DOI: 10.5061/dryad.rv15dv469
title: Integron array MICs and cassettes transcription data

description: 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.
description: 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 (OD<sub>595)</sub> 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.
description: MIC dataset: The 'MIC_ancestral_arrays' sheet
 corresponds to the raw gentamicin MIC values for the ancestral arrays. The
 'strain' column corresponds to the genotype of the strain being
 tested. Strains annotated A1 to A6 corresponds to PAO1:WTA1 to PAO1:WTA6
 strains. The 'experiment' column tracks the biological replicate
 from which the data was generated. The 'MIC_evolved_populations'
 sheet correspond to the gentamicin MIC values of the evolved populations
 at the x1024 MIC time-point. 'Integron array' corresponds to the
 prevalent integron array in that population and the 'population
 identifier' refer to the population numerotation used in the article.
 The 'MIC_evolved_plasmids' sheets corresponds to the MIC of the
 populations with an ancestral chromosomal background but evolved plasmids.
 Here the 'population identifier' column corresponds to the
 original population from which the plasmid was extracted.
 aadB transcription: a further description of the meaning of each
 column is indicated at the bottom of the file.