Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “ Bacillus cereus Group”

Abstract

ABSTRACT Bacillus anthracis , Bacillus cereus , Bacillus mycoides , Bacillus pseudomycoides , Bacillus thuringiensis , and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “ B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed 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. pseudomycoides . The second included B. cereus and B. thuringiensis , B. anthracis appeared as a lineage of B. cereus .