1 GPMS, Institut de Génétique et Microbiologie, Université Paris XI, 91405 Orsay cedex, France
2 Centre d'Etudes du Bouchet, 5 rue Lavoisier, 91710 Vert le Petit, France
Correspondence
G. Vergnaud
Gilles.Vergnaud{at}igmors.u-psud.fr
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ABSTRACT |
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INTRODUCTION |
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In Yersinia pestis, Jansen et al. (2002) reported the existence of three CRISPR elements with the same repeat sequence. Y. pestis strains are classified into three biovars according to their ability to reduce nitrate and to ferment glycerol (Devignat, 1951
). Since Y. pestis was first linked to plague by Yersin (1894)
, strains of biovar Antiqua have been generally isolated from Asia and Africa; Medievalis was found in Central Asia, and Orientalis worldwide. Pestoides are particular strains isolated in Central Asia, and have never been found associated with disease in humans (Anisimov et al., 2004
). The complete genomic sequences of Y. pestis CO92 (Parkhill et al., 2001
), biovar Orientalis, and of strain KIM (Deng et al., 2002
), biovar Medievalis, have been determined, as well as the sequence of a so-called microtus' strain (Zhou et al., 2004
). The deficiency of glycerol fermentation in CO92 was found to result from a microdeletion in the glpD gene, and all Orientalis strains investigated so far (Motin et al., 2002
; Pourcel et al., 2004
) have been shown to harbour the same defect. This confirms the initial proposition that the Orientalis phenotype is derived from the Antiqua phenotype. In contrast, the Medievalis phenotype has been associated with different mutation events in the napA gene (Pourcel et al., 2004
).
In the present work, the three CRISPR elements were analysed in a collection of Y. pestis strains from three biovars and different geographical origins and in nine Yersinia pseudotuberculosis strains. Members of the species Y. pseudotuberculosis, from which Y. pestis is thought to have recently evolved some 1500 to 20 000 years ago (Achtman et al., 1999), also possess the three CRISPRs. A total of 109 CRISPR alleles (mostly of the most polymorphic locus, CRISPR YP1) were sequenced. We describe a collection of new spacers, some of them probably recently acquired by Y. pestis strains in a clearly polarized fashion. The majority of these spacers correspond to fragments of a prophage.
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METHODS |
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For sequencing, a 60 µl PCR reaction was performed. Amplicon quality was checked on a 2 % agarose gel. Then the product was purified by PEG precipitation as described by Embley (1991). Sequencing was performed by MWG Biotech (Germany).
Sequence similarity analyses were performed using the NCBI BLAST with microbial genomes database (http://www.ncbi.nlm.nih.gov) and GenBank.
Data management.
The data produced were stored using the BioNumerics software package version 3.5 (Applied-Maths).
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RESULTS |
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At the CRISPR YP3 locus, KIM and CO92 strains have the same three motifs a3b3c3. No size polymorphism was observed in any of the Y. pestis strains tested by PCR. In contrast, the microtus strain possesses only motif a3, and the Pestoides strain has, in addition to motifs a3b3c3, motifs d3 and e3. In summary, partial sequence analysis reveals the existence of respectively 21, 9 and 3 different CRISPR YP1, YP2 and YP3 alleles.
CRISPR polymorphism in Y. pseudotuberculosis
Y. pseudotuberculosis represents an older, more diverged species from which Y. pestis probably emerged as a clone (Achtman et al., 1999). The three CRISPRs are present at least in some members of this species, and can be amplified using the Y. pestis primer pairs. We analysed the CRISPRs of nine strains selected for their relative genetic proximity to Y. pestis as estimated by MLVA (data not shown). An important polymorphism was seen in CRISPR YP1, the alleles being in general larger than in Y. pestis, except for strain IP32802, which only possesses three motifs. The sequencing of these alleles produced a large collection of new spacers, 132 for nine isolates (data not shown). In strain IP32952 the first three motifs are a, b, c and they are the only examples of motifs shared with Y. pestis. Five additional motifs were shared by two or three Y. pseudotuberculosis isolates, the rest being unique to a given strain. The polymorphism of CRISPR YP2 and YP3 is also high, and some alleles are very large. Interestingly, the Y. pseudotuberculosis strain IP32802 has small alleles at these two loci as well, with only one spacer at CRISPR YP2 and CRISPR YP3.
The origin of new spacers
We analysed all the spacers by BLAST against the sequences of the CO92 and KIM genomes, and against the complete GenBank database. In Fig. 3 the different CRISPR alleles are shown as a succession of boxes, where white boxes indicate spacers for which a homologous sequence was found at a locus different from the three CRISPR loci in the Y. pestis genome. For the first spacers of CRISPR YP1, a, b, c, d, e and f, no sequence homology was found, whereas with motifs g to z, one copy of a homologous sequence, consisting of a portion of a gene or intergenic sequence, could be found in another region of the Y. pestis genome (Table 2
). Some spacers in CRISPR YP2 and CRISPR YP3 also had matches elsewhere in the genome. Interestingly, 24 out of 31 Y. pestis spacers were found at a single locus, inside a 46 kb region corresponding to a defective lambdoid prophage. Spacers p, l and f2 were found in two contiguous genes, aceB and metA, separated by only 400 bp. Spacers j, r, t, y and z were found in four additional regions. In Y. pseudotuberculosis, only three spacers had a homologue in the Y. pestis genome at another locus, spacers psf and psg2 in the prophage, and spacer psc in the atpA gene.
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DISCUSSION |
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CRISPR YP1 was investigated in detail by measuring its length in 26 isolates of the Orientalis O1 group and in 134 isolates of the Orientalis O2a group (Pourcel et al., 2004), followed when relevant by sequencing of the locus. The vast majority of isolates have the YP1 allele abcdefgh, with six exceptions in the O1 group and 53 exceptions in the O2a group (44 of which are abcdefgho, observed in three genotypes, Fig. 4
). All these strains show the same glpD, Orientalis-specific deletion compared to the Antiqua phenotype (Motin et al., 2002
; Pourcel et al., 2004
), which inactivates the gene. Therefore they must result from a clonal expansion which subsequently produced the two groups, O1 and O2a, represented here. The abcdefgh allele is the only allele detected in both groups, and must have been present in a common ancestor. The other alleles differ from the abcdefgh structures by simple deletion or insertion events. abcd is missing in the closely related genotypes 11, 12, 13, and e is missing in genotype 37 (Fig. 4
). All eleven insertion events are terminal additions after motif h near the leader sequence. In two instances, new motifs were added to the frequent abcdefgho variant, in strains with an otherwise identical MLVA genotype (genotypes 46, 47, 48, 49; Fig. 4
). As in the DR of M. tuberculosis the order of the motifs is always the same and there is no duplication (van Embden et al., 2000
). In Y. pestis, the fact that the Orientalis O2a strains investigated here are of extremely limited geographical (Dalat, Vietnam) and temporal (years 19641967) origin clearly indicates that addition of spacers is an ongoing process.
These observations suggest the simple following rules for CRISPR evolution in general: (1) random deletions of one or more spacers and repeats may occur; (2) in contrast, the addition of new motifs is polarized and requires that the last constant region near the leader sequence is duplicated and that a piece of DNA about 32 bp in length (the spacer) is simultaneously copied and added; (3) the presence of identical spacers in a CRISPR allele reflects shared ancestry and does not result from independent events.
These rules have a predictive value. In the Pestoides strain, which can be considered as an outgroup according to MLVA analysis and current knowledge (Anisimov et al., 2004), motifs a, b, c, d and e are present, indicating that a common ancestor of all Y. pestis biovars and Pestoides had already acquired these five motifs. Similarly, the sequence of the microtus genome reveals the presence of motifs adf. Consequently, one might expect to find ancestor (Antiqua phenotype) strains with spacers a to f (Fig. 5
). Because most Orientalis isolates investigated contain abcdefgh, it is tempting to speculate that the Antiqua strain, progenitor of the Orientalis strains, had the same allele. The shorter alleles abcdj and abc observed in the Antiqua and Medievalis strains investigated here would be the result of deletion events. Additional Antiqua strains, from Africa and more importantly Central Asia, will need to be investigated in order to prove these predictions, summarized in Fig. 5
.
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Jansen et al. (2002) described a group of genes they called cas, present near one CRISPR of all the sequenced thermophilic archaea and in some bacteria. The cas1 and the cas3 genes are present at one side of the CRISPR YP1 (Fig. 1
). Cas1 belongs to the family of proteins COG1518 predicted to be involved in DNA repair and is probably a DNase. Cas3 is believed to be a helicase and belongs to COG1203 (Jansen et al., 2002
). The cas genes are members of a cluster that has been extensively described by Makarova et al. (2002)
independently of the study of CRISPRs, despite the fact that one CRISPR was always present close to this cluster. Makarova et al. (2002)
believe that these genes are part of a DNA repair system particularly important in thermophilic archaea. Mojica et al. (2000)
proposed that the CAS proteins might also be responsible for adding new repeats to the CRISPR loci. The fact that CRISPR YP1, the most active of the three elements, is the one associated with the cas genes supports this hypothesis. The adjacent leader sequence probably plays a role in this process.
More than two-thirds of the new spacers have a homologue in a region extending over 46 kb and corresponding to a prophage. A second preferential site extending over 2 kb contains the homologues of six spacers and corresponds to genes aceB and metA. The other spacers have homologues at different positions in the genome. This is believed to be the first time that the potential source of the CRISPR spacers has been identified. None of the spacers were found in the regions of instability described previously (Hinchliffe et al., 2003; Radnedge et al., 2002
). The observation that Y. pestis CRISPR loci acquire new spacers from a prophage DNA is quite striking. Most of these phage sequences are also present in the Yersinia enterocolitica genome, although having only 8090 % homology. It is believed that Y. pestis and Y. enterocolitica separated 41186 million years ago (Achtman et al., 1999
). This suggests that the prophage was present in an ancestor but that the CRISPRs in Y. pestis have acquired new motifs only recently. It is interesting to note that the CRISPR YP1 and the prophage are located near the replication terminus (Fig. 1
), a region in which levels of recombination are high.
We have found in the published sequence data additional evidence for a mechanism by which CRISPRs could acquire phage DNA. Hoe et al. (1999) described spacer sequences and organization in a CRISPR of Streptococcus pyogenes. Only one of the five sequenced S. pyogenes genomes, MIGAS, possesses a CRISPR. We performed a BLAST search with the S. pyogenes spacer sequences against the five sequenced genomes and found that seven out of the nine spacers described correspond to a phage-associated sequence, present in at least one of the genomes except that of MIGAS. Phage DNA constitutes up to 12·4 % of the S. pyogenes genome (Beres et al., 2002
) and is involved in recombination and horizontal transfer of new genes. There may be, in the case of strain MIGAS, a relationship between the presence of a CRISPR and the lack of a particular prophage. One possible explanation for that finding could be that CRISPRs are structures able to take up pieces of foreign DNA as part of a defence mechanism. In this view, it is tempting to further speculate that CRISPRs may represent a memory of past genetic aggressions. The fact that most of the spacers described in other bacteria have no homologue in the databases could still be explained by such a phage origin, as only a very small number of the existing bacteriophages have so far been sequenced.
The way in which the CRISPR loci appear to evolve in Y. pestis, and the frequency at which they acquire new motifs, at least within the Orientalis group of strains, are such that these loci may provide powerful and easy-to-use phylogenetic tools in complement to MLVA. It may be that the picking up of new spacers is not occurring at a uniform rate across the Y. pestis species, but rather that some unknown conditions are able to trigger an increased activity. In spite of the very limited number of Antiqua and Medievalis strains which could be investigated, MLVA data suggested the existence of two groups of Antiqua strains (Pourcel et al., 2004). The first group, from Asia, represented by genotypes 6, 7 and 8, holds an intermediate position between the Medievalis and the Orientalis group; the second group comprises the African Antiqua strains. This grouping is supported by sequence analysis of the CRISPR loci (Fig. 4
).
In addition to the biological relevance of their curious behaviour, analysis of the CRISPRs of Y. pestis provides a tool, comparable to spoligotyping for M. tuberculosis (Kamerbeek et al., 1997), which might open the way to strain typing of ancient, degraded DNA, since a perfectly conserved multi-copy sequence (the repeat) can be used to amplify a variable library of very short spacers and since this kind of assay is not sensitive to the occasional misincorporation of an incorrect nucleotide during the amplification process.
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ACKNOWLEDGEMENTS |
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Received 28 June 2004;
revised 8 November 2004;
accepted 12 November 2004.
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