Integron array MICs and cassettes transcription data

Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to ‘evolve on demand’ in response to antibiotic pressure. To test this hypothesis, we inserted a custom three cassette integron into P. aeruginosa, and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. Our results show that integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity, and they highlight the importance of semi-conservative cassette excision for integron dynamics.

Methods
MIC dataset

This dataset represents the raw gentamicin MICs values used in Figure 1, 2 and 3 and the cassette transcription data described in Figure S1 of the pre-print https://www.biorxiv.org/content/10.1101/2020.08.07.237602v2 and article (eLife 2021;10:e62474)

The minimum inhibitory concentrations (MIC) for each arrays were determined in cation-adjusted Mueller-Hinton Broth 2 (MH2), following the broth microdilution method from the Clinical and Laboratory Standards Institute guidelines (CLSI, 2017). Briefly 5x c.f.u bacteria inocula were prepared using individual colonies grown on selective agar in interlaced 2-fold-increasing concentrations of antibiotics and incubated for 20h in a shaking incubator at 37°C.

The next day, plates’ optical density (OD595) was read using a Biotek Synergy 2 plate reader. Wells were considered empty when the overall was under 0.1 and the MIC for each assay was defined as the minimal concentration in which growth was inhibited in all three technical replicates (separate wells, but grown on the same day from the same inoculum). The final MICs values are the average of two to four replicate assays (from separately prepared inocula, on different days).

All RNA samples were treated with the TURBO DNA-free Kit (ThermoFisher) to eliminate genomic DNA. Concentration of the RNA samples was quantified using the Quantifluor RNA system (Promega). cDNA was synthesized from 100ng of RNA templates using random primers from the GoScript Reverse Transcription Mix (Promega). qPCR was carried out on the StepOnePlus Real-time PCR platform (Applied Biosystems) using the iTaq Universal SYBR Green Supermix. The cassette, as well as two reference genes (actpA and acp), were amplified using the primers described in Supplementary Table S2 in two technical replicates for each extraction. Standard curves for the pair of cassette primers was included in each PCR using restriction enzyme-digested gDNA as template, and used to quantify the amount of target cDNA in each sample to control for inter-run variations. Melting curve analysis was included after each run to test for non-specific amplification products. For each biological replicate the cassette transcript levels were normalized based on the geometric means of the two internal reference genes, using the first array A1 as a reference, before division by its plasmid copy number.