IDENTIFICATION OF A REGION OF GENETIC-VARIABILITY AMONG BACILLUS-ANTHRACIS STRAINS AND RELATED SPECIES

The identification of a region of sequence variability among individua l isolates of Bacillus anthracis as well as the two closely related sp ecies, Bacillus cereus and Bacillus mycoides, has made a sequence base d approach for the rapid differentiation among members of this group p ossible, We have identified this region of sequence divergence by comp arison of arbitrarily primed (AP)-PCR ''fingerprints'' generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed, The 1,480-bp fragment deriv ed from genomic DNA of the Sterne strain of B. anthracis contained fou r consecutive repeats of CAATATCAACAA. The same fragment from the Voll um strain was identical except that two of these repeats were deleted, The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separ ated B. cereus and B. mycoides from B. anthracis in pairwise compariso ns. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kD a protein located between the carboxy terminus of an upstream ORF (des ignated orf1) and the amino terminus of a downstream ORF (designated l ytB). The sequence variation was primarily in vrrA, which was glutamin e- and proline-rich (30% of total) and contained repetitive regions, A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath prote in shp2 of the parasitic worm Litomosoides carinii.