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Applied and Environmental Microbiology, February 2004, p. 1068-1080, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.1068-1080.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

Karen K. Hill,1 Lawrence O. Ticknor,2 Richard T. Okinaka,1 Michelle Asay,1 Heather Blair,1 Katherine A. Bliss,1 Mariam Laker,1 Paige E. Pardington,1 Amber P. Richardson,1 Melinda Tonks,1 Douglas J. Beecher,3 John D. Kemp,4 Anne-Brit Kolstø,5 Amy C. Lee Wong,6 Paul Keim,7 and Paul J. Jackson1*

Bioscience Division,1 Decision Applications Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545,2 FBI Academy, Quantico, Virginia 22135,3 Department of Plant Pathology, Entomology, and Weed Science, New Mexico State University, Las Cruces, New Mexico 88003,4 Institute of Pharmacy, University of Oslo, Oslo, Norway,5 Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706,6 Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 860117

Received 18 June 2003/ Accepted 24 October 2003

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.

* Corresponding author. Mailing address: Los Alamos National Laboratory, Bioscience Division, Mail Stop M888, Los Alamos, NM 87545. Phone: (505) 667-2775. Fax: (505) 665-3024. E-mail: pjjackson{at}lanl.gov.

Applied and Environmental Microbiology, February 2004, p. 1068-1080, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.1068-1080.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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