Abstract
Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.
Highlights
Utilizing length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) as genetic markers, aka PCR ribotyping, was developed by Kostman and colleagues [1]
Footnote: *These are uncharacterized members of the Burkholderia cepacia complex. **These ITS types were discovered in this work. doi:10.1371/journal.pone.0029323.t002
Our ITS data indicate the existence of three major ITS types of B. pseudomallei
Summary
Utilizing length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) as genetic markers, aka PCR ribotyping, was developed by Kostman and colleagues [1]. This technique was first used as an alternative method to traditional 16S ribosomal DNA (rDNA) ribotyping for detecting epidemiological differences between Burkholderia cepacia strains [1]. The technique involves PCR of the 16S-23S internal transcribed spacer (ITS), aka the 16S-23S intergenic spacer region, and assigns phylogenetic groupings based on length polymorphisms, sequence differences or restriction patterns in the ITS [2,3,4]. High intraand interspecies homology of conserved blocks has led to the conclusion that the primary source of ITS variation is homologous recombination [9,13]
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