A streamlined approach for fluorescence labelling of low copy-number plasmids for determination of conjugation frequency by flow cytometry

Bacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of the conjugation frequency of plasmids between bacterial strains and species is therefore important to understand the transfer dynamics and epidemiology of conjugative plasmids. In this study, we present a streamlined experimental approach for fluorescence labelling of low copy-number conjugative plasmids that allows plasmid transfer frequency during filter mating to be measured by flow cytometry. A blue fluorescence gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescence gene with a toxin-antitoxin system that functions as a plasmid stability module, is used to label the recipient bacterial strain. This offers the dual advantage of circumventing chromosomal modifications of recipient strains and ensurin..., Flow Cytometry
Filter mating was performed in four biological replicates on four different days. Stationary phase cultures of the recipient strain J53Az + p_mCherry-stable and the donor strain UB5201Rf + pConj_blue-strong were grown in LB containing 8 µg/mL gentamicin and 50 µg/mL ampicillin respectively at 37°C with shaking. Each culture was washed in antibiotic-free LB broth and adjusted to the same OD600 in the 0.9±0.1 range. 1.6 mL of the donor and recipient strains were mixed, pelleted, and re-suspended in a 170 µL LB medium. Concentrated cultures of the single-color donor and recipient strains were prepared in an identical way. 40 µL drops of each cell suspension were transferred onto individual Whatman cellulose nitrate membranes (GE Healthcare, United States) placed on antibiotic-free 1.5% LB agar plates. At the start of the conjugation experiment, one membrane containing each cell suspension was re-suspended in 4 mL sterile-filtered PBS containing 0.2 mM EDTA and vortexed to di..., Flow cytometry workspace (WSP) files for each set of biological replicate should be opened with the FlowJo software (v10.7, FlowJo LLC): https://www.flowjo.com
Deposited plasmid maps were created using Geneious bioinformatic software (v10.2.6, Biomatters) according to the molecular cloning procedures described in the Materials & Methods section of the manuscript. The plasmid maps were exported in the GenBank format and can be opened with SnapGene Viewer (Dotmatics): https://www.snapgene.com/snapgene-viewer,

细菌接合（Bacterial conjugation）通过质粒的水平转移，在抗生素耐药性与毒力性状的传播中发挥核心作用。因此，可靠且精准地测量不同细菌菌株及物种间的质粒接合频率，对于理解接合性质粒的转移动力学与流行病学特征至关重要。本研究提出一种简化高效的低拷贝数接合性质粒荧光标记实验方案，可通过流式细胞术（Flow Cytometry）检测滤膜交配过程中的质粒转移频率。采用简便的同源重组工程（homologous recombineering）流程，将蓝色荧光基因插入目标接合性质粒。同时使用携带红色荧光基因且带有作为质粒稳定性模块的毒素-抗毒素系统（toxin-antitoxin system）的小型非接合性质粒，对受体细菌菌株进行标记。该方法兼具双重优势：无需对受体菌株进行染色体修饰，且确保……
滤膜交配实验设置4个生物学重复，于4个不同日期独立开展。受体菌株J53Az + p_mCherry-stable与供体菌株UB5201Rf + pConj_blue-strong的静止期培养物，分别在添加8 μg/mL庆大霉素与50 μg/mL氨苄青霉素的LB培养基中，于37℃振荡培养。将每株菌的培养液用无抗生素LB肉汤洗涤，并调整至OD600处于0.9±0.1的统一区间。取1.6 mL供体菌与受体菌混合，离心收集菌体后重悬于170 μL LB培养基中。单色供体、受体菌株的浓缩培养物制备流程完全一致。将每种细胞悬液滴加40 μL至置于无抗生素1.5% LB琼脂平板上的Whatman硝酸纤维素膜（GE Healthcare，美国）表面。接合实验启动时，将每张承载细胞悬液的膜重悬于4 mL经无菌过滤的含0.2 mM EDTA的PBS中，涡旋混匀……
每个生物学重复组的流式细胞术工作区（Flow Cytometry Workspace, WSP）文件需使用FlowJo软件（v10.7, FlowJo LLC）打开，下载地址：https://www.flowjo.com
已提交的质粒图谱使用Geneious生物信息学软件（v10.2.6, Biomatters），依据手稿材料与方法部分描述的分子克隆流程构建。质粒图谱以GenBank格式导出，可通过SnapGene Viewer（Dotmatics）打开，下载地址：https://www.snapgene.com/snapgene-viewer