Data for: Constitutive expression of the Type VI secretion system carries no measurable fitness cost in Vibrio cholerae

Bacterial strains and media: 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. Mutant Construction: 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. Liquid LB Competition Experiment: Overnight cultures of V. cholerae 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). Solid Agar Competition Experiment: Overnight cultures of V. cholerae 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. Wright-Fisher Model: 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. Statistics: 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. Killing assay: Overnight cultures of V. cholerae and E. coli (resistant to chloramphenicol) were normalized to an OD600 = 1 then mixed in a 10:1 ratio of V. cholerae to E. coli 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. RNA sequencing analysis: 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).

菌株与培养基：实验菌株于37℃有氧振荡条件下在LB肉汤（Lysogeny Broth, LB）中过夜培养，LB肉汤配方为1%（质量体积比）胰蛋白胨（Teknova公司，美国加利福尼亚州霍利斯特市）、0.5%（质量体积比）酵母提取物（Hardy Diagnostics公司，美国加利福尼亚州圣玛丽亚市）及1%（质量体积比）氯化钠（VWR生命科学公司，美国宾夕法尼亚州拉德诺市）；或于LB琼脂平板（含1.5%质量体积比琼脂；Genesee Scientific公司，美国加利福尼亚州圣迭戈市）上37℃静置培养。LB-X-gal平板的制备方法为：将40 μg/mL的X-gal（GoldBio公司，美国密苏里州圣路易斯市）加入液态LB琼脂中混匀。

突变体构建：所有霍乱弧菌（Vibrio cholerae, V. cholerae）突变株均采用Skorupski等人报道的pKAS等位基因交换系统（pKAS allelic exchange system），以pKAS32质粒完成构建（Skorupski & Taylor, 1996）。JT101与SN598[补充数据文件1]已在既往研究中被报道（Ng et al., 2022; Thomas et al., 2017）。CZ005与CZ006分别通过在JT101和SN598的lacZ基因中插入壮观霉素抗性基因盒构建获得。所有插入突变与表型变化均通过PCR、抗生素筛选及杀伤实验验证。

液体LB竞争实验：将霍乱弧菌的过夜培养物归一化至光密度600nm（Optical Density at 600 nm, OD600）=1，随后按体积比1:1混合。将混合体系进行梯度稀释，取10⁻³稀释液100 μL加入5 mL LB培养基中，37℃振荡培养过夜。取10⁻⁵稀释液100 μL涂布于LB X-gal平板，通过蓝白斑筛选进行定量。后续4天内，每日均将过夜混合培养物进行梯度稀释，取10⁻⁵稀释液100 μL加入5 mL LB培养基中，再取10⁻⁶稀释液涂布于LB X-gal平板完成定量。菌株适合度通过计算马尔萨斯参数（Malthusian parameters）的比值得到，具体方法参照Lenski等人（1991）的报道。

固体琼脂竞争实验：将霍乱弧菌的过夜培养物归一化至OD600=1，随后按体积比1:1混合。将混合体系进行梯度稀释，分别取10⁻⁵稀释液100 μL涂布于LB X-gal平板与LB平板。保留X-gal平板用于后续定量，将LB平板置于37℃静置培养。后续5天内每8小时取出LB平板，刮取平板上全部琼脂并转移至含10 mL LB培养基的50 mL离心管中。将离心管涡旋30秒，取上清液300 μL至96孔板并进行梯度稀释。取约含200~1000菌落形成单位（Colony-Forming Unit, CFU）/100 μL的稀释液100 μL涂布于LB平板，用于下一个时间点的传代。每3个时间点通过蓝白斑筛选完成定量：取约含30~300 CFU/100 μL的梯度稀释液100 μL涂布于X-gal平板。适合度计算方法与液体LB竞争实验一致。

赖特-费希尔模型（Wright-Fisher Model）是进化生物学中用于描述包括遗传漂变在内的随机等位基因进化过程的标准框架，本研究中用于分析克隆干扰（clonal interference）。在赖特-费希尔模拟中，细菌的离散世代通过从上一代的加权二项分布中抽样得到。为模拟克隆干扰效应，每个细胞每一代发生突变的概率为10⁻⁵。当细胞发生突变时，突变的整体适合度效应从以Good等人（2012）推导的参数β为参数的指数分布中抽样获得。每次模拟运行1000次，为便于结果展示，选取200次随机模拟结果进行绘图。

统计学分析：两种竞争实验的95%置信区间均通过两样本t检验计算两组均值差异得到，详细信息参见补充表格。图2中的P值通过统计5000次模拟世代后达到固定（fixation）或灭绝（extinction）的种群数量，使用卡方检验（χ²-test）验证原假设：T6SS+菌株有50%的概率达到固定。

杀伤实验：将霍乱弧菌与氯霉素抗性大肠杆菌（Escherichia coli, E. coli）的过夜培养物归一化至OD600=1，随后按体积比10:1（霍乱弧菌:大肠杆菌）混合。取该混合液5 μL点样于LB琼脂平板，室温晾干10分钟。将平板置于37℃培养3小时。切取平板上的点样区域，与5 mL LB培养基一同涡旋30秒。将培养基进行梯度稀释，取各稀释液5 μL点样于LB氯霉素平板。

RNA测序分析：将T6SS+、T6SS−与野生型（Wild Type, WT）霍乱弧菌的过夜培养物置于37℃ LB培养基中培养（n=2次生物学重复）。使用Qiagen RNeasy迷你试剂盒（Qiagen公司，德国希尔登市）提取上述培养物的RNA。使用NEB Ultra II定向RNA文库制备试剂盒（New England Biolabs公司，美国马萨诸塞州伊普斯维奇市）构建RNA文库，并使用QIASeq Fast Select -RNA HMR试剂盒（Qiagen公司，德国希尔登市）完成细菌核糖体RNA（ribosomal RNA, rRNA）的去除。将构建完成的细菌RNA文库在Illumina NovaSeq 6000测序平台（Illumina公司，美国加利福尼亚州圣迭戈市）上进行高通量测序。转录组数据分析流程如下：首先使用Trimmomatic（v0.39）对测序reads进行接头修剪与低质量reads过滤（Bolger et al., 2014）；随后使用STAR（v2.7.11a）将双端reads比对至霍乱弧菌C6706的参考序列（NCBI RefSeq组装号GCF_009763945.1），生成按基因组坐标排序的二进制比对文件（Binary Alignment Map, BAM）（Dobin et al., 2013）；使用featureCounts（v2.0.6）统计NCBI RefSeq注释中所有已注释基因座的比对片段数（Liao et al., 2014）；过滤低表达的片段计数后，使用R语言中的DESeq2包进行差异表达分析（Love et al., 2014）。