Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the "Bacillus cereus group"

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomyco ides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely r elated in phenotype and genotype, and their genetic relationship is still o pen to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to descr ibe the genetic relationships within the "B. cereus group," advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacryl amide gel electrophoresis and silver staining. One hundred forty-one strain s belonging to the six species were investigated, and 73 ITS-HHP pattern ty pes were distinguished by MDE, a polyacrylamide matrix specifically designe d to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. ps eudomycoides, The second included B. cereus and B. thuringiensis, B. anthra cis appeared as a lineage of B. cereus.