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      Insight into Microevolution of Yersinia pestis by Clustered Regularly Interspaced Short Palindromic Repeats


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          Yersinia pestis, the pathogen of plague, has greatly influenced human history on a global scale. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), an element participating in immunity against phages' invasion, is composed of short repeated sequences separated by unique spacers and provides the basis of the spoligotyping technology. In the present research, three CRISPR loci were analyzed in 125 strains of Y. pestis from 26 natural plague foci of China, the former Soviet Union and Mongolia were analyzed, for validating CRISPR-based genotyping method and better understanding adaptive microevolution of Y. pestis.

          Methodology/Principal Findings

          Using PCR amplification, sequencing and online data processing, a high degree of genetic diversity was revealed in all three CRISPR elements. The distribution of spacers and their arrays in Y. pestis strains is strongly region and focus-specific, allowing the construction of a hypothetic evolutionary model of Y. pestis. This model suggests transmission route of microtus strains that encircled Takla Makan Desert and ZhunGer Basin. Starting from Tadjikistan, one branch passed through the Kunlun Mountains, and moved to the Qinghai-Tibet Plateau. Another branch went north via the Pamirs Plateau, the Tianshan Mountains, the Altai Mountains and the Inner Mongolian Plateau. Other Y. pestis lineages might be originated from certain areas along those routes.


          CRISPR can provide important information for genotyping and evolutionary research of bacteria, which will help to trace the source of outbreaks. The resulting data will make possible the development of very low cost and high-resolution assays for the systematic typing of any new isolate.

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          Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.

          Prokaryotes contain short DN repeats known as CRISPR, recognizable by the regular spacing existing between the recurring units. They represent the most widely distributed family of repeats among prokaryotic genomes suggesting a biological function. The origin of the intervening sequences, at present unknown, could provide clues about their biological activities. Here we show that CRISPR spacers derive from preexisting sequences, either chromosomal or within transmissible genetic elements such as bacteriophages and conjugative plasmids. Remarkably, these extrachromosomal elements fail to infect the specific spacer-carrier strain, implying a relationship between CRISPR and immunity against targeted DNA. Bacteriophages and conjugative plasmids are involved in prokaryotic population control, evolution, and pathogenicity. All these biological traits could be influenced by the presence of specific spacers. CRISPR loci can be visualized as mosaics of a repeated unit, separated by sequences at some time present elsewhere in the cell.
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            Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

            Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
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              CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.

              The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or 'spacers'. CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair. In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic. The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate. In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y. pestis isolates. In Y. pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event. This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized. Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage. This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained. The CRISPR structure provides a new and robust identification tool.

                Author and article information

                Role: Editor
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                9 July 2008
                : 3
                : 7
                [1 ]State Key laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing, China
                [2 ]Univ. Paris-Sud 11, CNRS, UMR8621, Institut de Génétique et Microbiologie, Orsay, France
                [3 ]State Research Center for Applied Microbiology, Obolensk, Moscow Region, Russia
                [4 ]Antiplague Research Institute of Siberia and Far East, Irkutsk, Russia
                [5 ]DGA/D4S-Mission pour la Recherche et l'Innovation Scientifique, Bagneux, France
                Centre for DNA Fingerprinting and Diagnostics, India
                Author notes

                Conceived and designed the experiments: RY YL YC YS AA GV. Performed the experiments: YL YC OG MP CP SD. Analyzed the data: RY YL YC YS AA OG YY CP XW GV. Contributed reagents/materials/analysis tools: AA MP ZG SD SB. Wrote the paper: RY YL YC GV. Other: Modified the paper: YS GV AA RY.

                Cui et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 10
                Research Article
                Biotechnology/Applied Microbiology
                Computational Biology/Evolutionary Modeling
                Evolutionary Biology/Evolutionary and Comparative Genetics
                Evolutionary Biology/Microbial Evolution and Genomics
                Genetics and Genomics/Comparative Genomics
                Microbiology/Applied Microbiology
                Microbiology/Environmental Microbiology
                Microbiology/Medical Microbiology
                Microbiology/Microbial Evolution and Genomics
                Molecular Biology/Bioinformatics
                Molecular Biology/Molecular Evolution
                Infectious Diseases/Bacterial Infections
                Public Health and Epidemiology/Infectious Diseases



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