title: Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.
abstract: 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.
authors.affiliation: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi, 20133 Milan, Italy. daniele.daffonchio@unimi.it
authors.forename: Daniele
authors.initials: D
authors.lastname: Daffonchio
authors.forename: Ameur
authors.initials: A
authors.lastname: Cherif
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Brusetti
authors.forename: Aurora
authors.initials: A
authors.lastname: Rizzi
authors.forename: Diego
authors.initials: D
authors.lastname: Mora
authors.forename: Abdellatif
authors.initials: A
authors.lastname: Boudabous
authors.forename: Sara
authors.initials: S
authors.lastname: Borin
mesh_terms: D001407:Bacillus / Q000145:classification* / Q000235:genetics*; D003001:Cloning, Molecular; D016172:DNA Fingerprinting / Q000379:methods; D007438:Introns / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S