| Abstract: | The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms.The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, as well as the more distantly related species B. mycoides and B. weihenstephanensis. These gram-positive, spore-forming bacteria form a highly homogeneous subdivision of the genus Bacillus, which also contains several other organisms belonging to the B. subtilis group. The importance and public awareness of B. cereus group organisms are associated with their distinct phenotypes and pathological effects. B. anthracis is the causative agent of anthrax, a disease that affects humans and animals worldwide and has also been developed as a biological warfare agent (17, 25). B. cereus is an opportunistic human pathogen which is responsible mainly for gastrointestinal illnesses resulting from food contamination (9), whereas B. thuringiensis is an insect pathogen whose toxin is a biological pesticide widely used in global agriculture (38). The systematics of the members of the B. cereus group poses significant challenges due to very high level of chromosomal synteny and protein identity (33). Intense efforts have focused on overcoming these challenges, and there has been a particular focus on developing methods for specific detection of B. anthracis and for differentiating among strains of these closely related organisms.Biodefense and forensic needs prompted large-scale sequencing of multiple bacillus genomes in a search for polymorphic sites for use in typing procedures (33). One type of polymorphism involves variation in the number of repeating nucleotide units that are referred to as variable-number tandem repeats (VNTRs). The resulting variation in the length and mass of the PCR products of these units can be demonstrated by gel and capillary electrophoresis (20), mass spectrometry (29), or microchannel fluidics (30). To date, several different VNTRs have been identified and tested. For example, Keim et al. studied the genetic relationship among a large collection of B. anthracis isolates based on the VNTRs found in the vrr genes (19, 20). Using a similar approach, Valjevac et al. used VNTRs of Bcms loci as markers to assess the phylogeny of members of the B. cereus group (46). Finally, length variation of the collagen-like (CL) region of the bclA gene was employed to differentiate among B. anthracis strains (6, 42).The CL sequences, which are composed of Gly-Xaa-Yaa (i.e., a glycine followed by two additional residues; GXY) repeats, have been identified in silico in more than 100 prokaryotic proteins (34). Recent studies demonstrated that some bacterial CL proteins (CLPs), such as streptococcal protein Scl and BclA, can form the collagen triple helix (4, 14, 48). Bacterial CLPs are typically surface exposed and are found in microorganisms pathogenic to humans and animals. BclA (Bacillus CLP of B. anthracis) is a major spore surface protein (41) and is found in all members of the B. cereus group (6; this study). A second CLP, designated BclB (47), was identified as a component of the B. anthracis exosporium; however, its distribution and structural properties have not been well characterized. Likewise, two closely related proteins, ExsH and ExsJ, contain GXY CL repeats and are presumably located in the exosporium of Bacillus strains (45).In this work we investigated in silico the occurrence and distribution of the bcl genes, presumably encoding CLPs, in all members of the B. cereus group. A new classification of the resulting Bcl protein variants is proposed based on the domain composition and folding of these proteins. As many as 10 bcl genes were found in a single B. cereus strain. Five genes were consistently observed in B. anthracis strains and designated bclA to bclE. We further analyzed sequence polymorphisms among these bcl genes and assessed use of them for B. anthracis detection and strain fingerprinting. Representative members of the B. cereus group and less closely related control bacilli were used to demonstrate specific bclB gene-based detection of B. anthracis spores. Finally, a combination of experiments and mathematical modeling was used to demonstrate how combined use of the bclABCDE sequence polymorphisms can be a powerful tool for strain fingerprinting in biodefense and forensic applications. |
