Site-specific Recombinase Research Articles

An extensible vector toolkit and parts library for advanced engineering of plant genomes.

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

Recombineering 101: Making an in-Frame Deletion Mutant.

DNA recombineering uses phage λ Red recombination functions to promote integration of DNA fragments generated by polymerase chain reaction (PCR) into the bacterial chromosome. The PCR primers are designed to have the last 18-22 nt anneal on either side of the donor DNA and to carry 40- to 50-nt 5' extensions homologous to the sequences flanking the chosen insertion site. The simplest application of the method results in knockout mutants of nonessential genes. Deletions can be constructed by replacing a portion or the entirety of a target gene with an antibiotic-resistance cassette. In some commonly used template plasmids, the antibiotic-resistance gene can be coamplified with a pair of flanking FRT (Flp recombinase recognition target) sites that, following insertion of the fragment into the chromosome, allow excision of the antibiotic-resistance cassette via the activity of the site-specific Flp recombinase. The excision step leaves behind a "scar" sequence comprising an FRT site and flanking primer annealing sequences. Removal of the cassette minimizes undesired perturbations on the expression of neighboring genes. Even so, polarity effects can result from the occurrence of stop codons within, or downstream of, the scar sequence. These problems can be avoided by the appropriate choice of the template and by designing primers so that the reading frame of the target gene is maintained past the deletion end point. This protocol is optimized for use with Salmonella enterica and Escherichia coli.

An intersectional genetic approach for simultaneous cell type-specific labelling and gene knockout in the mouse.

Precise genome manipulation in specific cell types and subtypes in vivo is crucial for neurobiological research because of the cellular heterogeneity of the brain. Site-specific recombinase systems in the mouse, such as Cre-loxP, improve cell type-specific genome manipulation; however, undesirable expression of cell type-specific Cre can occur. This could be due to transient expression during early development, natural expression in more than one cell type, kinetics of recombinases, sensitivity of the Cre reporter, and disruption in cis-regulatory elements by transgene insertion. Moreover, cell subtypes cannot be distinguished in cell type-specific Cre mice. To address these issues, we applied an intersectional genetic approach in mouse using triple recombination systems (Cre-loxP, Flp-FRT and Dre-rox). As a proof of principle, we labelled heterogeneous cell subtypes and deleted target genes within given cell subtypes by labelling neuropeptide Y (NPY)-, calretinin (calbindin 2) (CR)- and cholecystokinin (CCK)-expressing GABAergic neurons in the brain followed by deletion of RNA-binding Fox-1 homolog 3 (Rbfox3) in our engineered mice. Together, our study applies an intersectional genetic approach in vivo to generate engineered mice serving dual purposes of simultaneous cell subtype-specific labelling and gene knockout.

Interspecies Horizontal Transfer and Specific Integration of the Mosquitocidal Toxin-Encoding Plasmid pTAND672-2 from Bacillus thuringiensis subsp. israelensis to Lysinibacillus sphaericus.

pTAND672-2, a 144-kb resident plasmid of Bacillus thuringiensis serovar israelensis strain TAND672, was sequenced and characterized. This extrachromosomal element carries mosquitocidal toxin-, conjugation-, and recombinase-encoding genes, together with a putative arbitrium system, a genetic module recently discovered in temperate phages controlling lysogeny-lysis transition and in mobile genetic elements (MGEs) where its function remains clarified. Using conjugation experiments, pTAND672-2 is shown to be a novel integrative and conjugative element (ICE), which can horizontally transfer from B. thuringiensis serovar israelensis to Lysinibacillus sphaericus, another mosquitocidal bacterium, where it integrates into the chromosome. Its integration and circularization are reversible and involve a single-cross recombination between 33-bp specific sites, attB in the chromosome of L. sphaericus and attP in pTAND672-2. CDS143, coding for the putative tyrosine integrase Int143 distantly related to site-specific tyrosine Xer recombinases and phage integrases, can mediate the integration of pTAND672-2 to attB. The B. thuringiensis mosquito-killing genes carried by pTAND672-2 are efficiently transcribed and expressed in L. sphaericus, displaying a slight increased toxicity in this bacterium against Aedes albopictus larvae. The occurrence of pTAND672-2-like plasmids within the Bacillus cereus group was also explored and indicated that they all share a similar genetic backbone with diverse plasmid sizes, ranging from 58 to 225 kb. Interestingly, among them, the pEFR-4-4 plasmid of Bacillus paranthracis EFR-4 and p5 of B. thuringiensis BT-59 also display conjugative capability; moreover, like pTAND672-2 displays a chimeric structure between the pCH_133-e- and pBtoxis-like plasmids, pBTHD789-3 also appears to be mosaic of two plasmids. IMPORTANCE Horizontal transfer of mobile genetic elements carrying mosquitocidal toxin genes may play a driving role in the diversity of mosquitocidal bacteria. Here, the 144-kb mosquitocidal toxin-encoding plasmid pTAND672-2 is the first verified integrative and conjugative element (ICE) identified in Bacillus thuringiensis serovar israelensis. The key tyrosine integrase Int143, involved in the specific integration, is distantly related to other tyrosine recombinases. The study also reports the occurrence and potential interspecies transmission of pTAND672-2-like plasmids with varied sizes in B. thuringiensis, Bacillus paranthracis, and Bacillus wiedmannii isolates belonging to the Bacillus cereus group. This study is important for further understanding the evolution and ecology of mosquitocidal bacteria, as well as for providing new direction for the genetic engineering of biopesticides in the control of disease-transmitting mosquitoes.

Conformational selection in the Cre CBD domain: Insights into intasome assembly.

During site-specific recombination tyrosine recombinases promote scission, insertion, and recombination of specific DNA sequences. The Cre/loxP system from P1 bacteriophage promotes viral DNA circularization after infection and equitable plasmid assortment during cell division. It acts through the formation of a tetramer (intasome) in which Cre monomers are bound in a head-to-tail fashion to a half loxP site, alternating between active and inactive conformations. Despite knowledge of the recombination mechanism and strand exchange order, information regarding target selection and intasome assembly is still missing. We used molecular dynamics simulations (MD) to assess the conformational flexibility of the CBD domain of Cre recombinase. The starting seeds for MD were structures with PDB ID 1q3u (an intasome) and 7rhy (a Cre monomer bound to a loxP half-site); these structures vary in the position of helix A. We simulated four replicas for each system for 5 microseconds in an explicit solvent model (TIP3P) with the software GROMACS using CHARMM36m at 300K and 1 atm. Van der Waals interactions were calculated using a 1.2 nm cutoff and PME for electrostatics, with a 4 fs timestep. Our results show that helix A can switch from an intasome-like to a monomer-like conformation, passing through distinct intermediate and unfolded states. These results suggest that the position of helix A is selected concomitantly with establishing monomer-monomer interactions during intasome assembly. Also, the flexibility of helix A could help to interpret NMR data in which the signals for this helix are missing.We acknowledge the computer resources provided by the LNS del Sureste de México through grant 202101023N, Laboratorio de Supercómputo y Visualización en Paralelo, and LANCAD through grants 99-2021 and 49-2022, and CONACyT (graduate school scholarship 858905, grant INF-2014-02-231504, grant A1-S-11842).

PASTE: The Way Forward for Large DNA Insertions.

PASTE: The Way Forward for Large DNA Insertions.

Simple and Rapid Site-Specific Integration of Multiple Heterologous DNAs into the Escherichia coli Chromosome.

Escherichia coli is the most studied and well understood microorganism, but research in this system can still be limited by available genetic tools, including the ability to rapidly integrate multiple DNA constructs efficiently into the chromosome. Site-specific, large serine-recombinases can be useful tools, catalyzing a single, unidirectional recombination event between 2 specific DNA sequences, attB and attP, without requiring host proteins for functionality. Using these recombinases, we have developed a system to integrate up to 12 genetic constructs sequentially and stably into in the E. coli chromosome. A cassette of attB sites was inserted into the chromosome and the corresponding recombinases were cloned onto temperature sensitive plasmids to mediate recombination between a non-replicating, attP-containing "cargo" plasmid and the corresponding attB site on the chromosome. The efficiency of DNA insertion into the E. coli chromosome was approximately 107 CFU/μg DNA for six of the recombinases when the competent cells already contained the recombinase-expressing plasmid and approximately 105 CFU/μg DNA or higher when the recombinase-expressing plasmid and "cargo" plasmid were co-transformed. The "cargo" plasmid contains ΦC31 recombination sites flanking the antibiotic gene, allowing for resistance markers to be removed and reused following transient expression of the ΦC31 recombinase. As an example of the utility of this system, eight DNA methyltransferases from Clostridium clariflavum 4-2a were inserted into the E. coli chromosome to methylate plasmid DNA for evasion of the C. clariflavum restriction systems, enabling the first demonstration of transformation of this cellulose-degrading species. IMPORTANCE More rapid genetic tools can help accelerate strain engineering, even in advanced hosts like Escherichia coli. Here, we adapt a suite of site-specific recombinases to enable simple, rapid, and highly efficient site-specific integration of heterologous DNA into the chromosome. This utility of this system was demonstrated by sequential insertion of eight DNA methyltransferases into the E. coli chromosome, allowing plasmid DNA to be protected from restriction in Clostridium clariflavum and enabling genetic transformation of this organism. This integration system should also be highly portable into non-model organisms.

Novel integrative elements and genomic plasticity in ocean ecosystems

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

Antibiotic Susceptibility, Resistance Gene Determinants and Corresponding Genomic Regions in Lactobacillus amylovorus Isolates Derived from Wild Boars and Domestic Pigs.

Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our study was to determine antibiotic susceptibility profiles in 28 Lactobacillus amylovorus isolates derived from the digestive tract of wild boars and farm pigs by means of the broth microdilution method and whole genome sequencing (WGS). We revealed genetic resistance determinants and examined sequences flanking resistance genes in these strains. Our findings indicate that L. amylovorus strains from domestic pigs are predominantly resistant to tetracycline, erythromycin and ampicillin. WGS analysis of horizontally transmissible genes revealed only three genetic determinants (tetW, ermB and aadE) of which all tetW and ermB genes were present only in strains derived from domestic pigs. Sequence analysis of coding sequences (CDS) in the neighborhood of the tetW gene revealed the presence of site-specific recombinase (xerC/D), site-specific DNA recombinase (spoIVCA) or DNA-binding transcriptional regulator (xre), usually directly downstream of the tetW gene. In the case of ermB, CDS for omega transcriptional repressor or mobilization protein were detected upstream of the ermB gene.

An RNA Interference (RNAi) Toolkit and Its Utility for Functional Genetic Analysis of Leishmania (Viannia).

RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis (Lbr). We developed vectors facilitating generation of long-hairpin or "stem-loop" (StL) RNAi knockdown constructs, using GatewayTM site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as quinonoid dihydropteridine reductase (QDPR), a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful 'hypomorphic' approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a finding that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

Prediction of designer-recombinases for DNA editing with generative deep learning

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. Here we present RecGen (Recombinase Generator), an algorithm for the intelligent generation of designer-recombinases. We gather the sequence information of over one million Cre-like recombinase sequences evolved for 89 different target sites with which we train Conditional Variational Autoencoders for recombinase generation. Experimental validation demonstrates that the algorithm can predict recombinase sequences with activity on novel target-sites, indicating that RecGen is useful to accelerate the development of future designer-recombinases.

The bacteriophage lambda integrase catalytic domain can be modified to act with the regulatory domain as a recombination-competent binary recombinase

Site-specific recombinase Int mediates integration of the bacteriophage λ genome into the E. coli chromosome. Integration occurs once the Int tetramer, assisted by the integration host factor IHF, forms the intasome, a higher order structure, within which Int, a heterobivalent protein, interacts with two non-homologous DNA sequences: the core recombination sites and the accessory arm sites. The binding to these sites is mediated by the catalytic C-terminal domain (CTD) and the regulatory N-terminal domain (NTD) of Int, respectively. Within Int, the NTD can activate or inhibit the recombination activity of the CTD depending on whether the NTD is bound to the arm sites. The CTD alone cannot mediate recombination, and even when the NTD and the CTD are mixed together as individual polypeptides, the NTD cannot trigger recombination in the CTD. In this work, we set to determine what modifications can unlock the recombination activity in the CTD alone and how the CTD can be modified to respond to recombination-triggering signals from the NTD. For this, we performed a series of genetic analyses which showed that a single mutation that stabilizes the CTD on DNA, E174K, allows the CTD to recombine the core DNA sequences. When the NTD is paired with the CTD(E174K) that also bears a short polypeptide from the C-terminus of the NTD, the resulting binary Int can recombine arm-bearing substrates. Our results provide insights into the molecular basis of the regulation of the Int activity and suggest how binary recombinases of the integrase type can be engineered.

Computational Methods for the Design of Recombinase Logic Circuits with Adaptable Circuit Specifications.

Synthetic biology aims at engineering new biological systems and functions that can be used to provide new technological solutions to worldwide challenges. Detection and processing of multiple signals are crucial for many synthetic biology applications. A variety of logic circuits operating in living cells have been implemented. One particular class of logic circuits uses site-specific recombinases mediating specific DNA inversion or excision. Recombinase logic offers many interesting features, including single-layer architectures, memory, low metabolic footprint, and portability in many species. Here, we present two automated design strategies for both Boolean and history-dependent recombinase-based logic circuits. One approach is based on the distribution of computation within multicellular consortia, and the other is a single-cell design. Both are complementary and adapted for non-expert users via a web design interface, called CALIN and RECOMBINATOR, for multicellular and single-cell design strategies, respectively. In this book chapter, we are guiding the reader step by step through recombinase logic circuit design, from selecting the design strategy fitting to their final system of interest to obtaining the final design using one of our design web interfaces.

Sharing of Antimicrobial Resistance Genes between Humans and Food Animals.

The prevalence and propagation of antimicrobial resistance (AMR) are serious global public health concerns. The large and the ever-increasing use of antibiotics in livestock is also considered a great concern. The extent of the similarity of acquired antibiotic resistance genes (ARGs) between humans and food animals and the driving factors underlying AMR transfer between them are not clear, although a link between ARGs in both hosts was proposed. To address this question, with swine and chicken as examples of food animals, we analyzed over 1,000 gut metagenomes of humans and food animals from over the world. A relatively high abundance and diversity of ARGs were observed in swine compared with those in humans as a whole. Commensal bacteria, particularly species from Clostridiales, contribute the most ARGs associated with mobile genetic elements (MGEs) and were found in both humans and food animals. Further studies demonstrate that overrepresented MGEs, namely, Tn4451/Tn4453 and TnAs3, are attributed mainly to the sharing between humans and food animals. A member of large resolvase family site-specific recombinases, TnpX, is found in Tn4451/Tn4453 which facilitates the insertions of the transient circular molecule. Although the variance in the transferability of ARGs in humans is higher than that in swine, a higher average transferability was observed in swine than that in humans. In conclusion, the potential antibiotic resistance hot spots with higher transferability in food animals observed in the present study emphasize the importance of surveillance for emerging resistance threats before they spread. IMPORTANCE Antimicrobial resistance (AMR) has proven to be a global public health concern. To conquer this increasingly worrying trend, an overarching, One Health approach has been used that brings together different sectors, but the fundamental knowledge of the relationship between humans, food animals, and their environments is not mature yet or is lacking in some aspect. With swine and chicken as examples of food animals, a large global data set of over 1,000 human and food animal gut metagenomes was analyzed with a focus on acquired antibiotic resistance genes (ARGs) associated with mobile genetic elements (MGEs) to answer this question. Outputs from this work open a new avenue to further our understanding of ARG transferability in food animals. It is a necessary milestone to better equip governmental agencies to monitor and pre-empt antibiotic resistance hot spots. This work will assist and give guidance on how to decipher other links within any One Health initiatives with expected positive feedback to human health.

SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome.

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field.

Versatile seamless DNA vector production in E. coli using enhanced phage lambda integrase.

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.

STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells

SummaryInserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.

A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution

Unlike eukaryotes and archaea, which have multiple replication origins on their chromosomes, bacterial chromosomes usually contain a single replication origin.1 Here, we discovered a dicentric bacterial chromosome with two replication origins, which has resulted from the fusion of the circular and linear chromosomes in Agrobacterium tumefaciens. The fused chromosome is well tolerated, stably maintained, and retains similar subcellular organization and genome-wide DNA interactions found for the bipartite chromosomes. Strikingly, the two replication origins and their partitioning systems are both functional and necessary for cell survival. Finally, we discovered that the site-specific recombinases XerC and XerD2 are essential in cells harboring the fused chromosome but not in cells with bipartite chromosomes. Analysis of actively dividing cells suggests a model in which XerC/D are required to recombine the sister fusion chromosomes when the two centromeres on the same chromosome are segregated to opposite cell poles. Thus, faithful segregation of dicentric chromosomes in bacteria can occur because of site-specific recombination between the sister chromatids during chromosome partitioning. Our study provides a natural comparative platform to examine a bacterial chromosome with multiple origins and a possible explanation for the fundamental difference in bacterial genome architecture relative to eukaryotes and archaea.1.

Speciation and adaptation research meets genome editing.

Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Activation of class 1 integron integrase is promoted in the intestinal environment.

Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.