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

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.The "Bacillus cereus group," one of the most homogeneous groups of the genus Bacillus, encompasses six validly described species, B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis, all of which have an important impact on human activity (18,39,44).These species are known to be strictly related phylogenetically, as has been shown by DNA-DNA hybridization studies (35,39,(42)(43)(44)57) and the sequencing of the ribosomal RNA genes (2-4, 39). On the other hand, a marked variability is always observed when large collections of strains are examined by DNA fingerprinting methods that target the whole genome (8-13, 17, 25-29, 36, 58) and/or discrete genes (15,16,38,54,55,61). Hence, the phylogenetic and taxonomic relationship among these species is still open to debate. It has been proposed previously that B. anthracis, B. cereus, and B. thuringiensis represent a single species, this conclusion having been reached through genome sizing and mapping (9-13) and, very recently, by multilocus enzyme electrophoresis and the sequencing of nine different DNA loci (27).The ribosomal operon is a classic molecular marker used to trace genetic relationships and to identify strains rapidly (1). Of all the different regions of the ribosomal operon, the internal transcribed spacers (ITS) between 16S and 23S ribosomal DNA are frequently used as molecular markers to identify microbial species and analyze the phylogenetic relationship between strains (see, for example, references 14, 22, and 24). ITS are generally found in multiple copies in most bacterial genomes (24, 33). Since ITS are hypervariable with respect to adjace...

16S–23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification

“…Length polymorphism analysis of amplified ITS regions has been used as an efficient tool for species differentiation by several authors (Mora et al, 1997;Tyrrell et al, 1997;Daffonchio et al, 2000) but it was not applicable for the differentiation of several species of dairy streptococci including those analysed in this study. A single ITS amplification product of about 350 bp was detected in S. thermophilus and S. salivarius strains using a standard agarose gel electrophoresis as previously reported by Tilsala-Timisjärvi & Alatossava (1997) and by Moschetti et al (1998).…”

Atomic force microscopy of Bacillus spore surface morphology

“…Principally distinguished genetically by plasmids, it has been suggested that B. cereus, B. thuringiensis, and B. anthracis could be considered one species (Daffonchio et al, 2000;Helgason et al, 2000), or at least, are very closely related (Vilas-Boas et al, 2002). These organisms may even occupy overlapping ecological niches.…”

Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis

“…It includes B. cereus, a species often associated with food-borne disease, Bacillus thuringiensis, which is used as a biological pesticide worldwide, and Bacillus anthracis, a pathogen of warm-blooded animals that can cause the often fatal disease anthrax. Members of the B. cereus group form a highly homogeneous subdivision within the genus Bacillus and it has been proposed that B. cereus, B. thuringiensis and B. anthracis are in fact varieties of the same species (Daffonchio et al, 2000;Helgason et al, 2000). However, B. anthracis and B. thuringiensis differ from B. cereus by containing plasmid-encoded specific toxins and a capsule (B. anthracis only) (Okinaka et al, 1999;Schnepf et al, 1998) and recent studies have shown that B. anthracis is rather monomorphic, whereas there is large diversity within B. cereus and B. thuringiensis (Bavykin et al, 2004;Hill et al, 2004;Priest et al, 2004).…”