Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia1
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK2
Author for correspondence: G. Kholodii. Tel: +7 095 196 0015. Fax: +7 095 196 0015. e-mail: kholodii{at}img.ras.ru
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ABSTRACT |
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Abbreviations: AC, accession number; PMA, phenylmercuric acetate
b The accession numbers for the nucleotide sequences reported in this work are given in the legends for Figs 1 and 2.
c A comparison of the sequence of INT5041C with other proteins is available as supplementary data on Microbiology Online (http://mic.sgmjournals.org).
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INTRODUCTION |
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METHODS |
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Origin of the probes used in RFLP analysis.
All probes are shown in Figs 1 and 2
. Probe 1 is the 0·45 kb Sau3AI fragment from RP1::Tn5041, which includes the flanking sequence of RP1. Probes 2 and 4 are the 1·7 and 2·45 kb PstI fragments from Tn5041, respectively. Probe 3 is the 0·73 kb NcoI fragment from the merA gene of Tn5041. Probe 5 is the 2·2 kb PstI fragment from RP1::Tn5041, which includes the flanking sequence of RP1 (shown not to scale in Fig. 1
). Probe 6 is the 1·02 kb AgeIPstI fragment from INT5041C. Probe 7 is the 0·93 kb BglII fragment from the mer2 cassette insertion of Tn5041D. Probe 8 is the 1·2 kb EcoRI fragment from the tnpA gene of Tn21.
Localization of mer transposons.
In nine strains, the localization of the mer transposon to a plasmid was identified by its ability to be transferred by conjugation to P. aeruginosa PAO-R or P. fluorescens P22-1-2 using mercury resistance as a selective marker (Tra+ plasmids in Table 1). The transfer of Tn5041-related elements was confirmed by hybridization of total DNA from the transconjugants with probes 3 and 4, originating from the Tn5041 merA and tnpA genes, respectively (Fig. 1
). The four strains that did not demonstrate an ability to transfer (Tra- plasmids in Table 1
) were investigated further by agarose gel electrophoresis of crude lysates prepared according to Eckhardt (1978)
and by hybridization of the plasmid DNA (transferred from the gel to Hybond-N filters) with probes 3 and 4. For those strains in which we did not detect HgR plasmids by either method, we hypothesize that these transposons are located on the chromosome (Table 1
).
PMA resistance.
Tn5041D-conferred resistance to organomercurials was determined by the method of bacterial culture titration on LB agar containing different concentrations of PMA. Inhibitory concentrations of PMA were determined according to complete growth inhibition. The PMA resistant strain, E. coli JM83(pBR322::Tn5041D), grew at 7·5 µg PMA ml-1, whereas the susceptible strains, E. coli JM83(pBR322) and E. coli JM83(pBR322::Tn5041), grew only at 2·5 µg PMA ml-1. Each experiment was repeated 34 times.
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RESULTS |
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Total DNA preparations isolated from all strains that contained Tn5041-related elements were digested with PstI and subjected to Southern hybridization using probe 4. All but two strains contained identical PstI fragments that hybridized to this probe (Table 2). The two strains that displayed a different hybridization pattern were found to contain DNA elements only distantly related to Tn5041 (data not shown) and were excluded from further analysis. On the basis of RFLP patterns observed with the left- and right-arm internal Tn5041 probes, all strains were categorized into eight types, AH (Table 2
, probe 2, PstI digestion; probe 4, EcoRI digestion). The prototype transposon Tn5041 (renamed hereafter as Tn5041A) belonged to RFLP type A (Table 1
). Four new mobilizable transposons belonged to RFLP types B, C and D (Table 1
). For all strains, the fragments that hybridized with mer probe 3 were identical in size to the fragments that hybridized with tnpA probe 4 (Table 2
, EcoRI digestion), indicating the physical linkage of the mer and tnp determinants. Additional hybridizations were designed to detect different insertions within these elements (Table 2
, probes 6, 7 and 8). The terminal sequences fused with host target sequences (either plasmid or chromosomal, see Table 1
and Methods) were characterized with the help of probes 1 and 5 (Table 1
, last two columns). In most cases the sizes of junction fragments were unique, suggesting insertion into unique targets. Only for some pairs of strains from the same locations (NC2-3 and NC2-4; TC24-3 and TC39-4; and KHP22 and KHP25) did our data indicate the insertion into the same target (plasmids) (Table 1
). In three strains (TC30-1, TC36-1 and TC97) additional junction fragments were observed, indicating the presence of additional full-sized or truncated Tn5041-like transposons (Table 1
and the footnotes).
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Tn5041 variants containing a group II intron
Restriction analysis detected a 2·2 kb insertion relative to Tn5041A at the left arm of the transposon Tn5041C mobilized by RP1 from the Carpathian strain TC29-1. DNA sequencing demonstrated that the 2206 bp insertion, named INT5041C, interrupted the left terminal inverted repeat of a small Tn3/Tn5044-subgroup mobile element (Figs 1
and 2
) and showed features characteristic of group II self-splicing introns that have been found in both organelles and bacteria (Martínez-Abarca & Toro, 2000
; Dai & Zimmerly, 2002
). INT5041C carried an ORF, orf494 (Fig. 2
), encoding a 494 aa polypeptide showing significant similarity to proteins encoded by cyanobacterial and chloroplast group II introns. The sequence alignment is available as supplementary data at http://mic.sgmjournals.org. This protein contained a clearly recognizable maturase (nucleic acid binding) domain X (Mohr et al., 1993
) and reverse transcriptase subdomains 1 to 7 (Xiong & Eickbush, 1990
). As is characteristic of most bacterial group II introns (Dai & Zimmerly, 2002
), Orf494 lacked the zinc finger-like/endonuclease subdomain (Ferat & Michel, 1993
; Martínez-Abarca & Toro, 2000
). The region downstream of orf494 was well conserved (Fig. 3a
) and the predicted INT5041C RNA sequence of this region can be folded to produce a secondary structure typical of group II introns (Michel et al., 1989
). This structure (Fig. 3b
) contained a well conserved domain V with a bulging dinucleotide and a purine-rich terminal loop, and a domain VI with a bulging adenine, 7 bases away from the splice site which constitutes a 2' to 5' lariat branch point in spliced introns. The sequences at the 5' and 3' ends of INT5041C (5'-GTGCG-3' and 5'-GAC-3', respectively) conformed to the consensus sequences 5'-GTGYG-3' and 5'-RAY-3' deduced for group II introns (Michel et al., 1989
). No target DNA duplication was found at the site of insertion of INT5041C and, as in the case of many other bacterial group II introns (Dai & Zimmerly, 2002
), this element did not interrupt any gene with proven or suggested function.
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A truncated homologue of intron INT5041C is apparently present in all Tn5041 variants
Re-examination of the Tn5041A sequence showed that it contained a 195 bp DNA fragment (nt 42244418) with 88% identity to the 3' end of INT5041C (Fig. 3a). The undamaged end of the element (named INT
5041) was oriented towards orfP (Fig. 1
), whereas the damaged one was found to be fused to a damaged end of
, a relic of an IS2-related element (Kholodii et al., 1997
). Restriction analysis of the mobilized Tn5041-like transposons demonstrated that the NcoI site within INT
5041 was conserved, thus indicating the presence of this element in all these transposons.
Tn5041 variants containing the mer2 cassette
The 7120 bp insertion (named the mer2 cassette) was completely sequenced in Tn5041D (Fig. 2). The insertion was bounded by rearranged regions of a mer operon (named mer2; white bars in Fig. 4b
) which was distinct from the mer operon (named mer1; hatched bars in Fig. 4b
) common to all full-sized Tn5041-like transposons. It occurred within the merT gene of a mer1 operon and resulted in the formation of two functional tandem operons, named mer1x2 and mer2x1, separated by a 2·35 kb spacer containing a truncated copy of a novel insertion sequence, named IS1015
(Fig. 4b
, bottom). This IS element encoded a protein 57% and 50%, respectively, identical to transposases from the IS1490 (AC U80795) and Tn4502 (AC U60645) elements belonging to the IS256 family (Mahillon & Chandler, 1998
). Both the order of mer genes from mer1 and mer2 found in the tandem (Fig. 4b
, bottom) and the structure of the junction fragments [where the corresponding merT sequences from the target and insertion transferred to each other (Fig. 4a
)] were strongly indicative of the integration of the circularized mer2-containing intermediate within mer1 via homologous recombination (Fig. 4b
). As in other cases of homologous recombination involving non-identical partners (Doherty et al., 1983
; Rossignol et al., 1984
), DNA sequence correction (gene conversion) was likely to accompany this recombination (see Fig. 4a
legend).
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The mer2 operon proved to be a complex mosaic. Here, the DNA sequences belonging to the five sequence types, denoted by Greek letters in Fig. 4(b) were identified, which subdivided the Tn5041D mer2 operon into eight segments. The 474 bp sequence at the beginning of this operon belonged to the pKLH2 sequence type (
), with 1 and 2 mismatches found with respect to the end sequences of Tn5036 (Yurieva et al., 1997
; AC Y09025) and the transposon from pKLH2 (Kholodii et al., 1993b
; AC AF213017). In this sequence, a Tn21 subgroup terminal repeat (black arrowhead in Figs 2
and 4b
) and the merR gene sequence were identified. Because no tnp and tnp-end sequences were detected in the Tn5041D insertion, it was concluded that the mer2 operon forms part of a one-ended transposon (named the mer2 transposon). The pKLH2-type sequence (177 bp) was encountered once more in the mer2 operon. Here, it covered the merT gene 3' end and was 100% identical to the equivalent sequence from pKLH2. The DNA fragments of two further sequence types,
and
, came between the
segments in the presumed donor of the mer2 operon (Fig. 4b
, top). The
sequence (78 bp), first detected in the Tn5041D1-containing strain TC97 (Yurieva et al., 1997
; AC Y09210), was most closely related (91% identity) to the equivalent sequences from pKLH2 and pDU1358; and the
sequence (194 bp) belonged to the inferred crossover fragment that was split into two subfragments during the integration of the cassette and was indistinguishable from the equivalent sequence of pDU1358 (Griffin et al., 1987
; AC M24940). A 133 bp DNA fragment of a previously unknown sequence type (
), differing from the equivalent fragments of pKLH2 and Tn5041 by 7% and 22·6% base substitutions, respectively, covered the merP gene 5' end. After this fragment, the continuation of the pDU1358-type sequence with 2 mismatches over 1045 bp was found, which was followed by the DNA sequence (1986 bp) of the extended version of the ß-type sequence first detected in pKLH272/Tn5036 (Yurieva et al., 1997
; ACs Y08992 and Y09025). In the merA region, the sequence of ß was
90% identical to that of pMR26 (Kiyono et al., 1997
; AC D83080) and Tn5058 (AC Y17897), and in the merGBD region, it was 80·7% identical to that of pMR26.
Hybridization to the Tn5041D mer2 cassette-specific probe 7 was found in 4 strains isolated from remote geographical areas of Eurasia and belonging to RFLP types D1, E and F (Tables 1 and 2
). Using appropriate primers, we demonstrated that all these strains contained transposons with the same insertion at the same site as in Tn5041D isolated from a USA strain (Figs 1
and 2
). DNA database searches showed that two HgR Sphingomonas paucimobilis strains isolated from monkeys faeces (USA; Liebert et al., 2000
) contained the mer1x2 and mer2x1 mosaic sequences, of 842 and 1255 bp (ACs AF120972, AF120973, AF120976 and AF120977), identical to the Tn5041D sequences at the boundaries of the mer2 cassette and target (Fig. 4a
). One could imagine repeated events of cassette integration at the same point, but in this case it is impossible to explain why the accompanying gene conversion (Fig. 4b
and the legend) was the same.
Tn5041 variants containing a close relative of Tn21 lacking the In2 integron
Two different strains of P. fluorescens (KHP22 and KHP25) from the same location, which were identified as RFLP type G, hybridized with the Tn21-specific probe 8 (Table 2) and, according to all our tests, contained indistinguishable transposons. Using primers designed for the Tn5041 and Tn21 sequences, the left and right junction fragments from KHP22 and KHP25, of
450 and 610 bp, were amplified and sequenced. These fragments contained (with one mismatch) the merR- and tnpA-proximal terminal sequences of Tn21, respectively (Fig. 2
). The adjacent Tn5041 core sequences formed parts of a single sequence which was interrupted by the insertion of a Tn21-like element (named Tn21
In2); and the target duplication of 5 bp characteristic of the Tn21 subgroup of Tn3 transposons (Grinsted et al., 1990
) was found. It should be noted that the right-arm junction fragment from KHP22 was cloned and partly sequenced previously (Yurieva et al., 1997
; Figs 1
and 2
, the intermittent thick lines). Addition of the sizes of the left-hand-arm and Tn21 fragments from the G type transposons (Table 2
, probes 2 and 8, after PstI) showed that the insertion in these elements was 8·7 kb in length, i.e., it corresponded to the size of Tn21 lacking the In2 integron. The absence of the In2 integron sequences in strains KHP22 and KHP25 was confirmed by Southern hybridization (data not shown). Although only part of the Tn21
In2 mer operon was sequenced, the presence of an additional mer fragment expected due to the Tn21
In2 insertion (Table 2
, the footnote to probe 3) indicated the presence of the other mer genes. Like other Tn21-subgroup transposons (Yurieva et al., 1997
), Tn21
In2 seems to be recombinant due to a cross-over in the res site, since it differs from Tn21 in its left arm (by the presence of the EcoRI and PstI sites and a base substitution) but not in its right arm (Fig. 2
).
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DISCUSSION |
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The fact that one of the ends of the divergent segments creating the mosaic genes (the left end of , and the right end of
and
1) was found to be present at the corresponding end of the predicted Tn5041 att site (att5041) suggested that the site-specific recombination system was involved in this process. The proposed dif-like structure for att5041 (Kholodii et al., 1997
; Fig. 6a
, b
) delineates the probable overlap region, where the strand cleavage and exchange by a XerC/XerD-like recombinase is expected to occur, in the centre of att5041 and predicts (Blakely et al., 2000
) an asymmetric location of the inserted DNA, from the sequence asymmetry in this region. If this structure is accepted, it is impossible to explain the location of the substitutions,
and
/
1, on either side of the att site; and it is difficult to explain why the diversity associated with
and
/
1 does not start just after the overlap region. Re-examination of the DNA sequence of att5041 showed a structure that avoids these discrepancies (Fig. 6c
). In each half of the strong palindrome forming the potential att site, a structure was identified that was similar to the structure (known as the simple site) of the att site from class 1 integrons, attI1 (Partridge et al., 2000
). Moreover, the sequences of the boundaries in the recombinants containing the
and
/
1 segments were attI1 core-site-like, i.e., similar to the boundaries of inserted integron cassettes (Stokes et al., 1997
), so that the putative cross-over position on either side of att5041 coincided with that inferred for
and
/
1 (Fig. 6c
). These data suggested that the segments under consideration could be captured by a site-specific mechanism similar to the class 1 integron IntI1 integrase-dependent mechanism that acquires mobile gene cassettes (Stokes et al., 1997
). Inspection of the att-distal ends of
and
1 (the corresponding end of
was not sequenced) showed no palindrome related to the consensus sequence inferred for the att site (59-base element) of the mobile gene cassette (Stokes et al., 1997
). From these data, a most probable mechanism underlying the formation of
and
/
1 might consist of the fusion between two partly homologous (orfI-att5041-orfQ-containing) replicons at the att5041 sites, mediated by an IntI1-like recombinase, followed by resolution of the co-integrate via homologous recombination in the region adjacent to att5041 (Fig. 6d
). Note that the formation of
could follow either this pathway or via formation of
1 according to the mentioned mechanism (Fig. 6d
) and subsequent shortening of
1 (
1
in Fig. 5a
) via the allelic exchange (Fig. 5b
).
The left-arm recombination events suggest that the partners must have encountered each other in the same cell. Three strains (TC30-1, TC36-1, TC97; Table 1) carried an additional closely related transposon, indicating that this important event does occur relatively frequently in the wild.
Movement of circular DNAs by homologous recombination as a source of mosaic and new genes
The view of the mer operon from Gram-negative bacteria which has emerged (Liebert et al., 1997 , 2000
) is of a dynamic mosaic locus with relatively constant (essential) genes such as merR, merT, merP and merA interspersed with incoming (accessory) genes of known (merB, merC, merF), partly defined [merD, merG, merE (urf-1)] or unknown functions (urf-2). For the functions of the genes, see Mukhopadhyay et al. (1991)
, Hobman & Brown (1997)
, Wilson et al. (2000)
and Liebert et al. (2000)
. Although the non-random location of the accessory genes seems to have been identified, with the hotspots on the immediate 5' and 3' proximal regions of merA (Liebert et al., 1997
, 2000
), little data concerning the mechanistic basis of the gains and/or losses of the mer genes (termed as unusual plug and play recombination events) has been presented so far. There is a fundamental difference between how an operon originally gained an additional gene in the hotspot regions flanking merA and how these arrangements subsequently become distributed into other operons. The primary acquisition implies the involvement of site-specific recombination, whereas the distribution (re-assortment, or secondary acquisition of genes) implies participation of homologous recombination. So far, no consideration has been given in the literature to the cases of acquisition of mer genes via specific sites, e.g., the secondary sites described for the integron DNA integrase (Francia et al., 1993
; Recchia et al., 1994
) or res-like sites permitting a relatively stable integration of circular DNA (Kholodii, 2001
). At the same time, there is indirect evidence for the secondary acquisition of mer genes due to homologous recombination between more conserved adjacent genes (Liebert et al., 2000
), like the process involved in module exchange in lambdoid phages (Campbell, 1994
). Clear evidence for the operation of such a mechanism in the mer system has come from the sequence of a variant of the pKLH2 mer operon (AC AJ251272) which has pKLH2 sequences at the flanks and a long divergent segment with a merB gene (absent in pKLH2) in the centre (G. Kholodii, unpublished data). In Tn5041D and its microderivatives, not only did we find one more example of the insertion of a mer determinant at a hotspot (5' of merA), but also obtained data strongly indicative of another secondary mechanism to acquire new mer gene(s). The specific features of the junction sequences (Fig. 4a
) clearly provide one more example of fusion of circular DNAs by homologous recombination but, for the first time, operating within mer loci. The integration of mer-containing circular DNA, like the one proposed for Tn5041D (Fig. 4b
), probably occurred in other cases too, where the head-to-tail homologous HgR loci were found in the same replicon, e.g., in pDU1358 (Griffin et al., 1987
), pMR26 (ACs D83080 and AB013925), Tn5056 (Mindlin et al., 2001
) and Tn5058 (AC Y17897).
The tandem of two mer operons, similar to the one found in Tn5041D, should be prone to further rearrangements resulting from deletion/excision of one copy due to illegitimate or homologous recombination. With illegitimate recombination, deletion of one of the tandem operons may lead to the emergence of a locus with a new set of mer genes. For instance, the deletion of mer2x1 (IL in Fig. 4b, bottom) could have led to the substitution of a narrow-spectrum HgR locus for a broad-spectrum one. (What is important in this case is that one of the junctions indicative of the participation of homologous recombination in the acquisition of mer genes would have been lost as well.) With homologous recombination, excision of one of the tandem operons would result in a series of both circular mosaic DNAs (D1, D2 and D3 in Fig. 4b
, bottom) and single mosaic operons with overlapping mosaic segments. Such a process may provide an explanation of the fact that the mer2 cassette itself is a genetic mosaic. The same process may account for the origin of different-length mosaic segments of sequence type
that were revealed in the pKLH2-type mer operons (Yurieva et al., 1997
).
It seems that it is not by chance that almost all the regions where we have observed or proposed homologous recombination were regions where hotspots for such a pathway (RecBCD), known as Chi or Chi-like sites, were found. They included the sites shown in Fig. 4(b) and a Chi-like site, 5'-ACTGGTGG-3', occupying nt 13941401 of Tn5041. The data concerning the Tn5041D tandem of the two different mer operons (Fig. 4b
) are especially demonstrative: the mer region contains five occurrences of the hotspots in 5 kb versus 1·4 occurrences in 5 kb inferred from 20 sequenced mer operons (Liebert et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 22 April 2002;
revised 9 July 2002;
accepted 16 July 2002.