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Applied and Environmental Microbiology, September 2003, p. 5128-5137, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5128-5137.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of Bacillus and Related Genera

Daniele Daffonchio,1* Ameur Cherif,1,2 Lorenzo Brusetti,1 Aurora Rizzi,1 Diego Mora,1 Abdellatif Boudabous,2 and Sara Borin1

Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi, 20133 Milan, Italy,1 Laboratoire de Microbiologie, Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia2

Received 11 June 2003/ Accepted 19 June 2003

The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus. We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.

* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy. Phone: 39-0250316730. Fax: 39-0250316694. E-mail: daniele.daffonchio{at}unimi.it.

Applied and Environmental Microbiology, September 2003, p. 5128-5137, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5128-5137.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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