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1 Departamento de Microbiología, Inmunología y Parasitología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
2 Centre de Recherche en Infectiologie, Université Laval, Québec, Canada
3 Département de Biochimie et de Microbiologie, Université Laval, Québec, Canada
Correspondence
Daniela Centrón
dcentron{at}gmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table showing the GII intron and E1 sequences used in this study, and two supplementary figures showing the structures used for intron self-splicing and the secondary structures of the target sites corresponding to class C GII introns, are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dai & Zimmerly, 2002). They possess an intron-encoded protein (IEP) with several activities that include a maturase, a reverse transcriptase and a site-specific DNA endonuclease (Endo) (Kennell et al., 1993; Zimmerly et al., 1995a; Matsuura et al., 1997). GII introns mediate their dissemination by the well-characterized target DNA primed reverse transcription (TPRT; Zimmerly et al., 1995b; Cousineau et al., 2000), which involves an RNA–DNA pairing. This pairing is achieved through two regions: the exon binding sites (EBSs) in the intron RNA, and the intron binding sites (IBSs) in the targeted DNA (Lambowitz & Belfort, 1993; Michel & Ferat, 1995). These regions vary in sequence and length based upon the GII intron classification (Dai & Zimmerly, 2002). Based on their IEP sequences, they have been classified into chloroplast-like classes, mitochondrial-like classes and bacterial classes A–E (Zimmerly et al., 2001). Their RNAs and IEPs have largely coevolved; thus, it is possible to follow the evolution of the ribozyme by understanding the evolution of the reverse transcriptase (Toor et al., 2001). It has been proposed that the class C introns are direct descendants of a common ancestor of GII introns (Rest & Mindell, 2003). The class C GII introns, which have a novel RNA secondary structure, lack the Endo domain of the IEP (Endo–), have a shorter EBS–IBS pairing, and are located downstream of transcriptional terminators (Granlund et al., 2001; Dai & Zimmerly, 2002). While Endo+ introns use the TPRT mechanism, introns lacking the Endo activity use the host replication machinery to invade novel target sites (Jiménez-Zurdo et al., 2003; Zhong & Lambowitz, 2003). RNA–DNA pairing in most GII introns involves EBS regions 1, 2 and 3, which constitute an 11–12 nt motif (Matsuura et al., 1997; Mohr et al., 2000); in contrast, the class C GII introns lack the EBS2 region, resulting in a shorter RNA–DNA pairing (Toor et al., 2001).
Previously, we reported the presence of a class C group II intron identified as S.ma.I2 (Figs 1 and 2a) in a class 1 integron in the multiresistant Serratia marcescens strain SCH909 (Sm909) (Centrón & Roy, 2002). The S.ma.I2 GII intron is inserted within the attC site of the ant(2'')-Ia gene cassette, in the opposite orientation to the cassette ORF (Fig. 2a). Cassettes are mobile elements typically composed of a promoterless structural gene and a recombination site known as a 59-base element or attC site (Recchia & Hall, 1997). attC sites are 57–141 bp long, and consist of two short regions of sequence similarity at their boundaries (1L–2L and 1R–2R) separated by a stretch (20–104 bp) of imperfect internal dyad symmetry (Stokes et al., 1997). Single-stranded forms of the attC sites have the potential to form a stem–loop structure (Stokes et al., 1997; Bouvier et al., 2005). The 1L (also known as the inverse core site) is located at the left-hand end of the attC and has the 5' RYYYAAC 3' consensus sequence (CS), while the 1R (or core site) is located at the right-hand end formed by the 5' GTTRRRY 3' CS (Collis & Hall, 1992; Stokes et al., 1997). Cassettes can be excised into circular intermediates and inserted into an integron by integrase-mediated site-specific recombination through the cleavage of the 1R region (Hall et al., 1991; Collis & Hall, 1992). It has recently been demonstrated that two extrahelicoidal residues, a G and a T, of the attC stem collaborate in the affinity of the integrase for the substrate (MacDonald et al., 2006). Here, we report that the ant(2'')-Ia : : S.ma.I2 gene cassette is capable of excision, although its 1L is disrupted. Also, the S.ma.I2 intron can self-splice and invade a wide variety of gene cassettes. The successful insertion of the intron into a novel DNA involves an adjacent secondary structure, which is provided by the cassette attC site. In addition, based on phylogenetic studies, we found that those introns that also occur within attC sites belong to a distinct clade within class C group II introns together with S.ma.I2.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, JM109 and JM107 (Table 2) were grown at 37 °C overnight with agitation in Luria–Bertani media (10 g tryptone, 5 g yeast extract, 10 g NaCl), in the presence of 10 µg ampicillin (Ap) ml–1 and/or 50 µg chloramphenicol (Cm) ml–1, when necessary. The pUCSmI clone was obtained by cloning a 2590 bp fragment from Sm909 with the restriction sites SphI and XmaI at positions 440 and 3033 (GenBank accession no. AF453998) at the SphI and BspEI sites of the pUC19 vector. The pAC
attC and pACIBS clones were obtained from the pLQ424 clone (Table 2) by PCR amplification using the primer combinations pACYC184-5'/attCdfr-SLR and pACYC184-5'/attCdfr-NSR, respectively (Table 1), and Taq polymerase (Promega). The first product carried the complete dfrA1 gene along with 34 bp of the left-hand end of the dfrA1 attC site from Tn7; the second product carried the same dfrA1 gene with only 17 bp of the dfrA1 attC. Both products were cloned into the pCR2.1 vector (ApR) following the manufacturer's procedures (Invitrogen). Briefly, 4 µl of the PCR product was incubated with 1 µl of the pCR2.1 vector in the presence of the manufacturer's buffer and salt solution for 30 min at room temperature. An aliquot was transformed into the E. coli Top10' strain. Subcloning of the inserted regions was done into the pACYC184 vector (CmR). The integrity of the inserts was confirmed by sequencing. The pUatt-dfr and pPatt-dfr clones were obtained by introducing a 95 bp fragment of each into the pCR2.1 vector and subcloning into the pACYC184 vector at the BamHI and EcoRV restriction sites. The fragment used for the pUatt-dfr plasmid was obtained using the primers dfr-att-open_F and dfr-att-open_R, and corresponds to a modification of the dfrA1 attC site that avoids stem–loop formation (Table 1). The fragment used for the pPatt-dfr clone was obtained using the dfr-att-open_F and dfr-att-comp_R primers, and restores the stem–loop complementarity of the modified dfrA1 attC from pUatt-dfr. All pLQ plasmids were obtained by cloning the different gene cassettes into the EcoRV restriction site of the pACYC184 vector (P. H. Roy, unpublished results; Table 2).
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IEP and pESmIDI-IV clones were generated by deletions from the wild-type (wt) S.ma.I2 intron (Table 2
). The pESmIIEP construct was obtained by removing 786 bp of the intron IEP from the pUCSmI plasmid with BamHI (20 U), and religating with T4 DNA ligase (New England BioLabs) (Supplementary Fig. S1a). The internally deleted intron with its exons was subcloned into the pET-3d vector using the EXL and Smtr-XbaI primers, followed by transformation into E. coli DH5, as specified by Sambrook et al. (1989). The pESmIDI-IV construct was obtained by digestion of the pESmIIEP clone with BamHI and NdeI, followed by filling in with Klenow enzyme and blunt-ended ligation (Sambrook et al., 1989).
The complete ant(2'')-Ia : : S.ma.I2 gene cassette was cloned within the pCR2.1 vector as described above and subcloned in the pACYC184 vector (clone pACIcass) (Fig. 2c). The insertion fragment was obtained by PCR amplification using the primers sulpro 3 and Smtr-XbaI (Table 1).
Nucleic acid extraction.
Total DNA isolation of Sm909 was done by using a phenol/chloroform purification method (Sambrook et al., 1989). Plasmid DNAs were prepared using either the Miniprep Plasmid Extraction kit from Qiagen or the plasmid preparation protocol described by Sambrook et al. (1989). Total RNA extraction was done using TRIzol Reagent (Invitrogen) following the manufacturer's procedures. Once extracted, the total RNA was treated with 10 U DNase I (RNase free; Epicenter) for 1 h at 37 °C and cleaned up with the RNeasy Mini Kit from Qiagen.
In vivo recombination.
The pACIcass clone was introduced by transformation into the E. coli DH5 strain (Table 2) along with the pLQ369 plasmid, which encodes the IntI1 protein fused to the MalE protein from the pMAL-c2 vector (Messier & Roy, 2001). Cells were grown overnight at 37 °C. Induction of the ant(2'')-Ia : : S.ma.I2 cassette excision was done on an OD600 0.4 culture by the addition of 0.3 mM IPTG and incubation for 3 h at 37 °C. After plasmid DNA preparation, the detection of cassette excision was done by PCR with 50 ng DNA template and primers pACYC184-5' and pACYC184-3' (Table 1). A shorter amplification product confirmed by sequencing using the ABI Prism 3300 sequencer was considered evidence of cassette excision.
Splicing in vitro.
A fragment containing the S.ma.I2 intron with its exons was amplified by PCR with Pfx polymerase (Invitrogen) and the IVT-up and IVT-PT7L primers (the latter carries the T7 promoter sequence at the 5' extremity). Of the resulting purified product, 500 ng was added to the in vitro transcription buffer from Invitrogen and incubated for 1 h at 37 °C with 50 U T7 RNA polymerase and 0.4 mM NTPs. The synthesized RNA was treated with 4 U DNase I (RNase free; Epicenter) and cleaned up with the RNeasy Mini Kit from Qiagen. Two microlitres were incubated in the corresponding splicing buffer (SB) (50 mM Tris/HCl, pH 7.5, 50 mM Mg2+ and 500 mM 
, 10 mM DTT and 40 U µl–1 RNaseout inhibitor; final volume 20 µl), as described elsewhere (Matsuura et al., 1997). Samples were incubated for 5 min at 65 °C and then reduced to 37 °C either for 20 min in a water bath or in the DNA Engine PTC-200 Dual thermal cycler from MJ Research (speed of ramping 3 °C s–1). Once splicing was completed, 2 µl of the reaction mixture was used for the RT-PCR assay.
RT-PCR assay.
RT-PCR was performed following the manufacturer's conditions for the Superscript II RNase H– reverse transcriptase from Invitrogen. We performed the RT-PCR method on total RNA from strain Sm909 and on the in vitro splicing products. For total RNA, we used a concentration of 1 µg µl–1 in each reaction. For the in vitro-synthesized RNA, a one-tenth aliquot of the reaction mixture was used. In both cases, the reverse transcription reaction was done by adding the in vitro or in vivo RNA to a mix containing 0.2 mM dNTPs, 0.4 µM of the corresponding primer, 1x manufacturer's buffer, 0.02 M DTT and 40 U Superscript II RNase H– reverse transcriptase in a final volume of 20 µl. Incubation was done in the presence of RNaseout inhibitor from Invitrogen at 45 °C for 1 h and inactivated at 70 °C for 15 min. RNase H (2 U) treatment was done for 15 min at 37 °C. The subsequent PCR reaction was done using 2 µl of the reverse-transcription product as template with 0.3 µM of specific primers, 0.2 mM dNTPs, 2 mM MgCl2 and 1.25 U Taq polymerase from Invitrogen for 94 °C/1 min; 94 °C/1 min, followed by 30 s at the annealing temperature (Ta) of the primers (Table 1), 72 °C/5 min for 35 cycles; then 72 °C/10 min in a final volume of 50 µl. The products of the amplification were sequenced and analysed with Sequencher (Gene Codes Corporation) and GCG (Genetics Computer Group, Accelrys) software.
Mobility assay.
The pUCSmI (ApR) clone was co-transformed into E. coli JM109 (recA–) and JM107 (recA+) strains with each pLQ clone (CmR) (Table 2). The competent cells were obtained using the CaCl2 technique (Ausubel et al., 1994). The different co-transformants obtained were grown in the presence of both antibiotics at 37 °C overnight. An aliquot from the preculture was inoculated in 5 ml LB medium with the addition of 1 mM IPTG after 1 h incubation at 37 °C to induce the expression of the S.ma.I2 GII intron, followed by incubation for 3 h. The co-transformant DNAs were extracted and the mobility tested by PCR with the following combination of primers: a specific primer for the intron (INU or INL for the donor plasmid) and a specific primer for the recipient plasmid (primers pACYC184-5' and pACYC184-3', and primer orf-COOH for the pLQ428 plasmid) (Table 2). The amplification reaction was performed as described above with the following cycling steps: 94 °C/1 min; 94 °C/1 min, 52 °C/1 min, 72 °C/2 min for 35 cycles; 72 °C/5 min. Amplification products were confirmed by sequencing.
Nucleic acid folding.
S.ma.I2 RNA secondary structure, the DNA secondary structures of the attC sites, and E1 region folding were done using the MFOLD program from Zuker (2003), available at the Rensselaer bioinformatics website (http://frontend.bioinfo.rpi.edu/applications/mfold/). We used previous intron structures as a reference for the S.ma.I2 RNA secondary structure folding (www.fp.ucalgary.ca/group2introns/).
For the attC sites, we considered each sequence as beginning with the inverse core site (5'-RYYYAAC-3') and ending with the core site (5'-GTTRRRY-3'). We selected the most probable structure for their folding based on previous descriptions of attC structures (Stokes et al., 1997). Conditions for the DNA folding used with the MFOLD program were a flat exterior loop and natural algorithm.
Phylogenetic tree reconstruction.
The phylogeny studies of the class C GII intron were done using the amino acid sequences of 45 intron proteins. Several introns were retrieved from the Mobile GII intron database (www.fp.ucalgary.ca/group2introns/) and others by performing a BLASTP search at the NCBI site (www.ncbi.nlm.nih.gov/BLAST). Supplementary Table S1 summarizes the introns used for this purpose. The first alignment of the sequences was done with the CLUSTAL_X version 1.83 software using the neighbour-joining method (Thompson et al., 1997). We refined the alignment using the MEGA version 3 software (Kumar et al., 2004). The conditions used for the alignment were a gap open penalty of 10 (gap extension penalty 0.1) for the pairwise alignment and a gap open penalty of 10 (gap extension penalty 0.2) for the multiple alignments using a Blosum matrix. Phylogenetic tree reconstruction was done using the neighbour-joining method with 1050 bootstraps and a seed of 71 829; the maximum-likelihood method and the maximum-parsimony method were also tested to validate the tree topology. The following introns were used as outgroups: the mitochondrial class Ll.LtrB intron (U50902), the class D RmInt1 intron (Y11597), the class D intron from E. coli (AB024946), the chloroplast–like intron from Xylella fastidiosa (AE003999), the class E intron from S. marcescens (BX664015) and the class B intron from Enterococcus faecium (AAAK03000117).
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). The insertion of S.ma.I2 at the 1L of the ant(3'')-Ik attC site modifies the well known 5' GCCTAAC 3' DNA CS to the 5' GCCTAAT 3' sequence (1L, top strand; Fig. 2b). This insertion occurs at the ant(2'')-Ia stop codon (Fig. 2b), but since the intron left-hand end is an A, the protein translation was not modified and the ant(2'')-Ia gene conferred resistance to gentamicin, as was confirmed by disc susceptibility assay of the pACIcass plasmid, which has the complete ant(2'')-Ia : : S.ma.I2 gene cassette cloned into the pACYC184 vector (zone diameter of 8 mm for gentamicin). In order to test the recombination activity of the ant(2'')-Ia : : S.ma.I2 gene cassette, we analysed its excision ability by co-transforming the pACIcass plasmid along with the pLQ369 plasmid that provides the IntI1 integrase in trans (Fig. 2c; Messier & Roy, 2001). PCR amplification resulted in two products of 3000 and 400 bp before and after excision of the ant(2'')-Ia : : S.ma.I2 gene cassette, respectively. Sequencing of the 400 bp excised band confirmed the junction between the attI1 site and the attC site of the second cassette inserted within the variable region, which corresponded to the ant(3'')-Ii-aac(6')-IId gene cassette (Fig. 2d); however, we did not test for the presence of the predicted ant(2'')-Ia : : S.ma.I2 gene cassette circle. From an integron functional perspective, these results showed that despite the disruption of the 1L of the ant(2'')-Ia gene cassette by the S.ma.I2 GII intron, the attC was still functional during cassette excision, indicating that the ant(2'')-Ia : : S.ma.I2 gene cassette had not been immobilized.
S.ma.I2 is an active intron capable of self-splicing
Splicing of the S.ma.I2 intron and transcription of the surrounding genes in the original host Sm909 were tested by RT-PCR. Transcription of the integron genes intI1, ant(2'')-Ia and ant(3'')-Ii_aac(6')-IId was detected using the primers intiF/intiR, EXU/SB4 and EXU/EX1B, respectively, in order to obtain the first-strand cDNA (Fig. 2a). The S.ma.I2 intron precursor mRNA or its religated exons were not detected with the EXU primer for the first-strand cDNA synthesis followed by PCR with primers EXU/EXL. These results suggest that the S.ma.I2 intron RNA is not transcribed in Sm909 under these conditions.
We analysed the self-splicing ability of the S.ma.I2 intron under regulated conditions (Robart et al., 2004). The study was carried out using three different constructs: (i) the wt intron obtained directly from strain Sm909; (ii) the clone pESmIIEP, which has the S.ma.I2 intron with 786 bp deleted (IEP; Supplementary Fig. S1b); and (iii) the clone pESmIDI-IV, which has the S.ma.I2 intron deleted from DId up to the DIV region (DI-IV; Supplementary Fig. S1c). First, each structure was amplified with the primers IVT-up and IVT-PT7L (Table 1). The latter carries the T7 promoter in its sequence so that transcription will be regulated by the T7 RNA polymerase. Purification of the in vitro-synthesized RNA was done independently for each assay. An aliquot was incubated in the presence of SB buffer, which contains high concentrations of Mg2+ and 
(50 and 500 mM, respectively). We then performed an RT-PCR assay using the primer IVT-PT7L for first strand synthesis, and the IVT-up and IVT-PT7L primers for the PCR reaction. As expected, the DI-IV construct showed no exon religation. The controls used in every self-splicing assay included a reaction containing an aliquot of the wt intron exposed to standard conditions but without the RNA polymerase, to ascertain that each amplification reaction was the product of transcription and not due to DNA contamination (Fig. 3, -pol lane); a DNA-free sample exposed to standard conditions (Fig. 3, -DNA lane); and an in vitro-transcribed wt aliquot whose RNA was also purified but for which no reverse transcriptase was added to the RT-PCR mixture (Fig. 3, -RT lane). We observed four bands of 1100 (not shown), 550, 400 and 350 bp for the wt and IEP constructs (Fig. 3). The products that corresponded to sizes 1100, 550 and 350 bp were sequenced, and this showed that they did not correspond to any cognate intermediate seen for other in vitro splicing events of class C GII introns (Toor et al., 2006). Consequently, we considered these bands as artefacts of the methodology (Fig. 3). Sequence analysis of the 400 bp fragment showed the ligation of exons E1 and E2, confirming the correct splicing of S.ma.I2 (Fig. 3).
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). Upon induction with 1 mM ITPG at 37 °C, plasmid DNA was used to detect the intron insertion event. The presence or absence of a PCR product and the corresponding sequences that provided the intron–exon junctions confirmed positive or negative intron insertion. Gene cassettes that showed a positive intron insertion were: ant(3'')-Ia, ant(3'')-Ic, aac(6')-Ia_orfG, blaIMP-1, blaOXA-10 (blaPSE-2), sat2 and dfrA1 (Fig. 4a).
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. One of these regions is formed by only 4 nt (5' TTGT 3') complementary to the proposed EBS1 site for the class C GII introns (5' AACA 3', residues 204–207; Fig. 1). We also observed a conserved region, 5' TAR 3', at the E2 (Fig. 4b and positions +1 to +3 from Fig. 1). The thymidine residue T+1 corresponds to the IBS3 site complementary to the adenosine at position 242 in S.ma.I2 that has been proposed as the EBS3 for class C GII introns (Figs 1 and 4b). The EBS2 region is apparently absent, and the IBS2 region was not seen in the CS obtained from the target sites invaded by the S.ma.I2 intron nor was a convincing EBS2 region observed in the intron sequence (Fig. 1). Only two gene cassettes [aac(6')-Ib and orfH] were negative for the insertion of the S.ma.I2 GII intron. Analysis of the potential targets showed that their attC sites have a modification in their IBS1 sequences. Instead of the conserved 5' TTGT 3', the aac(6')-Ib and orfH gene cassettes possess the 5' TGGT 3' and 5' AGGT 3' sequences, respectively (Fig. 4a). These findings suggest that modifications in this region disrupt the interaction between the IBS1 site and its complementary EBS1 in the intron RNA. Since the recognition site of intron S.ma.I2 provided by IBS1 and IBS3 is 5 nt long, we searched for other copies of the conserved sequence in the plasmid sequences using the Findpatterns program from the GCG software, including the 5' AR 3' nucleotides that were also conserved (5' TTGTTAR 3', Fig. 4b). We found that this region is present in every pLQ plasmid at position 1597 (in the pACYC184 vector backbone; Rose, 1988). PCR analysis showed no insertion into the recipient plasmid at this site. In addition, a second 5' TTGTTAR 3' sequence copy was seen only for two plasmids, pLQ425 and pLQ439 (Table 2), both carrying the blaIMP-1 gene cassette. Although they contain a second putative target site in the middle of the gene, using the PCR technique we observed that none of these putative DNA target sites showed intron invasion, which suggested that the short primary sequence provided by the IBS1–IBS3 DNA sequence (5' TTGTT 3') is not on its own sufficient for intron mobility.
A DNA secondary structure is required for S.ma.I2 mobility
Recently, it has been shown that stem–loop structures are involved in the self-splicing and in vitro mobility of the class C GII intron B.h.I1 from Bacillus halodurans (Toor et al., 2006; Robart et al., 2007). We addressed the same question for S.ma.I2 by testing in vivo the dependence of the S.ma.I2 intron upon the stem–loop structure of the attC site for invasion of novel target DNAs. We repeated the mobility assay described above using as recipient plasmids (i) the pACattC clone, which has 59 bp deleted from the left-hand end of the dfrAI attC site; (ii) the pACIBS clone, which has 76 bp deleted from the left-hand end of the dfrAI attC site; (iii) the pUatt-dfr clone, which has lost the stem–loop pairing; and (iv) the pPatt-dfr clone, which has a restored complementation of the modified dfrA1 attC from pUatt-dfr (Fig. 5a, b, Table 2) Each clone was used for the mobility assay in the presence of the pUCSmI plasmid, which carries the S.ma.I2 intron. Our results showed that both attC deletions were negative for intron mobility (Fig. 5c, lanes 3 and 4). Consistent with this result, when the attC stem–loop was unpaired (pUatt-dfr clone), the S.ma.I2 intron did not invade the target site (Fig. 5c, lane 2); on the contrary, when the stem–loop was recovered, the intron inserted itself in the attC site (Fig. 5c, lane 1).
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Bacterial introns that insert within attC target sites show a distinctive evolutionary lineage
The phylogenetic analysis of the IEPs from a representative set of class C GII introns revealed a tree topology with a number of well-supported subgroups (Fig. 6). Significantly, S.ma.I2 belonged to a strongly supported group by three independent methods (neighbour-joining, maximum-likelihood and maximum-parsimony). This group comprises introns inserted within attC sites (Fig. 6, thick black line; Supplementary Table S1). This implies that the capacity to recognize attC sites arose in the common ancestor of this clade, which has probably developed a specific strategy for the recognition of a particular target DNA (Supplementary Fig. S2a). We refer to this clade as the class C-attC GII introns. Other class C GII introns that are not inserted within attCs also showed strong bootstrap support in their evolutionary lineages (Fig. 6). However, in contrast to the class C attC intron lineage, their E1 showed a wide variety of DNA secondary structures, such as the Pseudomonas syringae and Azotobacter vinelandii intron target sites (Supplementary Fig. S2b).
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DISCUSSION |
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), which indicates that it is an active retrotransposon. Different deletions in key regions of the intron sequence, DIV and DId-DIV, suggest that S.ma.I2 functions similarly to other described GII introns (Noah & Lambowitz, 2003; Guo et al., 2000; Su et al., 2005).
In addition, GII introns have a common genetic organization, being inserted in the opposite direction to transcription, and after intrinsic transcriptional terminators that usually yield low levels of transcripts (Dai & Zimmerly, 2002; Robart et al., 2007). These properties probably favour their survival in bacterial genomes for a long period of time. Although our results provide evidence that the S.ma.I2 intron is not transcribed in its natural host, the possibility of a sporadic transcription from its right-hand boundary should be considered. Taking into account our results, it is likely that S.ma.I2 was acquired from a different and unknown genome, in which it is probably expressed and active. The passive transfer of S.ma.I2 is suggested by the ability of the ant(2'')-Ia : : S.ma.I2 gene cassette to excise from the integron (Fig. 2d). The disruption of the 1L region generated by the intron does not affect the excision mediated by the integrase. Different modifications within attC sites have been assayed in vitro and in vivo (Johansson et al., 2004; Stokes et al., 1997). Although a C
T substitution in the 1L has not been described, we can see here that the ant(2'')-Ia : : S.ma.I2 cassette is recombinationally active. Further studies should be done in order to determine whether this cassette can also integrate and be considered a fully functional element (Stokes et al., 1997); however, the ant(2'')-Ia : : S.ma.I2 gene cassette has guaranteed its own mobilization since it is inserted in a class 1 integron within a conjugative plasmid (Centrón & Roy, 2002).
When we analysed the mobility properties of the S.ma.I2 intron, we found that its DNA target sequence (5' TTGTT 3'), represented by the IBS1 and IBS3 regions, partially overlaps with the 1L region of the attC sites (5' GTTRRRY 3' at the bottom strand; Supplementary Fig S2a; Fig. 4b), which can be found in all gene cassettes (Orman et al., 2002; Ramírez et al., 2005; Senda et al., 1996; Rowe-Magnus et al., 1999). Retrohoming and retrotransposition of GII introns are carried out by DNA and RNA pairings between EBS1, EBS2 and EBS3 in the intron RNA and IBS1, IBS2 and IBS3, respectively, in the target DNA. Since the S.ma.I2 GII intron, like other class C GII introns, lacks EBS2 (Dai & Zimmerly, 2002; Toor et al., 2001), the interaction is reduced to the EBS1 (5' ACAA 3') and EBS3 regions (A+242; Fig. 1). Hence, the complete CS is formed by only 5 nt, 5' TTGTT 3'. Our results showed that there are three highly conserved nucleotides (5' TAR 3') at the 5' end of the E2 region (Fig. 4b), containing the T+533 (AF453998) of the putative IBS3 proposed by Toor et al. (2001), and two downstream nucleotides overlapping with the attC 1L. Since the EBS3–IBS3 pairing probably occurs between their respective A and T residues, the extra nucleotides of this E2 region are a simple consequence of sharing a common DNA structure with the integrase target site.
Our mobility assays showed negative results when the S.ma.I2 targeted a dfrA1 attC site that has been deleted or modified by unpairing its stem–loop. On the other hand, positive results were obtained when S.ma.I2 targeted attC sites that were different in sequence and length. The compensation of the mutations of the pUatt-dfr clone that restore the stem–loop gave positive results for intron invasion, confirming that the secondary structure provided by the attC site is required for recognition of the target site, regardless of the DNA sequence beyond IBS1 (Fig. 5). Toor et al. (2006) implicated a stem–loop structure in the recognition site for the B. halodurans class C GII intron B.h.I1, and in a subsequent work Robart et al. (2007) strongly suggested that this requirement is generic across class C GII introns. These studies are in agreement with our finding that S.ma.I2 requires not only the specific sequences of the IBS1 and IBS3 regions but also the presence of a stem–loop for intron invasion of a new target DNA.
Dependence on the secondary structure of DNA has recently been reported in several studies. The phage CTX requires a stem–loop for integration into the genome of Vibrio cholerae (Val et al., 2005), while the TnpA transposase of ISHp608 uses an imperfect stem–loop for transposon end recognition (Ronning et al., 2005). In this regard, the attC site belongs to this group of ssDNA structures that act as a signal for a DNA process. However, the attC site is the target not only of the site-specific recombination event mediated by the integrases but also for the insertion of the S.ma.I2 intron, by a process that involves an RNA–DNA pairing. The phylogeny of these introns also shows a recent branch formed by the class C attC clade, suggesting a recent acquisition through a horizontal gene transfer event. The selection of this event might be biased by the fact that the exons for these introns correspond to antimicrobial resistance gene cassettes. This evolutionary strategy, which enhances the dispersal of mobile elements to novel DNA niches by sharing the target site of other mobile elements, may be a common process in nature.
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ACKNOWLEDGEMENTS |
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Edited by: A. Holmes
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REFERENCES |
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Received 18 July 2007;
revised 3 January 2008;
accepted 13 January 2008.
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