Structural Features of Single-Stranded Integron Cassette attC Sites and Their Role in Strand Selection

We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attI×attC reaction context using the suicide conjugation assay we previously developed, but also in an attC×attC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.     

The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity

Integrons are able to incorporate exogenous genes embedded in mobile cassettes, by a site-specific recombination mechanism. Gene cassettes are collected at the attI site, via an integrase mediated recombination between the cassette recombination site, attC, and the attI site. Interestingly, only three nucleotides are conserved between attC and attI. Here, we have determined the requirements of these in recombination, using the recombination machinery from the paradigmatic class 1 integron. We found that, strikingly, the only requirement is to have identical first nucleotide in the two partner sites, but not the nature of this nucleotide. Furthermore, we showed that the reaction is close to wild-type efficiency when one of the nucleotides in the second or third position is mutated in either the attC or the attI1 site, while identical mutations can have drastic effects when both sites are mutated, resulting in a dramatic decrease of recombination frequency compared to that of the wild-type sites. Finally, we tested the functional role of the amino acids predicted from structural data to interact with the cleavage site. We found that, if the recombination site triplets are tolerant to mutation, the amino acids interacting with them are extremely constrained.     

Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.     

Intracellular Steady-State Concentration of Integron Recombination Products Varies with Integrase Level and Growth Phase

IntI1 mediates the recombination of antibiotic-resistant gene cassettes between different integrons in the same cell, facilitating the persistence and dissemination of these genes. Historically, integrase activity has been measured by conjugating recombinant products from donor cells overexpressing integrase and quantifying them in recipient cells. Here we report the first measurements of the steady-state intracellular abundance of integrase-mediated recombination products in strains expressing natural or high IntI1 levels. Recombination products in both high and natural integrase strains increased markedly through late log phase and continued to rise in stationary phase in the high integrase strain, but declined in the natural expression strain. Simple acquisition of gene cassettes was seen only in strains expressing high integrase; in strains with natural integrase levels, only cointegrates between the two integron-bearing plasmids were detectable at all growth stages. Unexpectedly, more attI × attI than attC × attI recombination products were seen in log phase for both strains; however, in stationary phase, the high integrase strain increased attC recombination, consistent with earlier observations of integrase crossover site preferences. Thus, direct quantification of the steady-state concentration of recombination products reveals that the integrase's intracellular concentration affects the amount and type of recombination events in a growth-phase-dependent manner.     

Cellular pathways controlling integron cassette site folding

By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single‐stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double‐stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non‐recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination.     

The Integron Integrase Efficiently Prevents the Melting Effect of Escherichia coli Single-Stranded DNA-Binding Protein on Folded attC Sites

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.     

Identification and analysis of integrons and cassette arrays in bacterial genomes

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome.     

Integrase-Mediated Recombination of the veb1 Gene Cassette Encoding an Extended-Spectrum β-Lactamase

The veb1 gene cassette encodes the extended spectrum β-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively.     

Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution

Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the ‘nick-counter-nick’ mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.     

Meiotic DNA joint molecule resolution depends on Nse5�CNse6 of the Smc5�CSmc6 holocomplex

Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81–Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81–Eme1 is unclear. In cells lacking Nse5–Nse6 of the Smc5–Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5–Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5–Smc6 complex in HJ resolution via Mus81–Eme1.     

GEN1 promotes Holliday junction resolution by a coordinated nick and counter-nick mechanism

Holliday junctions (HJs) that physically link sister chromatids or homologous chromosomes are formed as intermediates during DNA repair by homologous recombination. Persistent recombination intermediates are acted upon by structure-selective endonucleases that are required for proper chromosome segregation at mitosis. Here, we have purified full-length human GEN1 protein and show that it promotes Holliday junction resolution by a mechanism that is analogous to that exhibited by the prototypic HJ resolvase E. coli RuvC. We find that GEN1 cleaves HJs by a nick and counter-nick mechanism involving dual co-ordinated incisions that lead to the formation of ligatable nicked duplex products. As observed with RuvC, cleavage of the first strand is rate limiting, while second strand cleavage is rapid. In contrast to RuvC, however, GEN1 is largely monomeric in solution, but dimerizes on the HJ. Using HJs containing non-cleavable phosphorothioate-containing linkages in one strand, we show that the two incisions can be uncoupled and that the first nick occurs upon GEN1 dimerization at the junction. These results indicate that the mechanism of HJ resolution is largely conserved from bacteria to man, despite a lack of sequence homology between the resolvases.     

Marine integrons containing novel integrase genes,attachment sites,attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like β propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome.     

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.     

Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution

During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to selectively stabilize the "activated" phosphate conformation in order to promote cleavage.     

Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase,SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.     

First Gene Cassettes of Integrons as Targets in Finding Adaptive Genes in Metagenomes

The first gene cassettes of integrons are involved in the last adaptation response to changing conditions and are also the most expressed. We propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.Integrons, genetic elements discovered in clinical environments in 1989 (26), are known to carry gene cassettes encoding adaptive proteins in different environmental contexts (17, 20); environmental pressures may thus favor the propagation of cassettes conferring a selective advantage (21, 29). Integrons contain (Fig. ​(Fig.1)1) an integrase gene, intI (6, 7, 18); a recombination site, attI (23); a set of gene cassettes formed by a coding sequence and a recombination site, attC (14, 19); and one or two promoters, allowing gene cassette expression (4, 16). Different classes of integrons were defined according to the intI gene diversity. They were found in metagenomes from various environments (9, 15, 22). New metagenomic studies always discover new integron classes, showing the importance and the diversity of such genetic elements (9, 22).Open in a separate windowFIG. 1.Structure of integrons and positions of the primers used. The intI gene encodes an enzyme, allowing the integration of new gene cassettes at the attI recombination site. Thus, the first gene cassette is the last one integrated. Gene cassettes are formed by a recombination site, attC, and a coding sequence. The promoter Pc allows gene cassette expression. The positions of primers AJH72, ICC21, and ICC48 used in this study are indicated.As integrons are involved in bacterial adaptation, study of integrons would allow the finding of adaptive genes in metagenomes. But the detection of such genes among the huge abundance of gene cassettes associated with integrons is a challenge. The integration of a new gene cassette, catalyzed by the integrase, occurs by recombination between the attC site and the attI site of the integron (6, 8) (Fig. ​(Fig.1).1). The first gene cassette of an integron is, therefore, the last one integrated. As it is the closest gene to the promoter, its expression level is the highest in the integron (4). Thus, this gene cassette is a good target to find new adaptive genes in metagenomes. To amplify the first gene cassettes, a forward primer targeting the intI gene or attI site must be used. In previous studies, the determinations of gene cassette collection from environmental metagenomes did not target first gene cassettes, since they were performed by PCR methods targeting attC sites. Thus, we propose a method to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection.The method was developed by using DNA from Xanthomonas campestris ATCC 33913^T, a bacterial strain carrying an integron. DNA was extracted according to the work of Goñi-Urriza et al. (12). Coastal sediments maintained in the laboratory were used to validate this method. Total DNA (metagenomic) was extracted 1 week after addition of oil using the UltraClean soil DNA isolation kit (Mo Bio Laboratories), as previously described (24). PCR amplification, targeting the integrase gene intI in the forward direction (because attI sites are not well conserved enough to allow the good design of a primer) and the attC site in reverse, to amplify integron first gene cassettes was performed (Fig. ​(Fig.1).1). Forward primer AJH72 (10) was used for PCR of X. campestris DNA, and primer ICC48 (intB-inverted primer [25]), targeting the class 1 integron intI, was used for PCR of sediment metagenome. As the intI1 genes from environmental contexts exhibit considerable sequence diversity (11), ICC48 does not cover the entire spectrum of known intI1 genes but was chosen for its proximity to the attI site. Many primers used in previous studies targeting attC sites, such as HS286 (27), were unsuccessfully tested in the studied metagenome. Thus, ICC21, a less-degenerated primer, was designed to target the attC sites from class 1 and 2 integrons with the following sequence: 5′-GTCGGCTTGRAYGAATTGTTAGRC-3′. The PCR mixture contained DNA, 1× PCR buffer, 200 μM of each dNTP (deoxynucleoside triphosphate), 1.5 mM MgCl₂, 0.2 μM of each primer, and 5 U of Taq DNA polymerase (Eurobio). The PCRs consisted of 95°C for 10 min, 40 cycles of amplification (95°C for 45 s, 52°C or 51°C for 45 s, 72°C for 1.5 min), and 72°C for 10 min. PCR products were gel purified with the GFX PCR DNA and gel band purification kit (GE Healthcare). Purified products were cloned with the TOPO TA cloning kit (Invitrogen). Clones carrying first gene cassettes were selected by colony PCR. In order to minimize time spent on and the number of PCRs, the following three primers were concomitantly used: the two TOPO TA M13 primers and the primer targeting the intI gene used in the previous PCR (AJH72 or ICC48), and this primer was fluorescently labeled by HEX (6-carboxyhexafluorescein). PCR products were separated by gel electrophoresis, and the fluorescent DNA fragments were detected with a Typhoon 9200 scanner (Amersham). The selected inserts were sequenced by using the BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems). Sequences were analyzed with ORF Finder (28), BLAST (1), and ProDom (3) algorithms.The X. campestris (ATCC 33913^T) integron possesses 23 gene cassettes (10). Different concentrations of primers were tested to amplify the first gene cassette, but in all cases, several amplified fragments were obtained. Sequence analyses revealed that most of them were gene cassettes other than the first one, and in these cases, the reverse primer was also used in the forward direction. The particular structure of the attC site with inverted repeat sequences, allowing the attC primer to anneal with both strands, may explain this result. As there was no other way to amplify the first gene cassettes, their selection could not be performed by PCR only. Because sequencing all clones would represent too much work when studying metagenomes, a triplex PCR screening method was developed (Fig. ​(Fig.2A).2A). Since the forward primer sequence is found only in the fragment containing the first gene cassette, the corresponding clones produce two PCR products, with one that is labeled (Fig. ​(Fig.2).2). Sequence-labeled inserts confirmed that the integron first gene cassette of X. campestris was selected. As a control, the sequences of unlabeled inserts showed that they contained integron gene cassettes but not the first gene cassettes.Open in a separate windowFIG. 2.Screening strategy for first gene cassette inserts. (A) Schematic of the clone library PCR screening strategy. The inserts are amplified by PCR using three primers. Only inserts carrying a first gene cassette lead to labeled amplified fragments. (B, C) Gel electrophoresis of insert PCR products from clones of X. campestris gene cassettes. (1) Insert carrying the integron first gene cassette of X. campestris; (2, 3) inserts carrying integron gene cassettes of X. campestris other than the first gene cassette; (4) molecular weight marker (SmartLadder; Eurogentec). (B) Detection of fluorescence; (C) detection of all fragments by ethidium bromide staining of the same gel.This method was then applied to coastal mud metagenomes, in which we focused on class 1 integrons, most commonly involved in adaptive responses (13). After PCR products were cloned, among 100 clones screened, 23 fluorescent fragments were detected and sequenced. As the primer targeting intI binds at the beginning of the gene, it was nearly impossible to recognize the intI sequence, except that the intI gene is longer at the 5′ end. On the other side of the sequences, a part of the attC site must be present. Sequence analysis revealed that some fragments showed similarities to characteristic class 1 or 2 integron attC sites, but these sites could not be found in each case because of their large variability (5). A total of 29 open reading frames (ORF) were characterized as potentially transcribed by an integron promoter, but for 16 of the ORF, no similarity with any known amino acid sequences could be found. The 13 other ORF exhibited less than 40% similarity with known sequences, and no putative conserved domains were found. These observations are in accordance with previous studies showing that most of the environmental integron gene cassettes code for proteins with unknown functions (2, 17).The first-gene cassettes of integrons appear to be good candidates to find gene cassettes, which aid bacteria in effecting a rapid adaptive response. We are now able to reveal integron last gene acquisitions of environmental bacterial communities submitted to stressful conditions. This method presents two limiting steps when working with metagenomes, as follows. (i) The primers are critical to cover the largest number of integrons. In this study, we targeted class 1 integrons because they are known to be mobile and to carry adaptive genes (29). (ii) When fragments with a large size disparity are cloned, the smallest fragments are preferentially cloned. In order to obtain a complete library with metagenomes, the cloning should be performed after fragment size separation. The PCR method combined with the screening method leads to 100% of clones carrying a first gene cassette. Thus, this new method allows the focus to be on spreading first gene cassettes in metagenomes after a specific stress.     

Control of directionality in the DNA strand-exchange reaction catalysed by the tyrosine recombinase TnpI

In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1–IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1–DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed.