane proteins that may allow interaction with epithelial cells in the oral cavity. Adjacent to this shared region, pPER272 contained a unique additional 90 kb of sequence not found in pXO1 (Fig. and ). It would be difficult to determine whether pBC10987 has lost the pPER272 unique region over time in the environment or whether pPER272 acquired the additional region to adapt to other ecological niches. Alternatively, the shared pPER272/pBC10987 region may encode factors associated with periodontal virulence, and the categorization of pBC10987 as an environmental plasmid is misleading. Screening B. cereus
isolates for pXO1-like plasmid regions supports the latter hypothesis (Table ), since no periodontal B. cereus
isolates outside the ones carrying pPER272 contained the pPER272-specific sequence. In contrast, 15 of the 22 clinical isolates contain the pBC10987 region, a region shared with pPER272 (Table ). Further molecular analysis is required to determine whether any virulence determinants are encoded on pPER272 and/or pBC10987.
The pXO1-like plasmids have a highly conserved region that contains the putative origin and a replication initiation protein (42
) (Fig. ). RepX, a pXO1 protein required for initiation of plasmid replication is highly conserved among the pXO1-like plasmids (Fig. ). This observation suggests that these plasmids might have evolved from a common ancestral plasmid, forming a unique plasmid family and a distinct incompatibility group. Comparison of the RepX sequences revealed a high level of identity with only eight variable amino acid positions, further suggesting that the replication mechanism is highly conserved among the pXO1-like plasmids. Based on the conserved core coding sequences in this region, pBC10987 is more closely related to pXO1 (and pBCXO1) than the pathogenic B. cereus
plasmids pPER272 and pCER270 (Fig. ). Additional analysis using BLAST score ratio revealed that pBC10987, pPER272, and pCER270 share an extended core region that is not shared with pXO1 (Fig. ), suggesting that pXO1 may have either evolved further or may represent a more ancestral form of the plasmid. In either case, pXO1 is more distantly related to the other pXO1-like plasmids.
It has been previously demonstrated that B. cereus
isolates from clinical presentations form clonal groups (1
). The plasmids may play a major role in this observed clonality, even though chromosomal markers have exclusively been used to determine the level of clonality. Clonal radiation can be initiated by events such as genomic insertion, deletion, or acquisition of plasmids and represents an increased level of fitness over the rest of the population. One can envision that a B. cereus
isolate having acquired a pXO1-like plasmid may then contain a unique combination of plasmid and chromosomal factors, allowing successful exploitation of an environmental niche. Examples of highly successful B. cereus
group clones are (i) B. anthracis
that infects mammals (27
) and (ii) B. thuringiensis
that specifically infects lepidopteran worms (36
). It is tempting to speculate by analogy that pCER270 in emetic-toxin-producing isolates or pPER272 in periodontal isolates are solely responsible for the observed virulence and success of these clones. Emetic B. cereus
isolates, almost all of which have been shown to contain pCER270 and which belong to the same sequence type 26 group, most likely represent an example of a successful clone (1
). However, there must also be additional unidentified mechanisms of virulence since not all periodontal or emetic strains have been shown to contain pXO1-like plasmids.
The present study reveals that pXO1-like plasmids vary in size and copy number and are widespread throughout the B. cereus group. The pXO1-like plasmids in combination with the chromosome appear to form identifiable subgroups of B. cereus that can be associated with certain disease presentations. Although a conserved pXO1-like plasmid core has been identified, each plasmid studied contains an additional set of unique genes. Some genes have a readily identifiable role in virulence, such as the emetic toxin biosynthetic genes, whereas others cannot be linked to pathogenicity solely through sequence analysis, as is the case with pPER272. In addition, we suggest that the pXO1-like plasmids have coevolved with the chromosome to further improve pathogenesis and/or niche adaptation. Much like B. anthracis is a specialized clonal pathogen, it appears that pathogenic B. cereus may harbor specialized plasmids associated with its clinical and metabolic phenotypes.