16S–23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus

Daffonchio, Daniele

doi:10.1016/s0378-1097(98)00168-2

Cited by 25 publications

(30 citation statements)

References 15 publications

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“…The strains used in this study (Table 1) (14) were grown routinely, as previously described (6,11,13), and the DNA suitable for amplification was obtained through lysis by boiling (15) or by sodium dodecyl sulfate-proteinase K treatment (44), as already described (2,6,11,13). The ITS homoduplex-heteroduplex polymorphism profiles were obtained by PCR followed by electrophoresis in MDE gel (BioWhittaker Molecular Applications, Milan, Italy) and silver staining using the procedures described previously (14).…”

Section: Methodsmentioning

confidence: 99%

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

Chérif

Borin

Rizzi

et al. 2003

Appl Environ Microbiol

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Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified (45) and is widely used for the biological control of insects in crop protection; B. mycoides has been recognized as a plant growth-promoting bacterium associated with conifer roots (38); B. weihenstephanensis, a psychrotolerant species frequently found in pasteurized milk, is a potential cause of spoilage problems (32). The six species are not easily distinguished on the basis of phenotypic or genetic traits (48). Recently, B. anthracis, B. cereus, and B. thuringiensis were found to be very closely related, and it has been proposed that they belong to a single species (24,33). This proposal has been based on multilocus enzyme electrophoresis data and sequencing of discrete genetic loci (24) and on the presence of an S-layer on the cell surface (33). The model considering B. anthracis, B. cereus, and B. thuringiensis as subspecies of a phylogenetically monomorphic group, differing mainly in characters linked to mobile genetic elements such as plasmids, is supported by very high sequence homology in the conserved molecular chronometers of the ribosomal operons, the 16S and 23S ribosomal DNA (rDNA), and the short intergenic spacer between them (3-5, 7, 21, 30). Considering the dangerousness of B. anthracis and the wide in-field application of B. thuringiensis as a biological insecticide, it would be opportune to further evaluate the phylogenetic relationship between the different clades of the B. cereus group. Whole-genome sequencebased analysis could give a definitive view of the genetic relationship between these species (29). However, an approach that is economically feasible, given current technology, is possible for few strains in a given species (42). Hence, for phylogenetic surveys based on a relatively large number of isolates of each species, permitting an assessment of the amount of variability and overlap within a species, the best means of approach remains the use of highly conserved molecules with no, or a low, horizontal gene transfer rate such as the ribosomal operon.In the prokaryote genome, the ribosomal operon can be present in multiple copies, up to 15 copies in Clostridium paradoxum (41). The 16S-23S rDNA intergenic transcribed spacers (ITS) are the most variable regions of the ribosomal operon, and, apart from interoperonic nucleotide substitutions, insertions, and deletions, such ITS can be differentiated, given the presence of the different numbers and types of tRNA genes (9,10,31,49). Since the ITS have fewer functional constraints than the adjacent ribosomal genes, which undergo concerted evolution (17-19), their sequences can contain traces of ribosomal operon rearrangements and species-specific or even strain-specific traits that are useful for strain typing.An analysis of ITS homoduplex-heteroduplex polymorphisms has shown that wide variability exists in the strains of the six species of the B. cereus group (14), indicating widely different length and sequence polymorphisms among the 8 t...

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Section: Methodsmentioning

confidence: 99%

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

Chérif

Borin

Rizzi

et al. 2003

Appl Environ Microbiol

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“…The discriminatory power of the ITS -PCR method can be improved by digestion of PCR products, and analysis of restriction fragment length polymorphism (PCR -RFLP) after agarose or polyacrylamide electrophoresis. The differences in sequences of the amplified fragments of intergenic spacer regions of the same length can be alternatively analyzed by using the single sequence conformation polymorphism (SSCP) (Daffonchio et al, 1998).…”

Section: Fig 1 Organization Of Bacterial Rrna Operonmentioning

confidence: 99%

Genotyping Techniques for Determining the Diversity of Microorganisms

Wolska¹,

Szweda²

2012

Genetic Diversity in Microorganisms

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“…Amplifications were performed in a Mini Cycler System (MJ Research INC, USA). DNA primers used in the experiments were, forward, 5¢-AGAGTTTGATCCTGGCTCAG-3¢ (Mora et al 1998) and reverse, 5¢-CAAGGCATCCACCGT-3¢ (Jensen et al 1993). The forward primer is complementary to the upstream of 16S rDNA of E. coli K12 strain (Accession number: AE000452, nucleotides from 1 to 20), and the reverse is complementary to the upstream sequences of 23S rDNA of E. coli 278710 (Accession number: AJ278710, nucleotides from 18 to 32).…”

Section: Amplification Of the 16s Rdna -Its Regionmentioning

confidence: 99%

“…ITS is generally found in multiple copies in most bacterial genomes. Since ITS are hypervariable with respect to adjacent genes, they can differentiate between multiple operons in the cell (Daffonchio et al 1998). The size of the spacer has been found to differ considerably among different species, and even among the different operons within a single cell (Garcia-Martinez et al 1999).…”

Section: Introductionmentioning

confidence: 99%

RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

Yavuz

Güneş

Bulut

et al. 2004

World Journal of Microbiology and Biotechnology

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SummaryThe 16S-ITS (internal transcribed spacer) region of the rrn operon was amplified by polymerase chain reaction (PCR). The amplification products were analysed by restriction fragment length polymorphism (RFLP) using a set of restriction enzymes, AluI, HaeIII, and TaqI. Restriction pattern analyses revealed that TaqI restriction enzyme could clearly differentiate the nine reference strains of Lactobacillus used in the study.

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16S–23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus

Cited by 25 publications

References 15 publications

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

Genotyping Techniques for Determining the Diversity of Microorganisms

RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

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