Replicative resolution of integron cassette insertion

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC × attI reaction.     

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.     

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.     

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.     

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.     

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.     

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.     

Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites

The site-specific recombinase IntI1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron-associated attI1 site and the 59-base element (59-be) family of sites that are associated with gene cassettes. IntI1 includes the four conserved amino acids that are characteristic of members of the integrase family, and IntI1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. IntI1 was purified as a fusion protein and shown to bind to isolated attI1 or 59-be recombination sites. Binding to attI1 was considerably stronger than to a 59-be. Binding adjacent to the recombination cross-over point was not detected. A strong IntI1 binding site within attI1 was localized by both deletion and footprinting analysis to a 14 bp region 24–37 bp to the left of the recombination cross-over point, and this region is known to be critical for recombination in vivo ( Recchia et al ., 1994 ). An imperfect (13/15) direct repeat of this region, located 41–55 bp to the left of the recombination cross-over point, contains a weaker IntI1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.     

IntI2 integron integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.     

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.     

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.     

Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.     

Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination

The Cre recombinase mediates precise site-specific recombination between a pair of loxP sequences through an intermediate containing Holiday junction. The recombination junction in the loxP sequence is located within the asymmetric 8-nucleotide spacer region. To examine the role of each nucleotide sequence of the spacer region in the recombination process, we synthesized a complete set of 24 loxP spacer mutants with single-base substitutions and 30 loxP spacer mutants with double-base substitutions. Each synthesized loxP mutant was ligated at both ends of a linear DNA or to one end of a DNA-containing wild-type loxP at the other end and their recombination efficiencies were analyzed with an in vitro system. The sequence identity of the right two nucleotides and left four nucleotides in the central six bases of the spacer region was found to be essential for formation and resolution, respectively, of the intermediate product. Furthermore, even when homology was maintained, the recombination efficiencies were lower than that of wild-type loxP and varied among mutants. Based on this knowledge, we identified two loxP mutants with double-base substitutions, mutants 5171 and 2272, which recombine efficiently with an identical mutant but not with the other mutant or wild-type loxP.     

Using Genomic Data to Determine the Diversity and Distribution of Target Site Motifs Recognized by Class C-<Emphasis Type="Italic">attC</Emphasis> Group II Introns

The attC sites are well-known integrase-targeted elements involved in the insertion and excision of gene cassettes from integrons. Recently, functional analysis of Sma.I2, a class C-attC group II intron, showed that this mobile element invades the attC sites through a specific process. The analysis of genomic data indicates that class C-attC group II introns are independently acquired by their bacterial hosts and evolve in the recognition of a variety of target sites, including the attCs. In addition, adaptation of class C-attC group II introns seemed to be favourable for particular genera, such as Shewanella, suggesting a possible niche for the spread of class C-attC group II introns inserted at attC sites. This understanding suggests a functional role of short palindromic DNA sequences, such as the attCs, as important tools for the acquisition of mobile elements associated with horizontal gene transfer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2 % or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.     

Two Portable Recombination Enhancers Direct Donor Choice in Fission Yeast Heterochromatin

Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality.