Abstract
Bacillus anthracis plasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax. However, the extent of copy number variation within the species and how the plasmids are related to pXO1/pXO2-like plasmids in other species of the Bacillus cereus sensu lato group remain unclear. To gain new insights into these issues, we sequenced 412 B.anthracis strains representing the total phylogenetic and ecological diversity of the species. Our results revealed that B.anthracis genomes carried, on average, 3.86 and 2.29 copies of pXO1 and pXO2, respectively, and also revealed a positive linear correlation between the copy numbers of pXO1 and pXO2. No correlation between the plasmid copy number and the phylogenetic relatedness of the strains was observed. However, genomes of strains isolated from animal tissues generally maintained a higher plasmid copy number than genomes of strains from environmental sources (P < 0.05 [Welch two-sample ttest]). Comparisons against B.cereus genomes carrying complete or partial pXO1-like and pXO2-like plasmids showed that the plasmid-based phylogeny recapitulated that of the main chromosome, indicating limited plasmid horizontal transfer between or within these species. Comparisons of gene content revealed a closed pXO1 and pXO2 pangenome; e.g., plasmids encode <8 unique genes, on average, and a single large fragment deletion of pXO1 in one B.anthracis strain (2000031682) was detected. Collectively, our results provide a more complete view of the genomic diversity of B.anthracis plasmids, their copy number variation, and the virulence potential of other Bacillus species carrying pXO1/pXO2-like plasmids. IMPORTANCE Bacillus anthracis microorganisms are of historical and epidemiological importance and are among the most homogenous bacterial groups known, even though the B.anthracis genome is rich in mobile elements. Mobile elements can trigger the diversification of lineages; therefore, characterizing the extent of genomic variation in a large collection of strains is critical for a complete understanding of the diversity and evolution of the species. Here, we sequenced a large collection of B.anthracis strains (>400) that were recovered from human, animal, and environmental sources around the world. Our results confirmed the remarkable stability of gene content and synteny of the anthrax plasmids and revealed no signal of plasmid exchange between B.anthracis and pathogenic B.cereus isolates but rather predominantly vertical descent. These findings advance our understanding of the biology and pathogenomic evolution of B.anthracis and its plasmids.
Highlights
Bacillus anthracis plasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax
The major results from these studies have shown that B. anthracis isolates are highly clonal with remarkably stable genomes and low intraspecies diversity and can be placed into 1 of 12 conserved lineages defined by canonical single nucleotide polymorphisms (SNPs) (CanSNPs)
To estimate pXO1 and pXO2 copy numbers, we calculated the ratio of plasmid sequence depth to the average sequencing depth for the chromosome
Summary
Bacillus anthracis plasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax. The extent of copy number variation within the species and how the plasmids are related to pXO1/pXO2-like plasmids in other species of the Bacillus cereus sensu lato group remain unclear. Our results provide a more complete view of the genomic diversity of B. anthracis plasmids, their copy number variation, and the virulence potential of other Bacillus species carrying pXO1/pXO2-like plasmids. Previous studies have suggested that the virulence levels of B. anthracis strains carrying both plasmids can differ depending on the copy number of the plasmids [8] These results underscore the necessity to accurately quantify plasmid copy variation in a large collection of diverse B. anthracis isolates and evaluate whether plasmid copy number is a phylogenetically conserved trait. Gene transfer and deletion are important for classification since these species are typically classified based on their plasmid and virulent factor content (as opposed to phylogeny) in this group
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