CSIRO Molecular Science, Sydney Laboratory, PO Box 184, North Ryde, NSW 1670, Australia1
School of Biological Sciences, Macquarie University Sydney, NSW 2109, Australia2
Author for correspondence: Ruth M. Hall. Tel: +1 612 9490 5162. Fax: +1 612 9490 5005. e-mail: ruth.hall{at}molsci.csiro.au
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: attI, integron, integrase, site-specific recombination, gene cassettes
Abbreviations: 2°rs, secondary recombination site; 59-be, 59-base element; 5'-CS, 5' conserved segment; Ap, ampicillin; Cm, chloramphenicol; IntI1, integrase of class 1 integrons; Nx, nalidixic acid; Sm, streptomycin; Su, sulphamethoxazole; Tc, tetracycline; Tp, trimethoprim
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four classes of integron, each encoding a distinct IntI integrase, have been identified (Stokes & Hall, 1989 ; Sundström & Sköld, 1990
; Arakawa et al., 1995
; Clark et al., 1997
; Mazel et al., 1998
). However, only the reactions catalysed by the IntI1 integrase encoded by class 1 integrons have been studied in detail using an in vivo experimental system devised by Martinez & de la Cruz (1990
). As well as the integrative and excisive reactions between attI1 and a 59-be (Martinez & de la Cruz, 1990
; Recchia et al., 1994
; Hall et al., 1999
), IntI1 can also catalyse both integrative and excisive recombination events between two 59-be (Martinez & de la Cruz, 1990
; Hall et al., 1991
; Stokes et al., 1997
). Integrative recombination between two attI1 sites has also been documented (Recchia, 1996
; Hansson et al., 1997
). Recombination between secondary sites (2°rs) and 59-be (Francia et al., 1993
, 1997
; Recchia et al., 1994
; Stokes et al., 1997
) or attI1 (Hansson et al., 1997
) also occurs at a low frequency.
The architecture of the attI1 site differs from that of the 59-be sites (Fig. 1). The 59-be family comprises a large number of sites that have diverse sequences and lengths, but share some common features (Hall et al., 1991
; Collis & Hall, 1992b
; Stokes et al., 1997
). All include about 25 bp at each end that conform to consensus sequences that are imperfect inverted repeats of one another. The organization of each consensus region is similar to that of a simple site used by other integrases and comprises a pair of inversely oriented integrase-binding domains, separated by a spacer of 7 or 8 bp (Stokes et al., 1997
; Fig. 1b
). In contrast, the attI sites of different integron classes do not share most of these features, nor do they share substantial sequence identity with each other (Recchia et al., 1994
; Collis et al., 1998
; Hall et al., 1999
). For attI1, 2 and 3 only one potential simple-site region can be detected by inspection of the sequences, whereas for attI4 even this simple site is not obvious.
|
The length of the 5'-CS region (in upper case in Fig. 1a) that is required for maximal recombination activity has been delineated experimentally for the attI1 site (Recchia et al., 1994
) using an assay that was shown to measure only recombination between attI1 and a 59-be. Maximal activity of the attI1/qacE site was observed with fragments containing at least 64 bp of the 5'-CS, and smaller fragments which include 33 or 31 bp of the 5'-CS had greatly reduced activities (Recchia et al., 1994
). However, in a subsequent study where recombination between two attI1 sites was measured (Hansson et al., 1997
), it was found that no more than 14 bp of the 5'-CS, i.e. only the simple-site region of the attI1 site, was needed for maximal recombination efficiency with a complete attI1 site. More recent studies have examined the binding of IntI1 to DNA fragments that include all or part of the attI1 region. As expected, two molecules of IntI1 bind to the simple-site region (Gravel et al., 1998
). However, IntI1 binds to two further regions located to the left of the simple site (Collis et al., 1998
; Gravel et al., 1998
). These binding sites (Fig. 1a
), one of which is a strong IntI1-binding site and the other a weak binding site, overlap the direct repeats DR1 and DR2 (Collis et al., 1998
; Hall et al., 1999
).
In this study the differences in the reported length of the attI1 site have been re-examined. The features of the attI1 site essential for efficient recombination with either a 59-be or a second attI1 site have been assessed using conditions where only one of these reactions can occur. Rare recombinants have been used to more accurately locate the left-hand IntI1-binding domain of the attI1 simple site.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids.
Plasmids used in this study are listed in Table 1. Fragments containing various lengths of the attI1 recombination site were generated by PCR using pRMH232 as a template and the following primers: RH205, 5'-CGTTACGCCGTGGGTCGACGTTTGAT-3' (attI1 -78 to -53); RH206, 5'-GCTGTGAGCAATTATCAGCTGAGTGC-3' (qacE +7 to +32); RH211, 5'-GGAGCAGCAACGATGTTAC-3' (attI1 -47 to -29), where bases in bold type indicate changes from the published sequence. These fragments were treated with the Klenow fragment of DNA polymerase I and cloned directly into the unique EcoRV site (GenBank accession no. X06403, position 1680; Rose, 1988
) of pACYC184 (CmR TcR; Chang & Cohen, 1978
; Table 1
). The plasmids recovered were sequenced to confirm that they had the required inserts and to determine their orientation. Orientation 1 refers to plasmids in which the right-hand end of each composite site, as shown in Fig. 1
, is closest to the origin of replication of pACYC184, while orientation 2 is the opposite. One clone in each orientation was retained. pRMH653 and pRMH654 were identified in this process and presumably arose by spontaneous deletion of part of the RH211/RH206 PCR product. pRMH653 includes only 36 bp of the 5'-CS and 32 bp of qacE, while pRMH654 has 45 bp of the 5'-CS and none of the qacE sequence, with base 1 (G) of the 5'-CS adjacent to the A at position 1682 on the bottom strand of pACYC184 (accession no. X06403; Rose, 1988
).
|
The plasmid pRMH560, a derivative of R388 which has lost both the dfrB2 and orfA cassettes, was generated by IntI1-mediated excision of cassettes, as described previously (Collis & Hall, 1992a ).
DNA procedures.
Plasmid DNA was isolated using an alkaline lysis method (Birnboim & Doly, 1979 ) or a Wizard Miniprep kit (Promega). Restriction enzyme digests were carried out according to the manufacturers instructions and fragments were separated on 1% (w/v) or 0·8% (w/v) agarose gels. EcoRI-digested bacteriophage SPP-1 DNA (Bresatec) was used as size markers. BamHI-digested R388 or pRMH560 were also used as markers for mapping cointegrates. DNA fragments for cloning were isolated from agarose gels using a Geneclean II kit (Bio101). Plasmid DNA for sequencing was annealed to primers according to the method of Jones & Schofield (1990
). Double-stranded DNA sequencing was performed using a Sequenase kit, version 2.0 (United States Biochemical) with reaction mixtures containing dITP.
Conduction assays.
Recombination efficiency was determined using a conduction (mating-out) assay (Martinez & de la Cruz, 1988 , 1990
; Hall et al., 1991
; Stokes et al., 1997
) to measure conduction of a pACYC184-based test plasmid (containing a cloned attI1 site) from the donor strain UB1637 (RecA- SmR) to the recipient strain UB5201 (RecA- NxR). Donor cells containing the Tra+ plasmid R388 (TpR SuR) or its cassette-free derivative pRMH560 (SuR; Table 1
) were first transformed with the pACYC184-based test plasmid. pSU2056 (ApR; Martinez & de la Cruz, 1990
) was then introduced to supply IntI1 integrase in trans. Single colonies of donor strains grown on LB agar were screened for ApR and CmR to confirm that each isolate assayed had a full complement of plasmids after purification.
For matings, 0·1 ml portions of stationary-phase cultures of donor and recipient cells were mixed, spread over the surface of an LB agar plate, and incubated overnight at 37 °C. Cells were then harvested and transconjugants selected on agar containing Tp and Nx (R388) or Su and Nx (pRMH560), while cointegrates were selected on plates containing Cm and Nx. The conduction frequency was expressed as the fraction of the total transconjugants (TpR or SuR) that contained cointegrates (CmR). Where possible, three isolates of each donor strain were assayed on at least two occasions and the mean value calculated. For conduction frequencies below 10-6, where spontaneous mutation of the donor to NxR can contribute significantly to the number of CmR NxR colonies detected, all colonies were screened for SmR, which is characteristic of the donor. For a number of cointegrates, the site of insertion of the test plasmid was determined by restriction mapping of the cointegrate DNA with BamHI and HindIII, as described previously (Hall et al., 1991 ; Recchia et al., 1994
; Stokes et al., 1997
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A further plasmid, pRMH654, contains a fragment that includes bases -45 to -1 of the 5'-CS and hence the strong binding site, but has lost the part of the simple-site region that lies to the right of the recombination crossover (Fig. 1a). This plasmid supports recombination with a 59-be in R388 at a frequency of 7·4x10-5 (Table 2
), which is only slightly higher than that observed with pMAQ79 (-31 to +198) and pRMH751 (-25 to +32). This finding indicates that the presence of the strong binding site is at least as important in determining the efficiency of recombination with a 59-be site as the sequence of the core site (consensus GTTRRRY) surrounding the crossover point, which is replaced by GATCCCG in pRMH654. Mapping of 20 recombinants showed that in half of them recombination had occurred between attI1 in pRMH654 and the orfA/qacE 59-be, indicating that this truncated attI1 site is still recognized in preference to any 2°rs. Most of the remaining cointegrates appeared to be due to recombination between the orfA/qacE 59-be in R388 and 2°rs in pRMH654.
To investigate if the shorter length for attI1 determined by Hansson et al. (1997 ) is due to the fact that they analysed the attI1xattI1 reaction, the assay used here was modified to measure only recombination between two attI1 sites. Recombinants formed via this reaction are not seen if the fragments are cloned in orientation 1 in pACYC184 but are recovered if the fragment is cloned in the opposite orientation (Recchia, 1996
; C. M. Collis, G. D. Recchia & R. M. Hall, unpublished). Therefore fragments of attI1/qacE with 78, 64, 47 and 25 bp of the 5'-CS were recloned in the opposite orientation. To ensure that only attI1xattI1 recombination could occur, a derivative of R388 that has lost the dfrB2 and orfA cassettes (pRMH560), leaving only the attI1/qacE site, was used in place of R388 in the donor strains. The recombination frequency for fragments containing the full attI1 site was at least 20-fold lower than for recombination of the equivalent fragments with a 59-be (Table 3
), consistent with a lower efficiency for attI1xattI1 events. A small decrease in activity was observed when the fragment length was shortened to include only 25 bp of the 5'-CS (Table 3
). However, the recombination frequency for this fragment (pRMH752) was still 65-fold higher than that for the vector alone, which in this case represents recombination between the attI1 site in pRMH560 and 2°rs in the vector. Restriction mapping of 12 cointegrates formed with pRMH752 revealed that, in all of them, recombination had occurred between the two attI1 sites.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When Hansson et al. (1997 ) replaced R388
1, which is equivalent to pRMH560, with R388 and the attI1 region tested included 36 or more than 200 bp of the 5'-CS, they observed a substantially higher recombination frequency than for the reaction with R388
1. This is presumably because the more efficient attI1x59-be reaction can now occur as well as attI1xattI1 recombination. These data are therefore not equivalent to any of the data presented here, where only one of these events is tested at one time. Shorter fragments containing 27, 20 or 14 bp of the 5'-CS tested by Hansson et al. (1997
) were at least 30-fold less active. Although these results are in broad agreement with those presented here, the residual recombination level is higher than that described here and presumably represents attI1xattI1 recombination, rather than the residual level of attI1x59-be recombination.
Previous studies have shown that in recombination between two 59-be or between attI1 and a 59-be, the predominant reaction involves the 1R site in the 59-be and site 1 in attI1 (Martinez & de la Cruz, 1990 ; Hall et al., 1991
; Recchia et al., 1994
; Stokes et al., 1997
). Furthermore, only a single strand-exchange event occurs (Stokes et al., 1997
) and this feature distinguishes reactions catalysed by the IntI1 integrase from site-specific recombination events catalysed by other integrases, where two strand-exchange events staggered by 68 bp normally occur (Sadowski, 1993
; Hallet & Sherratt, 1997
). The preferential recognition of site 1 in attI1 and 1R in a 59-be indicates that features of both attI1 and 59-be type sites ensure that the two sites participating in a recombination event are correctly oriented. Those features appear to lie entirely within the simple-site region of attI1, as recombinants arising from a crossover in site 2 of the attI1 simple site, which can be identified by restriction mapping, were not recovered amongst 19 recombinants where the partner site was the orfA/qacE 59-be or 12 recombinants where the complete attI1 site was the partner.
However, rare recombinants that arose via strand exchange within the 2R site in one 59-be and the 1R site in a second 59-be (Stokes et al., 1997 ) or the 1L site of a 59-be and the attI1 site 1 (Hall et al., 1999
) have been observed previously. In both cases, the sequences of the junctions within the recombinants revealed that only a single strand-exchange event had occurred. This indicates that strand exchange can occur, albeit with very low efficiency, at sites other than 1R in a 59-be but in both of these cases the crossover is in the normal location in the partner site. Such rare recombinants have been used to locate the 2R core site of 59-be (Stokes et al., 1997
).
A further type of rare recombinant that has arisen via recombination between the left-hand IntI1-binding domain (inverse core site) of the attI1 simple site (2 in Fig. 1a) and a 2°rs in pACYC184 was recovered here. These recombinants allow a more precise definition of the attI1 simple site than was possible from inspection of the sequence. However, the sequences of the regions in pACYC184 that are involved in the recombination events do not conform to the Ga/tTa/ca/t (Francia et al., 1993
) or Ga/tT (Recchia et al., 1994
) consensus for 2°rs that participate in reactions with 59-be. Although five bases were conserved in each of the three sites identified here (Fig. 2d
), further examples are needed to establish if the conservation is significant. The reaction that gave rise to these recombinants is likely to be extremely rare as the frequency for all recombination events involving attI1 and 2°rs observed here is extremely low (Table 3
). It is approximately 20-fold lower than that for a 59-be with 2°rs (Table 2
) and similar background frequencies were observed by Hansson et al. (1997
). Only two 2°rs that recombined at the normal location in attI1 (1 in Fig. 1a
) have been located by sequencing and both include a region that is closely related to the 7 bp core site consensus (Hansson et al., 1997
). Further examples are needed to determine if the consensus for 2°rs that recombine with attI1 is the same as that for 2°rs that recombine with 59-be. The fraction of 2°rs recombinants that arise via a crossover at the core site (1) or inverse core site (2) of the attI1 simple site also remains to be determined.
The proposed route that leads to substitution of the attI1 simple-site spacer for the bulk of a 59-be is also likely to be extremely uncommon. The schematic model in Fig. 3(c) shows a requirement for an unusual recombination crossover between two core sites (the attI1 site 2 and 1L in the 59-be), neither of which is normally the location for strand exchange. Excision of a cassette from a class 1 integron via crossover between 1L in the 59-be and site 2 in attI1 would give rise in a single step to a circularized cassette containing the unusual hybrid simple site and leave behind the remainder of the 59-be fused to site 2 of attI1. Reintegration of this cassette into a complete attI1 site with the crossover at the normal position would lead to the configuration seen in the known examples. Alternatively, recombination between the 1L site of the 59-be in a cassette contained in one integron with the attI1 site 2 in a second integron would yield similar products in one step. It is likely that the simple site created as a consequence of these events can participate in IntI1-mediated recombination events. However, the efficiency of recombination with either the attI1 site or with a 59-be partner might be expected to be low (equivalent to pRMH751, i.e. -25 to +32 of 5'-CS) and this could provide a strong linkage between the cassette that ends with the simple site and the downstream cassette.
The fact that the architecture of attI1 differs from that of 59-be appears to play a role in ensuring that cassettes are preferentially integrated adjacent to the attI1 site of a class 1 integron. A similar effect is likely to pertain to the attI2, 3 and 4 sites, but the sequences of the known attI sites are not closely related and architecture of these sites has yet to be examined experimentally.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avila, P. & de la Cruz, F. (1988). Physical and genetic map of the IncW plasmid R388. Plasmid 20, 155-157.[Medline]
Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.[Abstract]
Cameron, F. H., Groot Obbink, D. J., Ackerman, V. P. & Hall, R. M. (1986). Nucleotide sequence of the AAD(2') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388. Nucleic Acids Res 14, 8625-8635.[Abstract]
Chang, A. C. Y. & Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J Bacteriol 134, 1141-1156.[Medline]
Clark, C. A., Purins, L., Kaewrakon, P. & Manning, P. A. (1997). VCR repetitive sequence elements in the Vibrio cholerae chromosome constitute a mega-integron. Mol Microbiol 26, 1137-1143.[Medline]
Collis, C. M. & Hall, R. M. (1992a). Site-specific deletion and rearrangement of integron insert genes catalysed by the integron DNA integrase. J Bacteriol 174, 1574-1585.[Abstract]
Collis, C. M. & Hall, R. M. (1992b). Gene cassettes from the insert region of integrons are excised as covalently closed circles. Mol Microbiol 6, 2875-2885.[Medline]
Collis, C. M., Grammaticopoulos, G., Briton, J., Stokes, H. W. & Hall, R. M. (1993). Site-specific insertion of gene cassettes into integrons. Mol Microbiol 9, 41-52.[Medline]
Collis, C. M., Kim, M.-J., Stokes, H. W. & Hall, R. M. (1998). Binding of the purified integron DNA integrase IntI1 to integron-and cassette-associated recombination sites. Mol Microbiol 29, 477-490.[Medline]
de la Cruz, F. & Grinsted, J. (1982). Genetic and molecular characterisation of Tn21, a multiple resistance transposon from R100-1. J Bacteriol 151, 222-228.[Medline]
Francia, M. V., de la Cruz, F. & García Lobo, M. (1993). Secondary sites for integration mediated by the Tn21 integrase. Mol Microbiol 10, 823-828.[Medline]
Francia, M. V., Avila, P., de la Cruz, F. & García Lobo, M. (1997). A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase. J Bacteriol 179, 4419-4425.[Abstract]
Gravel, A., Fournier, B. & Roy, P. H. (1998). DNA complexes obtained with the integron integrase IntI1 at the attI1 site. Nucleic Acids Res 26, 4347-4355.
Hall, R. M. & Collis, C. M. (1995). Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol 15, 593-600.[Medline]
Hall, R. M. & Collis, C. M. (1998). Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist Updates 1, 109-119.
Hall, R. M., Brookes, D. E. & Stokes, H. W. (1991). Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point. Mol Microbiol 5, 1941-1959.[Medline]
Hall, L. M. C., Livermore, D. M., Gur, D., Avoka, M. & Akalin, H. E. (1993). OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) ß-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother 37, 1637-1644.[Abstract]
Hall, R. M., Collis, C. M., Kim, M.-J., Partridge, S. R., Recchia, G. D. & Stokes, H. W. (1999). Mobile gene cassettes in evolution. Ann N Y Acad Sci 870, 68-80.
Hallet, B. & Sherratt, D. J. (1997). Transposition and site-specific recombination: adapting DNA cut-and-paste mechanisms to a variety of genetic rearrangements. FEMS Microbiol Rev 21, 157-178.[Medline]
Hansson, K., Sköld, O. & Sundström, L. (1997). Non-palindromic attI sites of integrons are capable of site-specific recombination with one another and with secondary targets. Mol Microbiol 26, 441-453.[Medline]
Jones, D. S. C. & Schofield, J. P. (1990). A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing. Nucleic Acids Res 18, 7463-7464.[Medline]
Martinez, E. & de la Cruz, F. (1988). Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol Gen Genet 211, 320-325.[Medline]
Martinez, E. & de la Cruz, F. (1990). Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J 9, 1275-1281.[Abstract]
Mazel, D., Dychinco, B., Webb, V. & Davies, J. (1998). A distinctive class of integron in the Vibrio cholerae genome. Science 280, 605-608.
Ouellette, M. & Roy, P. H. (1987). Homology of ORFs from Tn2603 and from R46 to site-specific recombinases. Nucleic Acids Res 15, 10055.[Medline]
Poirel, L., Le Thomas, I., Naas, T., Karim, A. & Nordmann, P. (2000). Biochemical sequence analyses of GES-1, a novel class A extended spectrum ß-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob Agents Chemother 44, 622-632.
Recchia, G. D. (1996). Mobile gene cassettes and integrons: evolutionary and recombinational studies. PhD thesis, Macquarie University Sydney, Australia.
Recchia, G. D. & Hall, R. M. (1995). Gene cassettes: a new class of mobile element. Microbiology 141, 3015-3027.[Medline]
Recchia, G. D., Stokes, H. W. & Hall, R. M. (1994). Characterisation of specific and secondary recombination sites recognised by the integron DNA integrase. Nucleic Acids Res 22, 2071-2078.[Abstract]
Rose, R. E. (1988). The nucleotide sequence of pACYC184. Nucleic Acids Res 16, 355.[Medline]
Sadowski, P. (1993). Site-specific genetic recombination: hops, flips, and flops. FASEB J 7, 760-767.
Stokes, H. W. & Hall, R. M. (1989). A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol 3, 1669-1683.[Medline]
Stokes, H. W., OGorman, D. B., Recchia, G. D., Parsekhian, M. & Hall, R. M. (1997). Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Mol Microbiol 26, 731-745.[Medline]
Sundström, L. & Sköld, O. (1990). The dhfrI trimethoprim resistance gene of Tn7 can be found at specific sites in other genetic surroundings. Antimicrob Agents Chemother 34, 642-650.[Medline]
Tolmasky, M. E. (1990). Sequencing and expression of aadA, bla, and tnpR from the multiresistance transposon Tn1331. Plasmid 24, 218-226.[Medline]
Received 31 May 2000;
revised 6 August 2000;
accepted 14 August 2000.