A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis

Abstract

Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.

FIG. 1

Identification of the SG-850 RAPD marker for the B. cereus group strains by RAPD fingerprinting of Bacillus strains obtained by amplifying total DNA with primer OPG-8 (A) and Southern hybridization of the RAPD patterns with the SG-850 DIG-labelled probe obtained from B. cereus 2896 (B). Lanes M, DNA size markers (EcoRI-, HindIII-digested lambda DNA, DIG labelled; Boehringer Mannheim). Lanes 1 to 22, B. cereus 31^T, 318, 336, 6127, 46321, CER4, 487, CER6, 360, 345, 351, 626, 2896, BC1, BC2, CER1, CER3, CER5, MY1, MYd, CO1, and CO2. Lanes 23 and 24, B. thuringiensis 2046^T and A1. Lane 25, B. mycoides 2048^T. Lane 26, B. cereus PO1. Lanes 27 to 39, B. sphaericus 461, B. coagulans 1^T, B. amyloliquefaciens 7^T, B. circulans 11^T, B. brevis 30^T, B. smithii 4216, B. megaterium 32^T, B. pasteurii 33^T, B. polymyxa 36^T, B. amyloliquefaciens BAM, B. subtilis 8633, B. licheniformis 14580^T, and a negative control.

FIG. 2

Nucleotide sequence of the SG-850 RAPD marker from B. cereus 336. Regions that are underlined represent the target sequences of primer OPG-8, SG-749f, or SG-749r. The recognition sites of the restriction endonuclease AluI (and those of others) are also indicated. The B. cereus (Bc) ORF366, spanning nt 404 to 773, encodes an amino acid sequence (shown below the nucleotide sequence in the single-letter code) homologous to the B. subtilis (Bs) YpuA protein. Hyphens represent residues identical in the B. cereus ORF366 and the B. subtilis YpuA protein; RBS indicates the putative ribosome binding site upstream from the ORF366.

FIG. 3

Characterization of the SG-749 fragment with the restriction enzyme AluI. (A) AluI restriction haplotypes of the SG-749 fragment found in the 61 strains of the B. cereus group listed in Table 1. Lanes M, 50-bp DNA size ladder; lane 1, SG-749 fragment; lanes A to K, AluI haplotypes (the capital letters refer to the haplotypes reported in Table 1). (B) AluI restriction pattern (haplotype K) of B. anthracis strains. Lanes M, 50-bp DNA size ladder; lanes 1 to 18, B. anthracis CEB 7700, 7702, 4229, 6602, Cepanzo, Davis TE702, 957, 227, 170, 300, 779, 832, 663, 376, 846, 256, 582, and 282; lane 19, SG-749 fragment.