Abstract
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. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 105 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution.
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Data availability
Fastq sequences of genomic cassette integration are publicly available in NCBI SRA (accession no. SRR23447848, SRR23447849 and SRR23447850). Bacterial genomes were directly downloaded from the NCBI RefSeq database (https://ftp.ncbi.nlm.nih.gov/genomes/refseq/). Source data are provided with this paper.
Code availability
All scripts used to quantify the ability of IntegronFinder 2.0 to detect SALINs are available at https://gitlab.pasteur.fr/hub/salin. The Nextflow pipeline used to analyse the raw fastq files and generate Fig. 4 and Extended Data Fig. 6 is available at https://gitlab.pasteur.fr/gmillot/14985_loot. The MarkDuplicates-Picard tool of GATK is available at https://github.com/broadinstitute/gatk. Bedtools used to extract sequence around integration sites is available at https://github.com/arq5x/bedtools2. IntegronFinder v.2.0.2 is available at https://github.com/gem-pasteur/Integron_Finder.
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Acknowledgements
We thank G. Macaux for experimental help; all the lab members for helpful discussion; M. Monot and L. Ma from the Biomics platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09) and IBISA. This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR 3525), the Fondation pour la Recherche Médicale (FRM Grant No. EQU202103012569; D.M.), ANR Chromintevol (ANR-21-CE12-0002-01; C.L.) and the French Government’s Investissement d’Avenir programme Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID; D.M.).
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C.L. and D.M. designed the research. C.L., E.R., C.V., B.D., D.L., V.P., F.L. and T.N. performed the experiments. G.A.M. and F.L. performed the computational analysis of deep sequencing data. E.L., J.C., B.N. and E.P.C.R. performed the bioinformatics genomics analysis. C.L. and G.A.M. wrote the draft of the manuscript. All authors read, amended and approved the final version of the manuscript.
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Extended data
Extended Data Fig. 1 Random PCR approach used to determine the genome integration sites.
The genome integrated attC cassette is represented by a light grey arrow (coding sequence) followed by a dark grey triangle (attC). The red arrow indicates the integration site to determine. Primers used for the PCR amplification and sequencing are shown. Electrophoresis analysis of PCR products is shown. MW: Molecular Weight Marker, kb: kilobases.
Extended Data Fig. 2 Cassette recombination events mediated by IntI2 and IntI3 integrases.
The graph represents the recombination frequencies of attCaadA7 donor sites into attI and attCereA2 sites mediated by IntI2 and IntI3 integrases. The expressed integrases (IntI2 or IntI3) are indicated above the graph and the receptor sites in the axis-x legends. Bar charts show the mean of three independent experiments (n = 3, individual plots) and error bars show the standard deviation. Statistical comparisons (Student’s t test) are as follow: na. (not applicable); Grey asterisk (*) indicates the recombination frequency was below detection level.
Extended Data Fig. 3 Cassette integration events in genome in presence of an integron.
a, Experimental setup of the cassette integration assay (as in Fig. 3a). b, Electrophoresis migration of PCR products. Several PCR products are sequenced and the integration sites are indicated (att sites and gene location). c, Recombination frequencies of attCaadA7 donor sites into the att sites and genome. Integration sites are indicated in the axis-x legends. Bar charts show the mean of four independent experiments (n = 4, individual plots) and error bars show the standard deviation. Statistical comparisons (Student’s t test) are as follow: na. (not applicable); Grey asterisk (*) indicates the recombination frequency was below detection level. ts: thermosensitive; MW: Molecular Weight Marker, kb: kilobases.
Extended Data Fig. 4 Excision events observed for two genome-integrated cassettes.
a, Excisions occurring at the integration point (1) and reconstituting the mltA and ilvD genes are shown. Excisions occurring outside the integration point (2) inducing either genome deletion (excision point located in the genome) or insertion (excision point located in the integrated cassette) are shown. b, Sequences of the mltA and ilvD integration sites and of excision sites are shown. All the sequenced excision events (that is 64) are shown. Sizes in bps of each induced genome deletion (-) and insertion (+) are indicated. Cassette sequences are highlighted in grey and cleavage points are indicated by head of arrows. bps: base pairs.
Extended Data Fig. 5 Library construction for Deep sequencing.
All the steps of the library construction are indicated. Adaptors are represented by dark and light green rectangles. attC cassettes are represented by light grey rectangles (coding sequence) followed by dark grey triangles (attC).
Extended Data Fig. 6 Computational analysis of Deep sequencing data.
The figure shows the previous results obtained with IntI1 grouped with the new results obtained with IntI2 and IntI3. The legend is the same as in Fig. 4. The red boxes show the ybhO hotspot sites.
Extended Data Fig. 7 Digital PCR analysis of the oriC copy number relative to terC in E. coli recipient strain during a conjugation mimicking assay.
DNA extraction and PCR were performed at 0, 30, 60, 180 minutes (min) and at an overnight (O/N) time of incubation after the beginning of the conjugation. Bar charts show the mean of three independent experiments (n = 3, individual plots) and error bars show the standard deviation. Grey asterisk (*) indicates the recombination frequency was below detection level.
Extended Data Fig. 8 Analysis of the attG integration site motifs.
a, Consensus sequence of the six highest hotspot integration sites. A total of 20 bases around the cleavage point was inputted into the WebLogo program (http://weblogo.berkeley.edu/) to generate the motif. The cleavage occurs between the -1 and 1 bases. Bits refers to the information content. b, Recombination frequencies of attCaadA7 donor sites into the mutated ybhO hotspot and attI1 sites. The 4 nucleotide sequences indicated in the axis-x legends correspond to the position 6 to 9 given that position -1 corresponds to the G of the cleaved triplet site. The wt ybhO and attI1 sites are indicated in red at the top of the bars. Bar charts show the mean of three independent experiments (n = 3, individual plots) except for ybhO wt CAGC (with IntI1) for which n = 8. Error bars show the standard deviation. Statistical comparisons (Student’s t test) are as follow: ns. (not significant); all two-sided. Grey asterisk (*) indicates the recombination frequency was below detection level.
Extended Data Fig. 9 Determination of the attG recombination nature.
a, Single or double cleavage of attG sites?. Double cleavage: we expect heterogeneity of products due to the repair process. Single cleavage: we expect homogeneity of products due to the replication process. b, Bottom or top strand cleavage of attG sites?. Bottom strand cleavage: we expect an orientation + (light brown arrow) and a functional attC site. Top strand cleavage: we expect an orientation - (dark brown arrow) and a non-functional attC site. attG containing replicons are represented by dark lines and attC cassettes by grey ones. The precise cleavage point is indicated by a red arrow. ss and ds: single and double-stranded; bs: bottom strand.
Supplementary information
Supplementary Table 1
Strains, transformed strains, plasmids and primers used in the present study.
Source data
Source Data Fig. 2
IntegronFinder data.
Source Data Fig. 3
Conjugation and excision assay results used for statistical analysis.
Source Data Fig. 5
Conjugation assay results used for statistical analysis.
Source Data Extended Data Fig. 2
Conjugation assay results used for statistical analysis.
Source Data Extended Data Fig. 3
Conjugation assay results used for statistical analysis.
Source Data Extended Data Fig. 7
Digital PCR data.
Source Data Extended Data Fig. 8
Conjugation assay results used for statistical analysis.
Unprocessed gels
Unprocessed gels from Extended Data Figs. 1 and 3.
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Loot, C., Millot, G.A., Richard, E. et al. Integron cassettes integrate into bacterial genomes via widespread non-classical attG sites. Nat Microbiol 9, 228–240 (2024). https://doi.org/10.1038/s41564-023-01548-y
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DOI: https://doi.org/10.1038/s41564-023-01548-y
