Overview

This repository contains the data, R code, statistical outputs, and plasmid sequences associated with the manuscript:

“DNA defense systems become a fitness liability during plasmid conjugation.”

Files

20260927_dryad.zip

Contains the experimental data and R scripts used to generate the figures and statistical analyses presented in the manuscript.

The archive is organized by manuscript figure:

• Figure_1
• Figure_2
• Figure_3_SuppFig_5
• Supplementary_Figure_1
• Supplementary_Figure_2
• Supplementary_Figure_3
• Supplementary_Figure_4

Within these directories, experiments contributing to individual figure panels are organized into separate analysis folders.

For experiments containing multiple biological replicates, processed data from each biological replicate are retained in subfolders. The main analysis directory aggregates these processed replicate-level data and contains the R script used for statistical analysis and figure generation.

Where applicable, output files containing statistical analyses, including ANOVA results, estimated marginal means, confidence intervals, and pairwise comparisons, are also included.

Each analysis is associated with its own RStudio Project (.Rproj) file. Analyses are intended to be run by opening the corresponding .Rproj file in RStudio and running the R script located in that project directory. Relative file paths are used, so the directory structure should be preserved.

vectors_genbank_format.zip

Contains GenBank-format sequence files and plasmid maps for plasmids constructed and used in this study. These files can be opened using standard plasmid visualization and sequence-analysis software that supports GenBank files.

Data organization

Raw experimental measurements were processed separately for independent biological replicates. When technical replicates were collected within a biological replicate, technical replicates were averaged before statistical analysis.

Processed biological-replicate data were then combined in the corresponding figure-level analysis directory for visualization and statistical testing.

Data files are provided primarily as comma-separated value (.csv) files. R scripts (.R) contain the code used for data processing, statistical analyses, and figure generation.

Data dictionary

The definitions below apply to variables with the same name throughout the repository. Not every variable is present in every dataset. Some CSV files are intermediate or statistical-output files generated by the R analysis scripts.

Experimental identifiers and conditions

• Repeat, biorep — Identifier for an independent biological replicate.
• replicate — Technical replicate identifier within the indicated experiment.
• combination — Identifier used to distinguish independent experimental combinations or replicate groups.
• strain_id — Laboratory strain identifier. See the manuscript Supplementary Tables for complete descriptions of strains and plasmids.
• alias — Human-readable strain, species, or treatment identifier used during analysis or plotting.
• description — Description of the strain, treatment, or experimental condition.
• role — Experimental role of a strain or population, such as donor or recipient.
• donor — Identifier for the donor strain.
• recipient — Identifier or species of the recipient strain.
• recipient_genotype — Recipient genotype relevant to the experiment; trbK+ indicates expression of the entry-exclusion gene and trbK- indicates its absence.
• trbK_y_n — Indicates whether the relevant recipient or editing vector carries/expresses trbK. Values yes or trbK+ indicate the presence of trbK; no or trbK- indicate its absence.
• donor_methylation — Methylation state of the RK2 plasmid carried by the donor (methylated or unmethylated).
• donor_recipient_ratio — Initial donor-to-recipient cell ratio. For example, a value of 10 represents a 10:1 donor:recipient ratio, whereas 0.1 represents a 1:10 ratio.
• OD, OD_factor — Relative starting cell-density condition based on culture optical density. Values are dimensionless experimental density factors.
• hours — Duration of conjugation/incubation in hours.
• temperature — Incubation temperature in degrees Celsius.
• induction — Induction state of the indicated expression system (induced or uninduced).
• CAST_promoter — Promoter controlling the VchCAST system (Plac or Ptet).
• treatment — Composite treatment label used to distinguish experimental conditions.
• notes — Additional annotation associated with an observation.

Colony-count and dilution variables

Variables ending in _count contain the number of colonies observed on the corresponding selective medium.

The prefix identifies the selective medium or antibiotic combination:

• LB_count — Colony count on LB medium.
• LB_DAP_count — Colony count on LB supplemented with diaminopimelic acid (DAP).
• Cm_count — Colony count on chloramphenicol-containing medium.
• Gm_count — Colony count on gentamicin-containing medium.
• Kan_count — Colony count on kanamycin-containing medium.
• Spec_count — Colony count on spectinomycin-containing medium.
• GmKan_count, Kan_Gm_count, Kan_Cm_count, Kan_Spec_count — Colony counts on medium containing the indicated combination of antibiotics.

Corresponding variables ending in _dil (LB_dil, LB_DAP_dil, Cm_dil, Gm_dil, Kan_dil, Spec_dil, GmKan_dil, Kan_Gm_dil, Kan_Cm_dil, and Kan_Spec_dil) give the dilution factor plated for the corresponding colony count.

• amt_plated_mL — Volume plated, in mL.
• Cm_cfu_mL, Gm_cfu_mL — Calculated colony-forming units per mL based on the corresponding colony count, dilution, and plated volume.
• Cm_total_cells, Gm_total_cells — Calculated total cells represented by the corresponding selective count.

Calculated cell abundances and fitness measurements

• recipient_cells — Number of viable recipient cells recovered from the conjugation spot (CFU).
• edited_cells — Number of transposon-edited cells recovered from the conjugation spot (CFU).
• mean_total_recipients — Mean viable recipient abundance across technical replicates within a biological replicate.
• mean_total_transposed — Mean abundance of transposon-edited cells across technical replicates within a biological replicate.
• recipient_ratio_t0 — Ratio between the indicated recipient populations at the beginning of the experiment.
• recipient_ratio_experimental — Ratio between the indicated recipient populations following experimental treatment.
• competitive_index — Competitive index calculated from the relative abundances of the two populations specified for the corresponding experiment.
• competitive_index_log10 — Base-10 logarithm of competitive_index.
• mean_ci_log10, mean_comp_index, mean_comp_index_log10 — Mean competitive-index measurements used for statistical analysis or plotting; variables containing log10 are base-10 transformed.
• fold_difference, fold_change — Fold difference between the indicated experimental groups.
• estimate_log10 — Estimated effect or group mean on a base-10 logarithmic scale.

For log10 competitive-index measurements, 0 indicates equal fitness of the compared populations. Negative values indicate lower fitness of the population specified as the numerator or focal population, whereas positive values indicate greater relative fitness.

Confidence intervals and summary statistics

Variables beginning with lower_CI_ and upper_CI_ are the lower and upper confidence limits, respectively, for the measurement indicated by the remainder of the variable name. These include:

• lower_CI_log10, upper_CI_log10
• lower_CI_competitive_index, upper_CI_competitive_index
• lower_CI_relative_fitness, upper_CI_relative_fitness
• lower_CI_recipient_cells, upper_CI_recipient_cells
• lower_CI_edited_cells, upper_CI_edited_cells

lower_CI and upper_CI similarly represent lower and upper confidence limits where the measured quantity is defined by the corresponding statistical-output file.

• fold_change_lower, fold_change_upper — Lower and upper confidence limits for the reported fold change.
• SE — Standard error.
• lower.CL, upper.CL — Lower and upper confidence limits generated by emmeans.

Statistical-analysis output

Several CSV files contain statistical results generated by the accompanying R scripts. These variables use their standard statistical meanings:

• effect, model term — Model term or experimental factor being tested.
• contrast — Pairwise statistical contrast being evaluated.
• estimate — Estimated difference for a statistical contrast.
• emmean — Estimated marginal mean calculated using the emmeans package.
• SE — Standard error.
• df, df1, df2 — Degrees of freedom.
• F, F.ratio — F statistic from an analysis of variance.
• t, t.ratio — t statistic associated with the indicated contrast.
• p.value, p_value — P value for the indicated statistical test.
• p_formatted — P value formatted for reporting or plotting.
• sum_sq — Sum of squares from analysis of variance.
• mean_sq — Mean square from analysis of variance.
• .group, letter — Compact-letter-display assignment from post hoc multiple comparisons. Groups sharing a letter are not significantly different at the significance threshold used in the corresponding analysis.
• donor_recipient_ratio_pairwise — Donor:recipient ratio contrast used for a pairwise comparison.
• trbK_y_n_pairwise — trbK treatment contrast used for a pairwise comparison.

Statistical significance was assessed at P < 0.05.

Software and R packages

All data processing, statistical analyses, and figure generation were performed in R using RStudio.

R version: 4.4.0

The primary R packages used were:

• tidyverse — Data manipulation and visualization.
• ggbeeswarm — Visualization of individual observations.
• emmeans — Estimated marginal means and post hoc comparisons.
• multcomp — Multiple-comparison procedures.

Additional package dependencies are documented within individual R scripts where applicable.

Reproducing the analyses

To reproduce an analysis:

1. Extract 20260927_dryad.zip while preserving its directory structure.
2. Navigate to the analysis corresponding to the figure or experiment of interest.
3. Open the associated .Rproj file in RStudio.
4. Install any required R packages that are not already installed.
5. Run the corresponding .R script.

Scripts read the data contained within their associated project directories and/or biological-replicate subdirectories and generate the statistical analyses and figure components used in the manuscript.