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Integrons are DNA elements that can mediate the dissemination of antibiotic resistance genes by a site-specific recombination system (37). They possess two conserved segments separated by a variable region which includes integrated antibiotic resistance genes or cassettes of unknown function (Fig. 1). The essential components of the integron are found within the 5' conserved segment and include an integrase gene intI (28) and an adjacent recombination site attI (13, 34).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   General structure of class 1 integrons. Cassettes are inserted in the variable region by the integrase using a site-specific recombination mechanism. The attI and attC sites are shown, respectively, by black and grey ovals and promoters are denoted by "P." intI1, integrase gene; qacE1, antiseptic resistance gene; sulI, sulfonamide resistance gene; orf5, gene of unknown function.

Four classes of integron have been described (25, 32), each of which codes for a distinct but related integrase enzyme. Class 1 integrons (Fig. 1) are the most widespread, with a 5' conserved segment that contains a promoter region from which integrated cassettes are expressed (7, 23). The 3' conserved segment contains a qacE1 gene encoding resistance to quaternary ammonium compounds and most also contain a sulI gene encoding resistance to sulfonamides, an open reading frame (ORF5) of unknown function, and other sequences that differ from one integron to another (1, 3, 30, 31). The gene cassettes, which may be found within the variable region of integrons, are mobile, nonreplicating elements which comprise an open reading frame (usually lacking a promoter region) associated with an integrase-specific recombination site, attC, also known as the 59-base element (17, 20, 32, 38).

IntI1 is a member of the tyrosine recombinase family (8, 27) and catalyzes the excision and integration of antibiotic resistance genes in class 1 integrons by a site-specific recombination system. These reactions are carried out by the integrase interacting with the two different recombination sites, the attI site in the 5' conserved segment of the integron and the attC sites of the gene cassettes. The attC site lengths and sequences vary considerably (from 57 to 141 bp) and their similarities are restricted to their boundaries, which correspond to the inverse core site (RYYYAAC) and the core site (GTTRRRY) (38). There is no inverse core site in the attI site so it cannot form a palindromic structure like attC sites. Cassettes are excised in a circular form and are integrated at core sites defined as GTTRRRY (6), with the crossover located between the G and the first T (20, 34). It is not yet clear if the cleavage occurs at the same place on both DNA strands or if it is a staggered cleavage. Some suppose that only one strand is cleaved by the integrase and the resulting Holliday junction is resolved by a cellular enzyme like RuvC (38). IntI1 can also act at secondary sites containing a degenerate core site (9, 33).

Site-specific recombination requires short and specific DNA sequences with only limited homology and involves the formation of a Holliday junction intermediate (10, 40). It is a conservative process in which all DNA strands that are broken are rejoined without ATP utilization or DNA synthesis. Site-specific recombinases are divided into two families, the resolvase family and the integrase family. The latter includes about 200 highly diversified members such as  integrase, Cre, Flp, and XerC-XerD (8, 27). The integrase nucleophile is a tyrosine located at the C-terminal end of the protein. This tyrosine is responsible for the cleavage and forms a covalent intermediate by esterification of a DNA 3'-phosphoryl group. The joining reaction is mediated by nucleophilic attack of the 5'-hydroxyl group from the same strand or another cleaved strand (12).

The integrase family definition is based on identification of conserved residues (RHRY) found in two boxes (I and II) located in the carboxyl half of the protein. Only the histidine is not invariant but is present in nearly 95% of the members (8). A recent analysis has identified three patches (I, II, and III) of residues which seem to play a role in the secondary structure of these enzymes (27), and a potentially essential fifth catalytic residue (lysine) has been identified (4). The following five tyrosine recombinases have been partially or totally crystallized: the  Int and HP1 Int catalytic domains, the XerD protein, and the Cre and Flp recombinases-DNA complexes (5, 11, 15, 16, 21, 22, 39, 40). Alignment of integron integrases with other tyrosine recombinases shows that they possess an additional group (about 35 residues) of amino acids near the patch III motif (14, 26, 27) containing several residues which are conserved among the integron integrases (Fig. 2). This special feature has led to the identification of several new integron integrases (26, 35) and some have been tested for the ability to excise class 1 integron cassettes (F. Drouin and P. H. Roy, Abstr. 101st Gen. Meet. Am. Soc. Microbiol., abstr. H-19, 2001).

View larger version (79K): [in this window] [in a new window]   FIG. 2.   Alignment of C-terminal integron integrase sequences with those of other tyrosine recombinases. IntI1, class 1 integron integrase from plasmid pVS1; IntI2, class 2 integron integrase from Tn7; IntI3, class 3 integron integrase from a Serratia marcescens plasmid; IntI4, class 4 integron integrase from the Vibrio cholerae super-integron; IntISpu, integron integrase from Shewanella putrefaciens; IntINeu, integron integrase from Nitrosomonas europaea; IntITde, integron integrase from Treponema denticola; IntIGsu, integron integrase from Geobacter sulfurreducens; XerC, recombinase from E. coli; XerD, recombinase from E. coli. There are two potential IntI's in N. europaea; the one shown is the most likely to be translated. Numbering is based on the IntI1 sequence and residues mutated in this study are underlined. The additional domain and the different boxes and motifs are indicated under the alignment. Residues from the conserved tetrad (RHRY) are shown in bold.

IntI1 possesses the four conserved residues (RHRY) of the family and other well-conserved residues and motifs (boxes I and II and patches I, II, and III) (8, 27). A study of IntI1 variants for the conserved RRY residues has shown that mutations of the conserved arginines by nonpositively charged residues abolish substrate recognition, while mutant proteins of the conserved tyrosine bind the attI1 site but are unable to catalyze recombination (14). In this study, we analyzed the properties of several mutants of conserved residues, potentially located in the active site of tyrosine recombinases (K171, H277, and G302) or within conserved, noncatalytic regions (E121, W229, and F233), and of additional residues conserved among integron integrases (ALER215 and K219) fused with the maltose binding protein (MBP) protein in in vivo recombination and in vitro substrate binding. We also show that the cellular enzyme RuvC is not responsible for the second DNA strand cleavage in the integron site-specific recombination reaction.

Construction of plasmids overexpressing mutant MBP-IntI1 fusion proteins. The plasmids encoding various mutants of MBP-IntI1 were constructed by PCR using the QuickChange site-directed mutagenesis kit of Stratagene with pLQ369 (50 ng) as a template (14). Two complementary primer pairs, designed with the OLIGO software package (version 4.1; National Biosciences, Plymouth, Minn.), were used to construct each mutant (Table 1). PCR conditions were 10 min at 95°C; 30 cycles consisting of 1 min at 95°C, 1 min at the appropriate annealing temperature (Table 1), and 15 min at 72°C; and a final elongation step of 20 min at 72°C. Since the PCR amplified the complete vector because of the complementary primers, the nonmutated methylated parental DNA was digested with the restriction enzyme DpnI. The uncut mutated DNA was then introduced into Escherichia coli XL1-Blue {recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lacI^qZM15 Tn10(Tet^r)]} from Stratagene and grown at 37°C on Luria-Bertani ampicillin plates. Mutant plasmids were purified and sequenced to determine the mutations (Table 2).

In vivo recombination. Mutant MBP-IntI1 clones (Table 2) were introduced by transformation into E. coli TB1 {F' ara (lac-proAB) rpsL(Str^r) [80dlac(lacZ)M15] hsdR(r_K m_K)} containing pLQ428, a pACYC184-based plasmid which possesses two excisable cassettes (aacA1-ORFG and ORFH) (14). We also introduced by transformation the clones pLQ369 and pLQ428 into the RuvC mutant E. coli CS85 [thr-1 araC14 leuB6 (gpt-proA)62 lacY1 tsx-33 qsr¹ glnV44(AS) galK2(Oc) Rac-O eda-51::Tn10 ruvC53 hisG4(Oc) frbD1 mgl-51 rpoS396(Am) rpsL31(Str^r) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1] from the E. coli Genetic Stock Center. Cells were grown at 37°C in Luria-Bertani medium and excision of the cassettes from pLQ428 was induced by using isopropyl--D-thiogalactopyranoside (IPTG) as previously described (14). Plasmid DNA was then prepared and the capacity of mutant MBP-IntI proteins to excise the cassettes was determined by PCR. We used the pACYC184-5' and pACYC184-3' primers (Table 1) to detect the reduction in length of pLQ428 (14). Figure 3 shows examples of different kinds of results obtained with PCR on some plasmids expressing mutants of the MBP-IntI1 fusion protein in the in vivo recombination assay with the pLQ428 vector. There is a major 2,499-bp PCR fragment in several lanes containing DNA preparations from mutant clones. This band represents the pLQ428 clone without any cassette excision and is also observed in the negative control, which is the pMAL-c2 vector without any gene fused to malE. In the reaction containing the wild-type MBP-IntI1-expressing clone pLQ369 in E. coli TB1 and in the E. coli ruvC mutant strain CS85, this 2,499-bp product was not obtained, indicating that the wild-type fusion protein is very efficient in site-specific recombination in these strains and that RuvC is not required to complete the reaction. In these PCRs, we observed two major bands, of 1,341 and 889 bp (Fig. 3). The 1,341-bp product represents a pLQ428 clone which has lost the aacA1-ORFG cassette and the 889-bp product represents a pLQ428 clone which has lost both the aacA1-ORFG and ORFH cassettes (14). One or both of the 1,341- and 889-bp PCR products are also observed, with variable intensity, in the reaction containing the mutant clones pLQ1101(E121D), pLQ1102(E121K), pLQ1103 (K171E), pLQ1104(K171I), pLQ1105(K171Q), pLQ1106 (K171R), pLQ1107(K171V), pLQ1110(K219I), pLQ1114 (F233L), pLQ1115(F233R), pLQ1116(F233Y), pLQ1120 (H277Y), and pLQ1121(G302A). These mutant proteins are equally efficient as or less efficient than the wild-type protein for the recombination activity, as seen by the presence and intensity of the PCR products. Some are able to excise only the first cassette (aacA1-ORFG), since the 1,341-bp product can be seen on the agarose gel and the 889-bp product is absent; others are able to excise both cassettes since the 889-bp product, but not the 1,341-bp product, is seen. Mutants pLQ1108 (ALER215), pLQ1109(K219E), pLQ1111(K219W), pLQ1112(W229G), pLQ1113(W229R), pLQ1117(H277D), pLQ1118(H277L), pLQ1119(H277R), and pLQ1122(G302R) did not show any recombinational activity and only the 2,499-bp PCR product is seen on the gel. As previously described (14), we were not able to detect a PCR product of 2,047 bp, corresponding to the rare event of excision of the ORFH cassette alone. We can see another apparent PCR product of 1,200 bp in the reactions containing active proteins. This PCR product has been sequenced and corresponds to an annealing of a DNA strand from the 1,341-bp product with a DNA strand from the 889-bp product.

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Electrophoresis of PCR products obtained with the pACYC184 primers and 100 ng of DNA preparation from overexpressed cultures on a 1% agarose gel. Lane 1, 1-kb-plus DNA ladder (Life Technologies); lane 2, DNA preparation of pLQ428-pLQ369 (wild type); lane 3, pLQ428-pMAL-c2 (MBP); lane 4, pLQ428-pLQ1101(E121D); lane 5, pLQ428-pLQ1102 (E121K); lane 6, pLQ428-pLQ1119 (H277R); lane 7, pLQ428-pLQ1120 (H277Y); lane 8, pLQ428-pLQ1121 (G302A); lane 9, pLQ428-pLQ1122 (G302R); lane 10, pLQ428-pLQ1109 (K219E); lane 11, pLQ428-pLQ1110 (K219I); lane 12, pLQ428-pLQ1114 (F233L); lane 13, pLQ428-pLQ1116 (F233Y); lane 14, pLQ428-pLQ369 (wild type) in a RuvC strain.

In vitro substrate binding. We used the purified fusion proteins and a gel retardation assay with the complete attI1 site of the integron to determine the binding properties of each mutant at the recombination site. MBP-IntI fusion proteins were overproduced and purified on amylose resin (New England BioLabs) as previously described (14). Binding reactions were done with a labeled attI1 site DNA fragment incubated with different concentrations of MBP-IntI1 as previously described (14). The wild-type fusion protein and native IntI1 were shown to lead to the same four distinct complexes with this DNA substrate (13). These complexes represent the binding of four IntI1 molecules to four different sites in the attI1 site. The binding specificity of the IntI1 molecules has been previously described (13). Figure 4 shows examples of different kinds of results obtained with some mutants of the MBP-IntI1 fusion protein in the gel retardation experiment with the attI1 site. We observed that mutants W229G and H277D have completely lost the ability to bind to the attI1 site, as no complexes are seen in the gel retardation assay. Mutants E121D, K171E, K171I, K171R, K171V, ALER215, K219I, F233Y, H277L, G302A, and G302R are fully active in binding activity and give a pattern of complexes similar to that of the wild-type fusion protein. Mutants E121K, K171Q, W229R, F233L, and F233R show a lower affinity for the attI1 site, with only some complexes seen in the gel retardation experiment. Mutants K219E, K219W, H277R, and H277Y seem to have more affinity than wild-type IntI1 for the recombination site, with as many as seven complexes formed with the attI1 site.

View larger version (113K): [in this window] [in a new window]   FIG. 4.   Gel retardation experiment with mutant MBP-IntI fusion proteins purified from E. coli TB1, on the complete attI1 site of the In2 integron (from nucleotide 96 to +71, relative to the G residue of the core site at position 0). A purified labeled fragment was incubated with a 200 nM concentration of mutant fusion proteins. Free DNA (F) and protein-DNA complexes (I to VII) were separated on 4% polyacrylamide gels and are indicated by arrows. Lane 1, free DNA; lane 2, wild-type MBP-IntI1 protein; lane 3, MBP-IntI1 (E121D) mutant; lane 4, MBP-IntI1 (H277R) mutant; lane 5, MBP-IntI1 (H277Y) mutant; lane 6, MBP-IntI1 (F233R) mutant; lane 7, MBP-IntI1 (E121K) mutant; lane 8, MBP-IntI1 (G302A) mutant; lane 9, MBP-IntI1 (G302R) mutant; lane 10, MBP-IntI1 (K171I) mutant; lane 11, MBP-IntI1 (K219I) mutant; lane 12, MBP-IntI1 (K219W) mutant.

Relationships with other tyrosine recombinases. The conserved lysine residue K171 has been proven to be a fifth essential catalytic residue of tyrosine recombinases and eukaryotic type IB topoisomerases (4). In crystal structures of  Int c170, HP1 Int, XerD, and Cre, this conserved lysine delineates one edge of the catalytic pocket (27). A mutation of the XerD recombinase at this position (K172A) conferred a severe defect in recombination at the dif site (4). This lysine seems to be in close contact with the substrates and may influence the reactivity of the phosphotyrosine intermediates formed during recombination reactions. Mutants K171E, K171R, K171I, and K171V were found to bind the recombination site similarly to the wild-type protein but showed an altered recombinational activity, excising both cassettes and not the first cassette alone as the wild-type protein does. Mutant K171Q was only able to form two complexes with the attI1 site and showed a weak recombination activity. These effects are probably due to the properties of the substitute residues. Glutamine contains a carboxyl group and might change the conformation of the protein and inhibit its binding capacity. The fact that only two cassettes are excised by those mutants might indicate that this residue is implicated in the recognition of the different attC sites and the mutant proteins were unable to recognize the aacA1-ORFG attC site, thus abolishing the excision of the first cassette alone. The wild-type protein recognizes this site efficiently.

Mutations in the IntI1 extra domain. ALER is a highly conserved sequence located in the additional domain, particular to integron integrases, near the patch III motif. We made a complete deletion of this sequence and the resulting fusion protein MBP-ALER215 showed a wild-type DNA binding activity but was deficient in recombination. This sequence does not seem to be implicated in the recognition of the recombination site attI1 but may play a role in positioning and stabilizing the active site or in recognition and binding to the diverse attC sites. K219 is located just after the ALER sequence and is conserved among all identified integron integrases. Fusion protein mutants K219E and K219W showed a stronger affinity to the attI1 site, forming up to seven complexes in the gel retardation assay, but these mutants failed to show any recombination activity. The K219I mutant was effective in binding to the attI1site and excising either the first cassette or both cassettes from the pLQ428 clone. This residue may play a role in the recognition of the recombination site, since it is positively charged and mutants K219E and K219W showed a greater affinity for it. The lack of recombination for these mutants may be due to the presence of too many monomers, resulting in interference and preventing the cleavage of the DNA. Alternatively, the substituted residues may cause a major change in the protein conformation and disturb the active site when the basic lysine is replaced by an acidic glutamate or an aromatic tryptophan. It is surprising that mutant K219I is still able to bind to the recombination site and to excise the cassettes since the amino acid substitution involves a change from a basic residue to a hydrophobic residue.

Recombination assay in a RuvC mutant strain. The crossover point of the integron site-specific recombination is located between the G and the first T in the core site GTTRRRY, but it is not yet clear whether the cleavage occurs at the same place on both DNA strands or whether there is a staggered cleavage. Because of the sequence differences between attI and the various attC sites, there appears to be a blunt crossing over at this site. Some authors hypothesized that only one strand (the bottom strand) is cleaved by the integrase and the resulting Holliday junction is resolved by a cellular enzyme like RuvC (38). We introduced the pLQ369 clone, coding for the MBP-IntI1 fusion protein, and the pLQ428 clone, containing two excisable cassettes (aacA1-ORFG and ORFH) into the E. coli ruvC mutant strain. The results showed that there is a high level of recombination in this strain (Fig. 3), so the cellular enzyme RuvC is not responsible for the cleavage of the second DNA strand in the recombination reaction. We believe that the cleavage is entirely mediated by the integrase and is staggered, with the bottom cleavage occurring between the C and the first A (corresponding to the G and the first T of the upper strand) and the upper cleavage occurring seven nucleotides upstream after two A's, giving the general binding motif TAAN₇TTR on attI sites for all integron integrases. This cleavage model is similar to that observed for the XerC-XerD recombinases for which the binding motif is TAAN_6-8TTR, with the cleavage occurring on either side of the central region immediately 3' of an AA dinucleotide on each strand (2). Studies of IntI1 binding sites have shown that this enzyme uses a recognition pattern similar to that used by XerC-XerD (13) and the C-terminal part of XerD shares about 50% similarity with the C-terminal part of IntI1.