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. 2009 Nov;75(22):7163-72.
doi: 10.1128/AEM.01069-09. Epub 2009 Sep 18.

Identification and classification of bcl genes and proteins of Bacillus cereus group organisms and their application in Bacillus anthracis detection and fingerprinting

Tomasz A Leski  1 , Clayton C Caswell, Marcin Pawlowski, David J Klinke, Janusz M Bujnicki, Sean J Hart, Slawomir Lukomski
Affiliations

Identification and classification of bcl genes and proteins of Bacillus cereus group organisms and their application in Bacillus anthracis detection and fingerprinting

Tomasz A Leski et al. Appl Environ Microbiol. 2009 Nov.

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.

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Figures

FIG. 1.
FIG. 1.
Two-dimensional projection of CLANS. Clustering results were obtained for the noncollagen regions of Bcl proteins. Connections between points represent the degree of pairwise sequence similarity, as quantified by BLAST P values (the darker the line, the higher the level of similarity).
FIG. 2.
FIG. 2.
Identification, detection, and characterization of the bcl genes and Bcl proteins of B. anthracis. Five genes designated bclA to bclE were identified in the genomes of B. anthracis strains. (A) Graphic representation of the B. anthracis strain Sterne chromosome showing the locations and orientations of the bclABCDE genes. Basic characteristics are shown in the table. (B) Detection of the bclABCDE genes in the genome of B. anthracis Sterne. The bclABCDE genes were PCR amplified, and resulting products were analyzed in a 2% agarose gel. Lanes M, 2-log DNA ladder. (C) Schematic representation (not to scale) and characterization of the BclABCDE proteins. The data are based on sequences obtained from the genomes of eight B. anthracis strains (Sterne, Ames, Australia 94, CNEVA-9066, A1055, Vollum, USA 6153, and Kruger). Unless indicated otherwise, Bcl protein regions were arbitrarily designated the N region (or amino-terminal region), L region, CL region, and CTD (or carboxyl-terminal region) (4). The table shows the ranges of the numbers of amino acids in the different regions of the BclABCDE proteins.
FIG. 3.
FIG. 3.
Specific detection of B. anthracis by PCR of the bclB gene. (A) Schematic representation (not to scale) of two main BclB variants of clan 2a and 2b proteins. Primers bclB F2 and bclB R4 (Table 2) were designed to differentiate between the bclB allele present in B. anthracis strains from the alleles present in other members of the B. cereus group, which are detected with primers bclB F3 and bclB R5. (B) PCR amplification using B. anthracis-specific primers. PCR products were analyzed by 2% agarose gel electrophoresis. The DNA templates used for PCR were genomic DNAs from B. anthracis (Ba) Sterne and Ames; B. cereus (Bc) strains ATCC 14579, ATCC 4342, and ATCC 13061; B. thuringiensis (Bt) strains ATCC 33679 and ATCC 33680; B. subtilis (Bs) strains ATCC 6051, ATCC 21332, and ATCC 31028; B. mycoides (Bmy) strain ATCC 6462; and B. megaterium (Bme) strain ATCC 14581. Lane M, 2-log DNA ladder.
FIG. 4.
FIG. 4.
Amplification of the bclB gene using Bacillus sp. spores. Spores were prepared from B. anthracis Sterne (Ba), B. cereus ATCC 13061 (Bc), B. thuringiensis ATCC 33679 (Bt), and B. mycoides ATCC 6462 (Bm). Specific detection of B. anthracis spores (∼104 spores per reaction) by PCR was performed with B. anthracis-specific bclB primers bclB F2 and bclB R4 (upper panel). A control positive PCR amplification was performed with primers bclB F3 and bclB R5, which amplify the bclB gene from the non-B. anthracis Bacillus species B. cereus, B. thuringiensis, and B. mycoides (lower panel). PCR products were analyzed by 2% agarose gel electrophoresis. Lane M, 2-log DNA ladder.
FIG. 5.
FIG. 5.
bcl-based fingerprinting of B. anthracis strains. Fragments of individual bclABCDE genes from B. anthracis strains Sterne and Ames were PCR amplified using primers flanking the CL regions (left panel). Expected sizes of PCR products are indicated below the gel. The individual PCR samples of bclABCDE for either strain Sterne or Ames were combined, and the sets were each loaded into single wells (middle panel). The solid squares indicate PCR bands in the gels. Multiplex PCR was performed with combined bclB primers using DNA from strain Sterne as the template (right panel). All PCR products were analyzed by 2% agarose gel electrophoresis. Lane M, 2-log DNA ladder.
FIG. 6.
FIG. 6.
Mathematical modeling of bclABCDE-based fingerprinting of B. anthracis strains. (A) Multidimensional scaling representation of the similarity among the bootstrap replicates of eight B. anthracis strains based on multivariate PCR measurement of bclABCDE gene fragments. (B) Dendrogram with confidence levels associated with correct classification of the eight B. anthracis strains.
FIG. 7.
FIG. 7.
bcl-based fingerprinting of non-B. anthracis Bacillus species. PCR amplification of the bclABCDE genes was performed using DNA templates from the following strains: B. cereus (Bc) strains ATCC 14579, ATCC 4342, and ATCC 13061; B. thuringiensis (Bt) ATCC 33679; and B. mycoides (Bm) ATCC 6462. Individually amplified bclABCDE gene products were combined and analyzed by 2% agarose gel electrophoresis. The estimated products sizes are indicated below the gel. Some primer pairs did not yield a detectable DNA product (ND). Lane M, 2-log DNA ladder.
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