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Applied and Environmental Microbiology, December 2000, p. 5460-5468, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Daniele Daffonchio,1,* Ameur Cherif,2 and Sara Borin1

Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi, 20133 Milan, Italy,1 and Laboratoire de Microbiologie, Faculté des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia2

Received 3 August 2000/Accepted 5 October 2000

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.

* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy. Phone: 39-02-23955845. Fax: 39-02-70630829. E-mail: daniele.daffonchio{at}unimi.it.

Applied and Environmental Microbiology, December 2000, p. 5460-5468, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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