The recombination efficiency of the bacterial integron depends on the mechanical stability of the synaptic complex.

Multiple antibiotic resistances are a major global health threat. The predominant tool for adaptation in Gram-negative bacteria is the integron. Under stress, it rearranges gene cassettes to offer an escape using the tyrosine recombinase IntI, recognizing folded DNA hairpins, the sites.

Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae.

Background: Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium.

Chromosomal integrons are genetically and functionally isolated units of genomes.

Integrons are genetic elements that increase the evolvability of bacteria by capturing new genes and stockpiling them in arrays. Sedentary chromosomal integrons (SCIs) can be massive and highly stabilized structures encoding hundreds of genes, whose function remains generally unknown. SCIs have co-evolved with the host for aeons and are highly intertwined with their physiology from a mechanistic point of view.

Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales.

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems.

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown.

Integron cassettes integrate into bacterial genomes via widespread non-classical attG sites.

Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites.

Unbridled Integrons: A Matter of Host Factors.

Integrons are powerful recombination systems found in bacteria, which act as platforms capable of capturing, stockpiling, excising and reordering mobile elements called cassettes. These dynamic genetic machineries confer a very high potential of adaptation to their host and have quickly found themselves at the forefront of antibiotic resistance, allowing for the quick emergence of multi-resistant phenotypes in a wide range of bacterial species. Part of the success of the integron is explained by its ability to integrate various environmental and biological signals in order to allow the host to respond to these optimally.

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.

Primary and promiscuous functions coexist during evolutionary innovation through whole protein domain acquisitions.

Molecular examples of evolutionary innovation are scarce and generally involve point mutations. Innovation can occur through larger rearrangements, but here experimental data is extremely limited. Integron integrases innovated from double-strand- toward single-strand-DNA recombination through the acquisition of the I2 α-helix.

Structure-specific DNA recombination sites: Design, validation, and machine learning-based refinement.

Recombination systems are widely used as bioengineering tools, but their sites have to be highly similar to a consensus sequence or to each other. To develop a recombination system free of these constraints, we turned toward sites from the bacterial integron system: single-stranded DNA hairpins specifically recombined by the integrase. Here, we present an algorithm that generates synthetic sites with conserved structural features and minimal sequence-level constraints.

Integron Identification in Bacterial Genomes and Cassette Recombination Assays.

Integrons are genetic elements involved in bacterial adaptation to the environment. Sedentary chromosomal integrons (SCIs) can stockpile and rearrange a myriad of different functions encoded in gene cassettes. Through their association with transposable elements and conjugative plasmids, some SCIs have acquired mobility and are now termed Mobile Integrons (MIs).

Structural heterogeneity of attC integron recombination sites revealed by optical tweezers.

A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete.

Recoding of synonymous genes to expand evolutionary landscapes requires control of secondary structure affecting translation.

Synthetic DNA design needs to harness the many information layers embedded in a DNA string. We previously developed the Evolutionary Landscape Painter (ELP), an algorithm that exploits the degeneracy of the code to increase protein evolvability. Here, we have used ELP to recode the integron integrase gene (intI1) in two alternative alleles.

Differences in Integron Cassette Excision Dynamics Shape a Trade-Off between Evolvability and Genetic Capacitance.

Integrons ensure a rapid and "on demand" response to environmental stresses driving bacterial adaptation. They are able to capture, store, and reorder functional gene cassettes due to site-specific recombination catalyzed by their integrase. Integrons can be either sedentary and chromosomally located or mobile when they are associated with transposons and plasmids.

Efficiency of integron cassette insertion in correct orientation is ensured by the interplay of the three unpaired features of attC recombination sites.

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins.

Adaptation to Global Change: A Transposable Element-Epigenetics Perspective.

Understanding how organisms cope with global change is a major scientific challenge. The molecular pathways underlying rapid adaptive phenotypic responses to global change remain poorly understood. Here, we highlight the relevance of two environment-sensitive molecular elements: transposable elements (TEs) and epigenetic components (ECs).

Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation.

Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases.

The Integron: Adaptation On Demand.

The integron is a powerful system which, by capturing, stockpiling, and rearranging new functions carried by gene encoding cassettes, confers upon bacteria a rapid adaptation capability in changing environments. Chromosomally located integrons (CI) have been identified in a large number of environmental Gram-negative bacteria. Integron evolutionary history suggests that these sedentary CIs acquired mobility among bacterial species through their association with transposable elements and conjugative plasmids.

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.

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.

Folded DNA in action: hairpin formation and biological functions in prokaryotes.

Structured forms of DNA with intrastrand pairing are generated in several cellular processes and are involved in biological functions. These structures may arise on single-stranded DNA (ssDNA) produced during replication, bacterial conjugation, natural transformation, or viral infections. Furthermore, negatively supercoiled DNA can extrude inverted repeats as hairpins in structures called cruciforms.

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.

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.

Different strategies to persist: the pogo-like Lemi1 transposon produces miniature inverted-repeat transposable elements or typical defective elements in different plant genomes.

Miniature inverted-repeat transposable elements (MITEs) are a particular type of defective class II elements present in genomes as high-copy-number populations of small and highly homogeneous elements. While virtually all class II transposon families contain non-autonomous defective transposon copies, only a subset of them have a related MITE family. At present it is not known in which circumstances MITEs are generated instead of typical class II defective transposons.

Control of IS911 target selection: how OrfA may ensure IS dispersion.

IS911 transposition involves a closed circular insertion sequence intermediate (IS-circle) and two IS-encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N-terminal region with OrfA.