DOI: 10.5061/dryad.v15dv4206
title: Data from: Prophage maintenance is determined by environment-dependent selective sweeps rather than mutational availability

description: Prophages, viral sequences integrated into bacterial genomes, can confer
 fitness benefits and costs. Despite the risk of prophage activation and
 subsequent bacterial death, active prophages are present in most bacterial
 genomes. However, our understanding about the selective forces that
 maintain prophages in bacterial populations is scarce. Combining
 experimental evolution with stochastic modelling we found that prophage
 maintenance and loss are primarily determined by environmental conditions
 that amplify the net fitness effect of the prophage. Whole genome
 sequencing revealed that prophage loss occurs through environment-specific
 sequences of selective sweeps, leading to rapid prophage loss when
 prophages are costly. However, conflicting selection pressures that select
 against the prophage but for a prophage-encoded accessory gene can prolong
 prophage maintenance. The extent of prolonged maintenance depends on the
 sociality of this accessory gene. Selection for non-cooperative genes is
 more effective for prophage maintenance as cooperative genes allow for
 protection of phage-free ‘cheaters’ that may emerge if prophage costs
 outweigh their benefits. Our mathematical model suggests that
 environmental variation plays a larger role than mutation rates in
 determining prophage maintenance. This challenges our understanding of the
 role of random chance events relative to environmental factors in shaping
 the evolutionary trajectory of bacterial populations.
description: We initiated a selection experiment to observe prophage
 maintenance over time in four different selection regimes using three
 bacterial genotypes, i.e., <em>n_lys, s_lys </em>and
 <em>r_lys</em> in a full-factorial design. With six biological
 replicates, each originating from single colonies, our design resulted in
 a total of 72 populations. We used the following selection regimes: (i)
 Amp at IC<sub>50</sub> (i.e., 6.4 ug/ml) selecting for the
 prophage encoded Amp<sup>R</sup> present in the
 <em>r_lys</em> population, (ii) Mitomycin C (MMC) at a
 concentration of 0.1 ug/ml which induces the lytic cycle of prophages
 causing massive cell death due to host lysis and increases the costs of
 prophage carriage for the <em>s_lys</em> and
 <em>r_lys</em> populations, (iii) a combination of Amp and MMC
 at the same concentrations as above, to create opposing selective
 pressures on the <em>r_lys </em>populations and two competing
 selective pressures on <em>s_lys </em>populations, and (iv) LB
 broth as control. Unless otherwise stated, all bacteria were grown at 37°
 C and constant shaking at 180 rpm. We used 10 µl of an
 overnight culture to inoculate each treatment. All 72 populations were
 grown in 96 deep-well plates at a total volume of 1 ml. Every 24 hours, we
 transferred 1% of each population to fresh medium for a total of 30
 transfers. After the first 24 hours, and subsequently every 7 days, we
 quantified bacterial and phage population dynamics. Additionally, we froze
 100 µl of each population every transfer in 25% glycerol at -80° C.
 Concurrently, every seven days, we made cryo stocks of 24 individual
 clones from each population which we used for follow-up analyses. At the
 end of the experiment, we detected contamination in two
 <em>s_lys</em> populations and excluded them from later
 analysis. <em>Bacterial dynamics:</em> We
 measured bacterial cell counts as cells/ml using a Novocyte Novosampler
 Pro using 1:1000 dilutions of our cell cultures, following a previously
 established protocol in Wendling et al., 2020 (7).
 <em>Phage dynamics: </em>We quantified the number of
 free phages as plaque forming units (PFU/ml) from each population using a
 plaque spot assay on a lawn of phage susceptible ancestral <em>E.
 coli</em> K-12 MG1655 as described in Wendling et al., 2020 (7). In
 populations exposed to MMC, we observed a decrease in free phage particles
 over time (Figure 1). This suggested either (i) a gradual reduction at the
 population level, resulting from lower burst size and phage productivity,
 or (ii) the presence of two different phenotypes, of which one stopped
 producing phages completely while the other produced phages at a rate
 comparable to the ancestral lysogen. To infer the underlying mechanism, we
 quantified phage production as PFU/ml, as described above, on days 0, 8,
 and 29 of 24 randomly selected clones from two <em>s_lys
 </em>and <em>r_lys </em>populations originating from
 either the MMC, or the Amp+MMC treatment. This assay confirmed that the
 observed decline in free phage particles observed at the population level
 results from individual clones that did not produce free phages. This
 suggested that these clones either lost or inactivated their
 prophages. 
description: # Data from: Prophage maintenance is determined by environment-dependent
 selective sweeps rather than mutational availability This database
 contains the data relevant to performing the analysis described in
 "Prophage maintenance is determined by environment-dependent
 selective sweeps rather than mutational availability" by Zachary M.
 Bailey, Claudia Igler, and Carolin C. Wendling Here is a brief description
 of the data files located in this dataset: ##
 01\_pfus\_phage\_dynamics\_Figure\_1.csv This file contains the data for
 figure 1 of the manuscript. genotype_treatment - the combination of both
 genotype and the treatment of the population replicate_population - the
 biological replicate numbered 1-6 for each combination of genotype and
 population time - the number of days since the beginning of the selection
 experiment total_pfus - the absolute number of plaque forming units (free
 phages) per population (in PFUs/ml) log10_pfus - the log base 10 value of
 the absolute values of phages (in PFUs/ml), this is what we show on the y
 axis in figure 1 ## 02\_wgs\_clonal\_mutations\_Figure\_2.csv This file
 contains the data for figure 2 position - the genomic position of each
 mutation (Genomic_Feature) in our E. coli chromosomes description - the
 type of mutation present ancestral_genotype - the genotype of each clone
 (nlys, slys, or rlys) treatment - the treatment applied to each population
 clone_id - the unique ID for each population with a combination of the
 genotype, treatment and the clone number day - the time in days since the
 beginning of the selection experiment (8 or 29) clone - the number of the
 clone from 1 to 24 of the clones we picked from our selection experiment
 for each population at each timepoint ##
 03\_muller\_plot\_frequencies\_Figure\_3.csv This file contains the data
 for figure 3 treatment - the treatment applied to each population (MMC or
 Amp+MMC) genotype - the genotype of each clone (nlys, slys, or rlys) day -
 the time in days since the beginning of the selection experiment
 (0,1,2,3,4,5,7 or 29) amp_resistance - whether the clone is resistant to
 Amp (Y/N) phage_resistance - whether the clone is resistant to phage
 infection based on TTZ data (Y/N) phage_presence - whether the prophage is
 present based on PCRs of individual clones (Y/N) n - the absolute number
 of clones with this combination of resistances and phage presence freq -
 the frequency of clones with that combination of resistance and phage
 presence identity - the combination of resistances used for ggmuller