CSIRO Molecular Science, Riverside Life Sciences Centre, Riverside Corporate Park, North Ryde, NSW 2113, Australia
Correspondence
Ruth Hall
Ruth.Hall{at}mmb.usyd.edu.au
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ABSTRACT |
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Present address: School of Molecular and Microbial Biosciences, Biochemistry and Microbiology Building G08, University of Sydney, Sydney 2006, Australia.
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INTRODUCTION |
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The IntI recombinases encoded by integrons form a distinct family within the tyrosine recombinase superfamily (Nunes-Düby et al., 1998) and contain an additional motif that appears to be a signature for IntI family members (Messier & Roy, 2001
; Nield et al., 2001
). Over 20 distinct IntI have been identified to date (Collis et al., 2002b
), and in most cases, the intI gene is in close proximity to one or more identifiable gene cassettes, indicating that it is indeed part of an integron. Some of these integrons are part of the mobile gene pool (Stokes & Hall, 1989
; Arakawa et al., 1995
; Recchia & Hall, 1995
; Hochhut et al., 2001
), some are a feature of a bacterial chromosome (Rowe-Magnus et al., 2001
; Drouin et al., 2002
) and the location of others has not been established (Nield et al., 2001
; Vaisvila et al., 2001
). Known members of the IntI family share as little as 35 % sequence identity (see Collis et al., 2002b
for a compilation), indicating a long evolutionary history for this natural gene cloning system.
Members of the tyrosine recombinase or integrase superfamily use a tyrosine residue as the nucleophile in their strand exchange reactions and share similarities in a large C-terminal domain of 180 aa which contains six conserved residues that are part of the catalytic site (Nunes-Düby et al., 1998
; Grindley, 1997
; Yang & Mizuuchi, 1997
; Grainge & Jayaram, 1999
). A few members of the tyrosine recombinase superfamily have been extensively studied. The Cre recombinase of phage P1, which circularizes the phage genome and resolves multimers, FLP, which inverts a segment of the 2 µm plasmid of yeast, and XerC and XerD, which act together to resolve chromosomal dimers formed during replication, each recombine two copies of the same simple site using two-strand exchange reactions that occur in an ordered fashion (Sadowski, 1986
; Stark et al., 1992
; Gopaul & Van Duyne, 1999
). The simple sites (also called core sites or the functional core) are approximately 30 bp long and consist of a pair of inverted repeats that are Int-binding sites, separated by a spacer (also called an overlap or central region) of 68 bp between the positions of the top and bottom strand exchanges (Sadowski, 1986
; Stark et al., 1992
; Nash, 1996
). In the case of the phage
and HK022 Int recombinases, a simple site in the bacterial chromosome recombines with a complex site in the phage genome on integration and two complex sites at the boundaries of the integrated phage genome recombine on excision (Nash, 1996
; Weisberg et al., 1999
). These reactions involve an additional domain in the Int protein and its binding sites as well as accessory binding factors and their sites. These additional binding sites are part of the complex site structure. Other examples where complex site arrangements and accessory factors are required include the resolution of plasmid multimers by XerCD, which is constrained to a resolution pathway by the accessory factors (Colloms et al., 1996
, 1998
; Alen et al., 1997
).
Both the 59-be sites in gene cassettes and the attI1 site in class 1 integrons are also complex sites that include both a discernable simple site and additional IntI-binding sites (Fig. 1a), suggesting that in this system further molecules of IntI act as accessory factors when bound to the additional sites. The cassette-associated 59-be sites have a set of common features that permit them to be identified, despite substantial sequence diversity and differences in length (Hall et al., 1991
; Collis & Hall, 1992a
; Stokes et al., 1997
). Each 59-be appears to consist of two pairs of inversely oriented IntI-binding sites, each making up a simple site, separated by a segment that has a variable length and sequence but generally includes an inverted repeat (Stokes et al., 1997
). These features (Fig. 1a
) appear to be generally recognized by IntI-type tyrosine recombinases, as the same 59-be act as sites for several different IntI that are less than 50 % identical at the amino acid sequence level (Martinez & de la Cruz, 1990
; Hall et al., 1991
; Stokes et al., 1997
; Collis et al., 2001
, 2002a
; Drouin et al., 2002
; Hansson et al., 2002
). However, IntI recombinases can distinguish between the two simple sites and recombination normally occurs exclusively within the right-hand simple site (Martinez & de la Cruz, 1990
; Hall et al., 1991
; Stokes et al., 1997
). IntI recombinases also appear to differ from other tyrosine recombinases in that they catalyse only a single strand exchange which occurs either between the G and TT in the right hand core site (vertical arrow in Fig. 1
) or between the AA and C on the complementary strand (Hansson et al., 1997
; Stokes et al., 1997
). By analogy with the XerCD system (Colloms et al., 1996
), strand exchange on the complementary strand seems more likely.
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We have recently examined the properties of the only known example of a class 3 integron (Arakawa et al., 1995) and demonstrated that both integrative and excisive recombination between two 59-be or between attI3 and a 59-be is catalysed by the class 3 recombinase, IntI3 (Collis et al., 2002a
). The general properties of IntI3 were the same as those of IntI1. However, we have also shown that IntI1 and IntI3 do not recognize the non-cognate attI2 and attI3 or attI1 and attI2 sites in integrative reactions with a 59-be (Collis et al., 2002b
). The stringent requirement of each IntI for the cognate attI site contrasts with their shared ability to recognize many different 59-be. This and the complex and quite distinct architecture of attI1 and 59-be recombination sites raises questions as to how these sites are recognized by the IntI recombinases and how they come together to participate in productive recombinational exchanges. Here, we have sought to identify the general features of attI sites by examining the features of a second attI site. The structureactivity relationships of the attI3 site from the class 3 integron were determined using IntI3-catalysed cointegration assays and binding of purified IntI3 to attI3-containing fragments of different lengths. The binding sites were further localized by examining interference with binding caused by methylation of bases in the major groove.
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METHODS |
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pRMH860, pRMH861, pRMH862 and pRMH872, which contain attI3 linked to the 198-bp qacE fragment (Table 1), were recovered from cointegrates formed between pRMH841, pRMH842, pRMH843 or pRMH871, respectively and the orfA/qacE 59-be element in pMAQ495 (Collis et al., 1998
) by religation of the appropriate HindIII fragment. pRMH962 and pRMH963 were constructed by end-filling the small BamHIClaI fragments from pRMH862 and pRMH872, respectively, and cloning them into the EcoRV site of pACYC184 in orientation 2 (Collis et al., 2001
). To make pRMH894 and pACYC184
, the large AvaI fragment of pRMH836 or the large BamHIEcoRV fragment of pACYC184, respectively, were religated after end-filling.
Culture conditions.
Bacteria were routinely cultured as described previously (Collis et al., 2001) in LB medium or on LB agar supplemented as appropriate with ampicillin (100 µg ml1), chloramphenicol (Cm, 25 µg ml1), kanamycin (5 µg ml1), nalidixic acid (Nx, 25 µg ml1), spectinomycin (25 µg ml1), streptomycin (Sm, 25 µg ml1); or sulphamethoxazole (Su, 25 µg ml1). Antibiotics were obtained from SigmaAldrich.
Conduction assays.
The frequency with which a pACYC184-based plasmid (CmR) containing a cloned attI3 fragment was transferred from the donor strain UB1637 (RecA SmR) to the recipient DH5 (RecA NxR) after IntI3-catalysed cointegration with a conjugative plasmid based on R388 (Fig. 2
) was determined as described previously (Collis et al., 2001
). IntI3 was supplied in trans using pRMH851. Transconjugants were selected on agar containing Su and Nx and recombinants on Cm and Nx, and the conduction frequency, which is equivalent to the cointegration frequency, was expressed as the ratio of CmR to SuR transconjugants. Cointegrates were mapped using digestion with either BamHI+HindIII or with NcoI.
Oligonucleotides.
Oligonucleotides were synthesized by Geneworks (Adelaide). Oligonucleotides from the class 3 integron conserved segment are listed below with positions in GenBank accession no. AF416297 in parentheses, extensions are shown in bold type and relevant restriction sites (BamHI or HindIII) are underlined. HS245, 5'-CGGGATCCCGGTGCCGTGCGACTTTGTTTAAC-3' (17781800); HS273, 5'-CGGGATCCATTTACAGGATTGATTTCAAAC-3' (17471767); HS274, 5'-CGGGATCCATTTGTGGGTATCCGGTGTTTG-3' (18091828); RH235, 5'-CGGGATCCGGTGTTTGGTCAGAT-3' (18171835); RH238, 5'-GATACCCACAACCGTGGTCGTTAAAC-3' (18191794); RH245, 5'-CGGGATCCCGACCTGTTCATGCATCCAGT-3' (17211739); RH263, 5'-CGAAGCTTGAACACCGGATACCCACAACCGTGGTCGTTAAACAAAGTCGCA-3' (18261785). RH69, complementary to sequence in the qacE cassette, is 5'-GCTGTGAGCAATTATaaGCTTAGTGC-3', equivalent to 53945369 in M95287 where aa is GT. Oligonucleotides complementary to the pACYC184 sequence are RH220 (5'-ACCATACCCACGCCGAAACAAGCGCT-3') and RH244 (5'-TTCTCATGTTTGACAGCTTATCATCG-3') found at positions 20141989 and 14961521, respectively, in X06403.
DNA procedures.
DNA for gel shift assays was prepared using the Wizard maxipreps (Promega) or Jetstar (Genomed) DNA purification systems. DNA fragments labelled at both ends were prepared by digesting 1·5 µg DNA from a pUC-derived plasmid containing the cloned fragment with BamHI+HindIII and end-filling with (-32P) dATP (Amersham Biosciences) and 100 µM dGTP, dCTP and dTTP and Klenow fragment of DNA polymerase I (Roche Diagnostics). The appropriate fragment was then recovered by electrophoresis through a 2·5 % agarose gel and purified using a Geneclean II kit (Bio 101). Plasmid DNA for automated sequencing was purified using Wizard minicolumns (Promega). Sequencing was carried out at the Macquarie Sequencing Facility, Department of Biological Sciences, Macquarie University Sydney, Australia, using an ABI Prism 377 DNA sequencer (PE Biosystems) and Big Dye terminator mixes. Cointegrate DNA for restriction analysis was isolated using an alkaline lysis method as described previously (Collis et al., 2001
).
DNA binding assays.
IntI3 with an N-terminal FLAG extension (F-IntI3) was purified as described previously for F-IntI1 (Collis et al., 1998, 2002b
). Briefly, F-IntI3 expression was induced in a 200 ml culture of DH5
containing pMAQ487 by the addition of IPTG to 0·5 mM. After growth for a further 2 h, a cell lysate was made and the soluble fraction passed through a column containing the M2 anti-FLAG affinity resin (International Biotechnologies), washed and eluted using the FLAG peptide (International Biotechnologies). After further purification using DEAE-Sephacel, F-IntI3 was concentrated using a Centricon 10 filter (Amicon) and stored at 70 °C in 20 mM Tris/HCl, pH 7·5, 0·1 M KCl, 4 mM dithiothreitol, 0·05 % (v/v) Tween 20, 10 % glycerol. The preparation included some GroEL, but the unfolded fraction of IntI3 was estimated to be less than 10 %.
Each 15 µl binding reaction contained 33 mM Tris/HCl, pH 7·5, 50 mM KCl, 9 % (v/v) glycerol, 7 µg poly(dG-dC) ml1 (Amersham Biosciences), 100 nM (approx.) F-IntI3 and 0·22 ng end-labelled DNA fragment. After incubation at room temperature for 15 min, the samples were electrophoresed through a 5 % non-denaturing polyacrylamide gel at 200 V for 1·5 h at room temperature, using 23 mM Tris-borate, 0·25 mM EDTA, pH 8, in the gel and as running buffer. After fixing and drying, gels were exposed to X-ray film overnight with an intensifying screen or to a PhosphorImager screen (Molecular Dynamics).
Methylation interference.
The attI3 fragments were labelled at one 3' end, partially methylated at guanine residues using dimethyl sulphate and purified as described previously (Collis et al., 1998). This DNA was used as the substrate for a binding reaction with F-IntI3 as described above, scaled up four- to fivefold. After electrophoresis the bands were excised from the gel and the DNA was recovered by elution into water and precipitated with ethanol. Pellets were heated in 100 µl 1 M piperidine at 90 °C for 30 min and the DNA fragments were separated by electrophoresis on a 12 % acrylamide/7 M urea denaturing gel.
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RESULTS |
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Recombination of two attI3 sites
Two attI1 sites are able to recombine (Hansson et al., 1997; Partridge et al., 2000
), but the frequency is about 100-fold lower than for attI1 with 59-be events. To facilitate detection of these events with attI3, a conjugative plasmid that contains only the full-length attI3 site (pRMH961; Fig. 2
) was constructed and used together with plasmids that have attI3 fragments cloned in orientation 2 (as defined by Collis et al., 2001
) to permit recombination with the attI3 site in pRMH961. Using the full-length attI3 site, attI3 with attI3 recombination occurred (Table 3
) at a frequency equivalent to approximately 1 % of that observed for attI3 recombination with a 59-be (Table 2
). Mapping of 12 cointegrates confirmed that the recombination observed involved two attI3 sites. A fragment containing only the simple site portion of the attI3 site also recombined with the complete site in pRMH961 at a similar, though slightly lower, frequency. The background level of recombination between the attI3 site in pRMH961 and secondary sites in pACYC184 was also at least 10-fold lower than for a 59-be with secondary sites.
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The 29 to +15 fragment, which includes only the simple site, shows only very weak retarded bands potentially corresponding to one and two IntI3 monomers bound. This is consistent with the loss of a further binding site, at least part of which is located in the 37 to 30 span, which appears to be important for the much stronger binding seen with longer fragments. Binding to a fragment containing only the left-hand portion of attI3 was also examined. A fragment that includes the 125 to 27 span formed two retarded products (Fig. 4c) and a 68 to 20 fragment gave a similar pattern (data not shown). Binding to these fragments was stronger than binding to the right-hand fragment and a strong band (one IntI3 bound) and a weaker band (two IntI3 bound) were observed, consistent with the presence of a strong and a weak binding site. Together these results localize site 3 to between positions 37 and 27 and suggest that it is the strongest binding site.
Correlating these gel shift data with the activity data in Table 2, it is possible to conclude that, as for attI1, at least three binding sites corresponding to the simple site (sites 1 and 2) and the adjacent binding site 3 are absolutely required for attI3 to recombine with a 59-be site. The fourth site corresponds to site 4 of attI1 and enhances activity a further 10-fold.
Methylation interference of IntI3 binding
Methylation interference was used to confirm the binding stoichiometry and to localize the IntI3-binding sites more accurately. DNA fragments were labelled at one end, G residues were methylated using dimethyl sulphate and DNA recovered from retarded bands was examined for underrepresentation of residues where methylation interferes with the binding of IntI3. Complexes I and II formed with the 99 to +15 fragment were examined using the bottom strand only (Fig. 5a). In complex I, G residues at 42, 43 and 46 were underrepresented in comparison to the free DNA, indicating that this complex consists mainly of one IntI3 monomer bound to site 4. The G residues that interfered with binding are adjacent to a discernable 7 bp core site, GTTTAAC, in the sequence at 52 to 46 that is likely to form the left end of site 4 (Fig. 5c
) and defines its orientation, as a 7-bp core site is normally found at the beginning of IntI-binding sites (Collis et al., 1998
; Gravel et al., 1998
; Stokes et al., 1997
). In complex II, residues that were underrepresented were in the vicinity of site 2, the predicted inversely oriented site, and site 3, indicating that binding to these two sites predominates in this species, which is normally far more abundant than complex I (Fig. 4
). No complex III was recovered.
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DISCUSSION |
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It remains to be established whether binding of IntI1 to attI1 proceeds in a similar way. In our study of IntI1 binding (Collis et al., 1998), only binding to sites 3 and 4 was observed, though patterns equivalent to those seen here with IntI3/attI3 were seen occasionally (C. M. Collis, unpublished). A pattern of four bands with IntI1/attI1 has also been reported by others (Gravel et al., 1998
). As IntI1 binds only weakly to the attI3 site and IntI3 does not bind significantly to the attI1 site (Collis et al., 2002b
), the ability of IntI3 and IntI1 to distinguish between the attI1 and attI3 sites and therefore to catalyse integrative recombination between a 59-be and the cognate attI site (Collis et al., 2002b
) appears to involve differences in the sequences of one or more of the binding sites that differentially influence their affinity for the IntI enzymes. These differences are likely to reside in sites 2 and 3.
A similar arrangement of four integrase-binding sites, a simple site and two directly oriented sites, has also been found in the site recognized by the TnpI tyrosine recombinase of Tn5401 which is responsible for resolution of cointegrates formed during transposition (Baum, 1995). However, the Tn5401 resolution site is longer than the attI sites and the position of the directly oriented accessory sites with respect to the simple site is not the same (Fig. 6
). In att5401 a binding site is not found at the position corresponding to site 3 of attI1 and attI3 and the fourth site is located further away from the simple site. Further differences are that the reaction catalysed involves two att5401 sites and that all four binding sites are required for resolution and to form a complex detectable in gel shift experiments. With the full-length site, a single complex corresponding to four bound TnpI is seen, indicating that TnpI binding is cooperative. Thus while the TnpI5401 recombinase is weakly related to members of both the IntI and Xer families (2027 % identity) and the IntI and TnpI systems share the feature of using IntI binding to additional sites as accessory functions, comparisons to this system appear to shed little light on the structureactivity relationships for attI sites.
The site-specific recombination systems specified by integrons are already known to differ from the well-studied paradigm systems in a number of quite fundamental ways. One is the complexity of the recombination sites described here, which contrasts with the simple sites normally recognized by Cre, FLP and XerCD, and the Int attB site. Another difference is that while the recombination events catalysed by
Int, Cre, FLP, XerCD and Int916 (Int1545) all involve two strand exchanges (top and bottom) that occur in an ordered fashion at positions staggered by 6, 7 or 8 bp, there is good evidence that IntI1 catalyses only a single strand exchange (Hansson et al., 1997
; Stokes et al., 1997
). IntI recombinases also include an additional conserved motif that is required for activity (Messier & Roy, 2001
) but whose role in the recombination process is yet to be determined. Studies of further members of this important recombinase family as well as further in-depth studies on the better-characterized IntI1 and IntI3 are needed to determine how productive synaptic complexes are formed and how the reaction is constrained to a single strand exchange.
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REFERENCES |
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Received 25 June 2003;
revised 8 December 2003;
accepted 2 February 2004.
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