Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence

Abstract

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

FIG. 1

MEE analysis. Genetic relationships between 239 strains of B. cereus, B. thuringiensis, and B. anthracis. The dendrogram was generated by the average-linkage method of clustering (unweighted-pair group matrix analysis) (19), from a matrix of genetic-distance coefficients based on 13 enzyme loci, using the Molecular Evolutionary Genetics Analysis package (12). The dendrogram generates two main clusters, I and II, with a genetic distance of 0.65. Isolates were placed on the same branch when the genetic distance was less than 0.1. Sources of the isolated strains are indicated with the following symbols: red triangles, patients (B. cereus); black circles, soil samples (B. cereus and B. thuringiensis); blue boxes, dairies (B. cereus and B. thuringiensis); yellow circles, B. anthracis; green triangles, Ba813-positive B. cereus strains isolated from B. anthracis outbreak areas; star 1, B. cereus ATCC 4342; star 2, B. thuringiensis subsp. thuringiensis (HD2); star 3, B. cereus ATCC 10987; star 4, B. thuringiensis subsp. kurstaki (HD1); star 5, B. thuringiensis subsp. subtoxicus (HD109); star 6, B. thuringiensis subsp. entomocidus (HD9); star 7, B. cereus ATCC 14579. Arrows indicate strains analyzed for the results shown in Fig. 2. Arrow a, B. cereus periodontitis strain; arrow b, B. anthracis 7700; arrow c, B. cereus ATCC 10987; arrow d, B. thuringiensis subsp. kurstaki (HD1); arrow e, B. cereus type strain ATCC 14579.

FIG. 2

Sequence analysis of genes. (a) Locations of genes used for the sequence analysis on a physical map (NotI restriction fragments) of the B. cereus ATCC 10987 chromosome (14). (b) Single-gene dendrograms based on DNA sequences from nine genes (sizes of sequences in base pairs are in parentheses): fumA (354), cmk (271), ykvW (415), mbl (568), glpT (309), ansB (414), pycA (437), purH (336), and ymcB (299) from B. cereus ATCC 10987, B. anthracis 7700, B. cereus periodontitis strain, B. thuringiensis subsp. kurstaki (HD1), and B. cereus type strain ATCC 14579. (c) Dendrogram based on DNA sequences from seven genes, cmk, ymcB, ykvW, mbl, glpT, ansB, and purH, including homologous gene sequences from B. subtilis 168 forming an outgroup in the analysis. Neither the fumA nor pycA gene was included in this dendrogram, since no fumA sequence homolog exists in B. subtilis 168 and pycA was not amplified from B. thuringiensis subsp. kurstaki. All dendrograms were constructed with the Molecular Evolutionary Genetics Analysis package (12) and show proportional divergence between strains by the unweighted-pair group matrix analysis (19).