Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

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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...

Section: Signature Nucleotides For the B Cereus Group Species In Itsmentioning

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

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“…On the other hand, a marked variability is always observed when large collections of strains are examined by DNA fingerprinting methods that target the whole genome (8-13, 17, 25-29, 36, 58) and/or discrete genes (15,16,38,54,55,61). Hence, the phylogenetic and taxonomic relationship among these species is still open to debate.…”

mentioning

confidence: 99%

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

Daffonchio

Chérif

Borin

2000

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142

155

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.The "Bacillus cereus group," one of the most homogeneous groups of the genus Bacillus, encompasses six validly described species, B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis, all of which have an important impact on human activity (18,39,44).These species are known to be strictly related phylogenetically, as has been shown by DNA-DNA hybridization studies (35,39,(42)(43)(44)57) and the sequencing of the ribosomal RNA genes (2-4, 39). On the other hand, a marked variability is always observed when large collections of strains are examined by DNA fingerprinting methods that target the whole genome (8-13, 17, 25-29, 36, 58) and/or discrete genes (15,16,38,54,55,61). Hence, the phylogenetic and taxonomic relationship among these species is still open to debate. It has been proposed previously that B. anthracis, B. cereus, and B. thuringiensis represent a single species, this conclusion having been reached through genome sizing and mapping (9-13) and, very recently, by multilocus enzyme electrophoresis and the sequencing of nine different DNA loci (27).The ribosomal operon is a classic molecular marker used to trace genetic relationships and to identify strains rapidly (1). Of all the different regions of the ribosomal operon, the internal transcribed spacers (ITS) between 16S and 23S ribosomal DNA are frequently used as molecular markers to identify microbial species and analyze the phylogenetic relationship between strains (see, for example, references 14, 22, and 24). ITS are generally found in multiple copies in most bacterial genomes (24, 33). Since ITS are hypervariable with respect to adjace...

“…By aligning the sequences of B. anthracis and the nearneighbor B. cereus and B. thuringiensis strains DSMZ 336, DSMZ 318, AH818, and Sam2 and the B. cereus strains AH812, AH817, and AH831 obtained for the five loci used in the MLST, signature nucleotides in SG-749, plcR, and cerA genes were identified, offering the opportunity for designing species-specific probes or primers for the rapid identification of B. anthracis, e.g., by RSI-PCR (13). By referring to the sequence coordinates of the three loci SG-749, plcR, and cerA (accession no.…”

Section: Resultsmentioning

confidence: 99%

“…The criteria used for the primer design were those reported by Daffonchio et al (13), i.e., leaving the 3Ј end nucleotide unmodified with respect to the target sequence and making a minimal number of primer-template mismatches to insert restriction sites for six base-pair-recognizing restriction enzymes that do not cut the original target sequence. Primer set AB was designed to simultaneously detect both of the SNP using the appropriate restriction enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…Primer set AB was designed to simultaneously detect both of the SNP using the appropriate restriction enzyme. a The cut primer is defined as the primer that inserts a restriction site in the target sequence during PCR and is eliminated by the following restriction digestion (13). Underlined in the primer and the target sequences are the nucleotides modified in the primer to insert the appropriate restriction site with respect to a specific SNP in the target sequence.…”

Section: Resultsmentioning

confidence: 99%

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Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Daffonchio

Raddadi

Merabishvili

et al. 2006

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Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are rather important microorganisms that interfere with or are related to human activities. B. anthracis is the active agent of anthrax disease (41), B. cereus causes food-borne disease syndromes associated with enterotoxin and emetic toxin (17, 27), and B. thuringiensis is an insect pathogen (39) currently used for the biological control of insects in crop protection.On the basis of genetic evidence, it has been proposed that B. anthracis, B. cereus, and B. thuringiensis belong to the same species (B. cereus sensu lato), but the status of separate species has been retained due to the remarkably different virulence phenotype (23). It has been hypothesized that B. anthracis derives from B. cereus-B. thuringiensis by the acquisition of the virulence pXO plasmids (29) anthracis that were absent in these closely related strains (34). In population genetics studies among a strain collection of the B. cereus group species, it was found by multilocus enzyme electrophoresis (23) and multilocus sequence typing (MLST) (24) that the strains could be divided into two main groups, the first including soil and dairy isolates and the second including strains with pathogenic potential. This second group included, besides B. anthracis, most of the strains isolated from patients in clinical environments and, among these, B. cereus strains isolated from periodontitis in humans. Helgason et al. (22,24) clearly showed that in the species B. cereus and B. thuringiensis some genetic types exist having a close relationship with B. anthracis. Very recently, a human isolate that had been identified as B. cereus on the basis of phenotypic and molecular data caused clinical symptoms similar to those of inhalation anthrax. It was shown to harbor a virulence plasmid very similar to pXO1, together with a capsule plasmid was completely different than pXO2 (25).Considering all of these data and the potential virulence shown by strains that are genetically similar to B. anthracis, approaches that allow the rapid identification of such strains are great interest to gain further insight into B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development.The most common strategies to analyze the genetic relationship between B. anthracis, B. cereus, and B. thuringiensis are based on genome fingerprinting, e.g., AFLP (29,34,40) or repetitive element polymorphism-PCR (rep-PCR) (11). Alternatively, the analysis of conserved molecular chronometers