## Data files associated with *Extremely fast amelioration of plasmid fitness costs by multiple functionally-diverse pathways* 

**Authors:** James P. J. Hall^1,2,3^* ORCID 0000-0002-4896-4592, Rosanna C. T. Wright^2^, David Guymer^3^, Ellie Harrison^2^ ORCID 0000-0002-2050-4631, Michael A. Brockhurst^2^ ORCID 0000-0003-0362-820X

**Affiliations:**

^1^Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB

^2^Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, S10 2TN

^3^Department of Biology, University of York, Wentworth Way, York, YO10 5DD 

*Correspondence to: j.p.j.hall@liverpool.ac.uk, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom. Tel: +44 (0)151 79 57691

Available at https://doi.org/10.17638/datacat.liverpool.ac.uk/953

### Abstract

The acquisition of plasmids is often accompanied by high fitness costs such that compensatory evolution is required to allow plasmid survival. The mercury-resistance plasmid, pQBR55, drastically reduced the growth of its host, *Pseudomonas fluorescens* SBW25, immediately after acquisition, causing a small colony phenotype. We observed rapid restoration of the ancestral large colony morphology within just 48 hours of growth on agar plates, suggesting that compensatory mutations had occurred. Using genome sequencing we identified that restoration of the large colony phenotype was associated with chromosomal mutations in either an uncharacterised hypothetical protein PFLU4242, RNA polymerase, or the GacA/S two-component system. Targeted deletions in *PFLU4242*, *gacA*, or *gacS* recapitulated the ameliorated phenotype upon plasmid-acquisition. These data show that compensatory evolution can occur through three distinct mutational pathways and is fast enough to allow the survival of a plasmid despite it imposing very high fitness costs upon its host.   

### Details of data files

**Movie S1.** Breakout of compensated mutants from a small morphology colony. Small colonies restreaked from lineage 15 were maintained at room temperature and photographed every 30 minutes. Multiple independent mutants can be seen emerging from each colony. Movie in animated .gif format.

**Table S1.** Mutations detected in whole genome resequenced lineages. Results are shown from the bwa/GATK pipeline, similar results were obtained using breseq.

**Table S2.** Rifampicin minimum inhibitory concentrations (MIC) for sequenced evolved lineages. Values are in µg/ml.

**Table S3.** Sequences of primers used in this study. 

**Table S4.** Relative fitness plotted in Figure 1B. 

**Table S5.** Gac assay results plotted in Figure 1B. 

**Table S6.** Summary of genetics information plotted in Figure 1B.

**Table S7.** Colony size measurements plotted in Figure 2.

**Table S8.** Growth curve data plotted in Figure S2. Time is in seconds and rate is in ∆OD/s.

**Table S9.** Relative fitness of knockout strains plotted in Figure S4. Two replicate experiments were performed, data from experiment 1 was plotted.