Microbiologia/Universita di Siena, Via Laterina 8, 53100 Siena, Italy1
Laboratoire de Microbiologie et Génétique Moléculaire CNRS-UPR 9007, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France2
Author for correspondence: Jean-Pierre Claverys. Tel: +33 561 33 59 11. Fax: +33 561 33 58 86. e-mail: claverys{at}ibcg.biotoul.fr
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ABSTRACT |
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Keywords: chromosome rearrangements, genetic flexibility, intergenic regions, repeated elements, Streptococcus pneumoniae
Abbreviations: IS, insertion sequence; IR, inverted repeat; IRL, left IR; IRR, right IR
The GenBank/EMBL accession numbers of the sequences determined in this work are AJ242695AJ242698
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INTRODUCTION |
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The presence of a highly repeated extragenic element, called BOX, was also reported in the Gram-positive bacterium Streptococcus pneumoniae (Martin et al., 1992 ). Although unrelated to the families of repetitive elements found in enterobacteria, BOX elements exhibit a similar architecture. They are modular and have the potential to form stable stemloop structures (Martin et al., 1992
). Recently, we reported the presence of two copies of another 108-bp-long repeated element that we called RUP (for repeat unit of pneumococcus) in the recAdinF intergenic region of S. pneumoniae (Claverys & Martin, 1998
). The aim of this study was to examine the distribution of RUPs in the chromosome of S. pneumoniae. The identification and analysis of RUPs present in all S. pneumoniae GenBank/EMBL entries and in the partial public domain S. pneumoniae type 4 genome allowed us to draw inferences about the origin and the dynamics of the element. This work strongly suggested that RUP was an IS-derivative that could still be mobile. Examination of sequences flanking RUPs led to the conclusion that RUP may preferentially target ISs and possibly DNA of foreign origin, and that sequence rearrangements could take place at RUPs. Collectively, these observations suggest a direct link between mobile genetic elements, repeated sequences and genome flexibility.
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METHODS |
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Dendrograms and trees were generated with TreeView 1.5.2 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html) from CLUSTAL W-based sequence alignments. Multiple alignments were displayed using GeneDoc (http://www.cris.com/~ketchup/genedoc.shtml). Secondary structures were predicted with PCfold and displayed with RNA_d2 (Perochon-Dorisse et al., 1994 ).
Contigs of the S. pneumoniae type 4 genome sequence were obtained from the anonymous FTP site at The Institute for Genomic Research (TIGR) (http://www.tigr.org/). Analysis of sequences flanking RUPs was carried out by the combined use of BLASTN, BLASTP, BLASTX and TBLASTN, together with the aid of WIT, to distinguish intergenic regions from potentially coding regions. Significant homology between translation products and proven (or putative) transposases was the primary criterion used for identification of new putative ISs of S. pneumoniae. DNA sequences of previously identified ISs of S. pneumoniae (reviewed by Mahillon & Chandler, 1998 ) were also used to screen RUP-containing contigs to reveal potential IS-RUP links.
Four RUP primary sequences exhibiting the highest degree of identity to the RUP consensus generated in this study have been deposited in the GenBank/EMBL databases under accession numbers AJ242695AJ242698. The four RUP consensus sequences can be obtained at http://www-is.biotoul.fr/rup.htm.
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RESULTS AND DISCUSSION |
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A consensus RUP sequence was derived from multiple alignment of the 19 different RUPs (RUP-gbk in Fig. 1a). RUP appeared to be 107 rather than 108 bp long as initially proposed based on the analysis of 68 sequences (Claverys & Martin, 1998
). Examination of the dendrogram generated from multiple alignment of these elements suggested the existence of subtypes (not shown). This was confirmed by examination of S. pneumoniae type 4 sequences (representing more than 90% of the genome) obtained through early release from TIGR. An extensive search for RUP elements (see Methods) retrieved a total of 108 RUPs in the entire genome. It is worth mentioning that this set included the above-mentioned 19 elements which were found to be also present in the type 4 genome. The latter observation strongly suggested that RUPs are stably maintained in the species. The dendrogram generated from multiple sequence alignment (not shown) revealed the existence of three (A, B, C) to four (if B is resolved into B1 and B2) subtypes (Fig. 2
). The 108 elements were distributed among 54 RUPA, 25 RUPB1, 10 RUPB2 and 19 RUPC. Consensus sequences derived for each RUP subtype are shown in Fig. 1(a)
. The most distant consensus subtypes, RUPB1 and RUPC, differed by 28%. Only RUPA could potentially form a stable stemloop structure [-21·0 kcal mol-1 (-88·2 kJ mol-1); Fig. 1b
] as suggested for STEM (Krauss & Hakenbeck, 1997
), while the calculated stability of structures predicted for the other subtypes is rather low [-5·5 to -7·6 kcal mol-1 (-23·1 to -31·9 kJ mol-1)].
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RUP elements insert preferentially within ISs
An exhaustive analysis of sequences flanking RUP was first carried out for the 19 elements present in GenBank/EMBL entries. It revealed that eleven RUPs were inserted within intergenic regions and showed no preferential orientation with respect to flanking ORFs, and that one RUP was inserted in a BOX element. Most intriguingly, seven RUPs were found to target ISs (Table A at http://www-is.biotoul.fr/rup.htm). The latter location seems unique to RUPs since we failed to detect any insertion of BOX within an IS (our unpublished observations).
A careful examination of RUP flanking sequences for the specific presence of ISs was then carried out for the entire set of RUPs. It revealed that at least 25 out of the 108 RUPs (23%) were flanked on one or both sides by IS DNA sequences (Table 1 and data not shown). We concluded that RUP insertion occurred preferentially within ISs. A random insertion hypothesis would be untenable as it would lead to the conclusion that at least 520 kb of the S. pneumoniae genome (23% of 2·27 Mb; Gasc et al., 1991
) would correspond to IS DNA, which obviously is not the case.
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Homology between the IRs of RUPs and of IS630-Spn1, a new putative IS of S. pneumoniae
In the course of our analysis, a new putative IS of S. pneumoniae, IS630-Spn1, was identified (Fig. 3). IS630-Spn1 is a 895899 bp element (deposited in the IS database; http://www-is.biotoul.fr) which harbours potential IRs (Fig. 3b
) and has a calculated GC content close to that of S. pneumoniae (35·9%). It appears to contain two potential ORFs, orf1 and orf2, separated by an in-frame TAG stop codon (Fig. 3a
). This intriguing situation raised the question of the functionality of the IS, a point that is discussed further below. The stop codon was present in all full-size or truncated copies of the sequence (in GenBank entries and in TIGR contigs), except in one contig in which a TAT codon replaced the stop codon. A unique ORF was therefore reconstituted by substituting TAT for the stop codon. A BLASTP search carried out with the sequence translated from this unique ORF revealed that the product belonged to the IS630 family of transposases, with the closest homologue being the transposase of ISTcsa from Synechocystis sp. strain PCC6803 (36·7% identity; Fig. 3c
). A similarity between the product of orf1 and Synechocystis sp. transposases had already been noticed (Iannelli et al., 1999
). Interestingly the TAT-encoded Tyr was the exact match to the ISTcsa transposase residue (not shown).
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The striking observation that IS630-Spn1 was very frequently targeted by RUP (Fig. 3 and data not shown) prompted us to compare the IRs of RUP and of IS630-Spn1. The comparison revealed the existence of highly significant homology (Table 2
). Interestingly, the percentage identity between the left and right IRs (IRL and IRR) of RUPs and those of IS630-Spn1 was greater than that between the IS IRL and IRR themselves. This finding strongly suggested that the putative IS tranposase could readily recognize the terminal IRs of RUP elements. These observations lead us to propose that RUP could be trans-activated by the transposase of IS630-Spn1.
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RUP insertion occurs at a TA dinucleotide
To document the trans-mobilization hypothesis, we searched for sequences that would be the RUP-free counterpart of RUP-occupied sites. Obvious candidates consisted of repeated sequences in which some of the RUPs had been identified, i.e. IS630-Spn1 and other ISs, and BOX elements. As anticipated, several RUP-free sites were found among these repeated sequences (data not shown, but see Table B at http://www-is.biotoul.fr/rup.htm). Comparison of RUP-free and RUP-containing sequence pairs revealed that RUP systematically inserted at a TA dinucleotide (Fig. 4 and data not shown). There was no other apparent sequence preference. The existence of a TA dinucleotide target and the presence of TA dinucleotides flanking the different RUP consensus sequences (Fig. 1a
) would be totally consistent with the hypothesis that RUP insertion results in a duplication of the dinucleotide target. The RUP core sequence would then be 103 bp long, flanked by TA dinucleotides representing the duplicated target. Together with the homologies detected between the IRs of RUPs and of IS630-Spn1, this observation strongly suggests that the putative transposase of IS630-Spn1 could be responsible for trans-mobilization of RUP.
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A scenario for the existence of RUP subtypes
A recent introduction of the element into S. pneumoniae would not by itself account for the observation of different RUP subfamilies that exhibit strikingly different sequence homogeneity (Fig. 2). However, these differences could be accounted for by the occurrence of successive eras of RUP mobility and non-mobility during the evolution of the species. If, as hypothesized, RUP movement requires trans-mobilization by the IS630-Spn1 transposase, inactivation of the transposase gene through RUP insertion (see Fig. 3a
) can result in a RUP non-mobility era. During this era, mutations can accumulate, thus leading to an increase in sequence heterogeneity of the existing RUP families.
Subsequent reacquisition of an active transposase gene would reactivate RUP. This could occur through reintroduction of an intact copy of IS630-Spn1 by natural transformation, a very efficient mechanism of DNA transfer in this species (Mortier-Barrière et al., 1997 ). Selection of the most active RUP element, i.e. the one harbouring the most conserved IRs, through interaction with the transposase would result in the appearance and expansion of a new homogeneous RUP subfamily. According to this scenario, which would explain the clonality of RUP subtypes, the RUPA subfamily would correspond to the most recent expansion of the element within the species, as it is much more homogeneous than the other three (the most distant RUPAs differed by only 16%; Fig. 2
). The fact that the IRs of RUPA exhibit the highest degree of identity to the IRs of IS630-Spn1 (Table 2
) fits nicely with this scenario.
RUP promotes sequence rearrangements
Multiple insertions of RUP may contribute to genetic plasticity by favouring chromosomal rearrangements. RUPs could promote inversion of segments between two RUPs present in opposite orientation, or deletion of sequences located between directly repeated RUPs. The latter event can be predicted to generate jun-RUPs, i.e. junction elements flanked by DNA segments of different origin (Fig. 5). Several jun-RUPs have been identified among IS-targeting elements, which gives some credit to this hypothesis (Table 1
, Fig. 3
and data not shown).
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Evaluation of this hypothesis would require prior demonstration that some RUPs are still mobile. Experiments aimed at demonstrating RUP mobility are in progress. It will also be interesting to see if our model for the origin and dynamics of the RUP element of S. pneumoniae applies to other highly repeated elements that will undoubtedly be discovered as prokaryotic genome sequences accumulate.
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ACKNOWLEDGEMENTS |
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Received 19 March 1999;
revised 14 June 1999;
accepted 18 June 1999.