MOLECULAR EVOLUTION AND DIVERSITY IN BACILLUS-ANTHRACIS AS DETECTED BY AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS

Bacillus anthracis causes anthrax and represents one of the most molec ularly monomorphic bacteria known. We have used AFLP (amplified fragme nt length polymorphism) DNA markers to analyze 78 B. anthracis isolate s and six related Bacillus species for molecular variation. AFLP marke rs are extremely sensitive to even small sequence variation, using PCR and high-resolution electrophoresis to examine restriction fragments. Using this approach, we examined ca, 6.3% of the Bacillus genome for length mutations and ca. 0.36% for point mutations, Extensive variatio n was observed among taxa, and both cladistic and phenetic analyses we re used to construct a phylogeny of B. anthracis and its closest relat ives. This genome-wide analysis of 357 AFLP characters (polymorphic fr agments) indicates that B. cereus and B. thuringiensis are the closest taxa to B. anthracis, with B. mycoides slightly more distant. B. subt ilis, B. polymyxa, and B. stearothermophilus shared few AFLP markers w ith B. anthracis and were used as outgroups to root the analysis. In c ontrast to the variation among taxa, only rare AFLP marker variation w as observed within B. anthracis, which may be the most genetically uni form bacterial species known. However, AFLP markers did establish the presence or absence of the pXO1 and pX02 plasmids and detected 31 poly morphic chromosomal regions among the 79 B. anthracis isolates. Cluste r analysis identified two very distinct genetic lineages among the B. anthracis isolates. The level of variation and its geographic distribu tion are consistent with a historically recent African origin for this pathogenic organism. Based on AFLP marker similarity, the ongoing ant hrax epidemic in Canada and the northern United States is due to a sin gle strain introduction that has remained stable over at least 30 year s and a 1,000-mile distribution.