DOI: 10.5061/dryad.9cnp5hqqc
title: Data for: Constitutive expression of the Type VI secretion system carries no measurable fitness cost in Vibrio cholerae

description: The Type VI Secretion System (T6SS) is a widespread and highly effective
 mechanism of microbial warfare; it confers the ability to efficiently kill
 susceptible cells within close proximity. Due to its large physical size,
 complexity, and ballistic basis for intoxication, it has widely been
 assumed to incur significant growth costs in the absence of improved
 competitive outcomes. In this study, we precisely examine the fitness
 costs of constitutive T6SS firing in the bacterium Vibrio cholerae. We
 find that, contrary to expectations, constitutive expression of the T6SS
 has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5%
 (95% CI) relative to a T6SS- control. Mathematical modeling of microbial
 populations demonstrates that, due to clonal interference, constitutive
 expression of the T6SS will often be neutral, with little impact on
 evolutionary outcomes. Our findings underscore the importance of precisely
 measuring the fitness costs of microbial social behaviors, and help
 explain the prevalence of the T6SS across Gram negative bacteria.
description: <strong>Bacterial strains and
 media</strong>: Bacterial strains were grown aerobically at 37°C
 overnight in lysogeny broth (LB) (1% w/v tryptone (Teknova, Hollister CA,
 USA), 0.5% w/v yeast extract (Hardy Diagnostics, Santa Maria CA, USA), 1%
 w/v NaCl (VWR Life Sciences, Radnor PA, USA)) with constant shaking or on
 LB agar (1.5% w/v agar; Genesee Scientific, San Diego CA, USA) standing at
 37°C. LB-X-gal was made by mixing in 40 μg/mL of X-gal (GoldBio, St. Louis
 MO, USA) to LB agar while it is liquid.
 <strong>Mutant Construction:</strong> All V.
 cholerae mutant strains were made using the pKAS allelic exchange system
 described by Skorupski et al using pKAS32 (Skorupski & Taylor,
 1996). JT101 and SN598 [Supplemental Data File 1] were described in
 previous studies (Ng et al., 2022; Thomas et al., 2017). CZ005 and CZ006
 were generated through an insertion of a spectinomycin resistance cassette
 into the lacZ gene of JT101 and SN598 respectively. All insertions and
 changes to phenotype were confirmed with PCR, antibiotic screening, and
 killing assays. <strong>Liquid LB Competition
 Experiment</strong>: Overnight cultures of <em>V.
 cholerae</em> were normalized to an OD600 = 1 then mixed in a 1:1
 ratio by volume. Each mixture was then serially diluted and 100 μL of the
 10-3 dilution was mixed into 5mL of LB and incubated overnight shaking at
 37°C. 100 μL of the 10-5 dilution was plated onto an LB X-gal plate for
 quantification with blue-white screening. For the subsequent 4 days, each
 overnight mixture was serially diluted and 100 μL of the 10-5 dilution was
 mixed into 5mL of LB and the 10-6 dilution was plated onto an LB X-gal
 plate for quantification. Fitness was calculated by finding the ratio of
 Malthusian parameters as described in Lenski et al (1991).
 <strong>Solid Agar Competition Experiment</strong>:
 Overnight cultures of <em>V. cholerae</em> were normalized to
 an OD600 = 1 then mixed in a 1:1 ratio by volume. Each mixture was then
 serially diluted and 100 μL of the 10-5 dilution was plated onto LB X-gal
 as well as on LB. The X-gal plate was saved for quantification. The LB
 plate was incubated standing at 37°C. Every following 8 hours for 5 days,
 the LB plate was taken out of the standing incubator and all of the agar
 was scraped off of the plate and transferred into a 50 mL conical tube
 containing 10mL of LB. This conical tube was vortexed for 30 seconds and
 300 μL of the supernatant was transferred into a 96 well plate and
 serially diluted. 100 μL that contained approximately 200-1000 CFU per
 100uL was transferred onto an LB plate for the next time point. Every
 three time points were recorded via blue-white screening by plating 100uL
 of the serial dilution that contained between 30-300 CFU per 100uL onto
 X-gal for quantification. Fitness was calculated with the same calculation
 as the Liquid LB competition experiment.
 <strong>Wright-Fisher Model</strong>: The
 Wright-Fisher model is a standard framework used in evolutionary biology
 to describe stochastic allelic evolutionary processes including genetic
 drift, and in our paper, clonal interference. In a Wright-Fisher
 simulation, discrete generations of bacteria are calculated by drawing
 from a weighted binomial distribution from the previous generation. To
 simulate the effects of clonal interference, each cell had a 10-5 chance
 of mutation every generation. When a cell was mutated, the overall fitness
 effect of the mutation was drawn from an exponential distribution with the
 parameter ꞵ derived from Good et al (2012). Each simulation was run 1000
 times and 200 random runs were plotted for clarity.
 <strong>Statistics</strong>: The 95% confidence
 interval for both competition assays was calculated with a two sample
 t-test for the difference in two means. See supplemental table for
 details. The p values in Figure 2 were calculated by counting the quantity
 of populations that reached fixation or extinction after 5000 simulated
 generations and using a ?2-test to test the null hypothesis that the T6SS+
 strain will go to fixation 50% of the time.
 <strong>Killing assay</strong>: Overnight cultures of
 <em>V. cholerae</em> and <em>E. coli</em>
 (resistant to chloramphenicol) were normalized to an OD600 = 1 then mixed
 in a 10:1 ratio of <em>V. cholerae</em> to <em>E. coli
 </em>by volume. 5 μL of this mixture was spotted onto an LB agarose
 plate and left to dry for 10 minutes. The plate was then incubated at 37°C
 for 3 hours. The spot on the plate was cut out and vortexed with 5 mL of
 LB broth for 30 seconds. The broth was then serially diluted and 5uL of
 each of the serial dilution mixtures were spotted onto an LB
 chloramphenicol plate. <strong>RNA sequencing
 analysis:</strong> Overnight cultures of T6SS+, T6SS−, and WT Vibrio
 cholerae were grown at 37°C in lysogeny broth (LB) (n = 2 biological
 replicates). RNA was extracted from these cultures using an Qiagen RNeasy
 mini kit (Qiagen, Hilden, Germany). An RNA library was prepared using the
 NEB Ultra II directional RNA library prep kit (New England Biolabs,
 Ipswitch, MA, USA) and the QIASeq Fast select -RNA HMR kit (Qiagen,
 Hilden, Germany) was used for bacterial rRNA depletion. The completed
 bacterial RNA library was sequenced on the Illumina NovaSeq 6000
 (Illumina, San Diego, CA, USA). To analyze the transcriptomic data, we
 first trimmed adapters and filtered low-quality reads using Trimmonatic
 (v0.39) (Bolger et al., 2014). We then perform alignment of paired reads
 to reference contigs of V. cholerae C6706 (NCBI RefSeq assembly
 GCF_009763945.1) using STAR (v 2.7.11a) (Dobin et al., 2013) to create
 binary alignment files (BAM) sorted by genomic coordinates. We counted
 aligned fragments to all annotated loci in NCBI Refseq annotation using
 featureCounts (v2.0.6) (Liao et al., 2014). Fragment counts were filtered
 for low expression and used for differential expression analysis using
 DESeq2 in R (Love et al., 2014).
description: # Data for: Constitutive expression of the Type VI secretion system
 carries no measurable fitness cost in Vibrio cholerae
 [https://doi.org/10.5061/dryad.9cnp5hqqc](https://doi.org/10.5061/dryad.9cnp5hqqc) Public repository for model and data collected by Christopher Zhang as a part of a manuscript describing the cost of the Type 6 Secretion System. Manuscript Preprint may be found here: [https://www.biorxiv.org/content/10.1101/2023.03.24.534098v1](https://www.biorxiv.org/content/10.1101/2023.03.24.534098v1) ## Description of the data and file structure **Supplementary Data Table 1:** .ods file containing list of strains used, measured results, values, and calculations. May be accessed with some spreadsheet editors like LibreOffice Calc. * **Strain List Tab**: A list of strains used in the publication * **Metabolic Cost Competition Experiment Tab:** For the metabolic cost experiment run on plates; raw CFU counts per day, calculated hypothetical bacterial quantities, calculated growth rates, summary statistics, calculations for a 95% confidence interval, and calculations for the cost of the antibiotic marker. * **Social Cost Competition Experiment Tab:** For the metabolic cost experiment run in liquid culture; raw CFU counts per day, calculated hypothetical bacterial quantities, calculated growth rates, summary statistics, and calculations for a 95% confidence interval. * **Descriptive statistics on proportion of bacteria per timepoint Tab**: Additional statistics on the metabolic cost and social cost experiments. **Supplementary Data Table 1:** .xlsx file containing list of strains used, measured results, values, and calculations. This file is an excel file copy of the .ods file. All of the contents are the same but optimized for usage with Microsoft Excel. * Tabs are same as above **Supplementary Data Table 2-6:** .csv files containing all of the raw outputs of the models generated for Figure 2. Title of each supplementary data table indicates the model parameters used. Column headers indicate the replicate number. Row headers indicate bacterial generation number. Contents in the matrix (indices i, j) indicate the quantity of positive bacteria in that particular replicate (i) and generation (j). Data may be accessed with most spreadsheet editors including Microsoft Excel, LibreOffice Calc, and Notepad. **Supplementary Data Table 7-9:** .csv files containing post processed outputs from the RNA-sequencing analysis generated for Supplementary Figure 2. Files were processed as outlined in the methodology. May be accessed with most spreadsheet editors including Microsoft Excel, LibreOffice Calc, and Notepad. **Supplementary Figure 1 (Zenodo):** .tiff file containing assays to confirm phenotype of CZ005 and CZ006. May be opened with any image reader. **Supplementary Figure 2 (Zenodo):** .pdf file containing results of an RNA-sequencing assay to confirm the expression levels of T6SS related genes. May be opened with any PDF reader. ## Sharing/Access information Other publicly accessible locations of these and related data: * [https://github.com/Sacrozhangt/T6SS-cost](https://github.com/Sacrozhangt/T6SS-cost) * Raw RNA sequencing data may be accessed at NCBI: [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1079255](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1079255) ## Code/Software **Supplementary Code 1:** .ipynb file containing code used for the model as well as code for visualization of the Wright-Fisher models (Figure 2). May be opened with Jupyter notebook or Google Colab. This code requires the following packages: numpy, matplotlib, seaborn, random, pandas, and tqdm. It was run with python 3.11