Data from: Prophage maintenance is determined by environment-dependent selective sweeps rather than mutational availability
Data files
Apr 04, 2024 version files 24.34 KB
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01_pfus_phage_dynamics_Figure_1.csv
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02_wgs_clonal_mutations_Figure_2.csv
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03_muller_plot_frequencies_Figure_3.csv
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README.md
Abstract
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.
README: 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
Methods
We initiated a selection experiment to observe prophage maintenance over time in four different selection regimes using three bacterial genotypes, i.e., n_lys, s_lys and r_lys 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 IC50 (i.e., 6.4 ug/ml) selecting for the prophage encoded AmpR present in the r_lys 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 s_lys and r_lys populations, (iii) a combination of Amp and MMC at the same concentrations as above, to create opposing selective pressures on the r_lys populations and two competing selective pressures on s_lys 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 s_lys populations and excluded them from later analysis.
Bacterial dynamics: 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).
Phage dynamics: 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 E. coli 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 s_lys and r_lys 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.