A streamlined approach for fluorescence labelling of low copy-number plasmids for determination of conjugation frequency by flow cytometry
Data files
Mar 22, 2023 version files 1.63 GB
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Biological_replicate_1.zip
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Biological_replicate_2.zip
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Biological_replicate_3.zip
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Biological_replicate_4.zip
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p_mCherry-stable.gb
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pConj_blue-strong.gb
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pJIMK46.gb
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README.md
Abstract
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 ensuring that the red fluorescence gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescence genes to be strongly and constitutively expressed from the plasmids, thus allowing flow cytometers to clearly distinguish between donor, recipient and transconjugant populations in a conjugation mix for monitoring conjugation frequencies more precisely over time.
Methods
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 dislodge bacterial cells. This procedure was repeated for the remaining membranes every 2 hours of incubation on LB agar plates at 37°C for 6 hours. The recovered cell suspensions were further diluted 1:250 in 1 mL PBS + 0.2 mM EDTA. A culture of the non-fluorescent J53Az strain was also diluted at 1:1000 in PBS + 0.2 mM EDTA to be used as the non-fluorescent control strain.
Data Analysis
The diluted bacterial suspensions were well vortexed prior to analysis on the BD FACSymphony flow cytometer (BD Biosciences, United States). The mCherry fluorophore was excited by the yellow laser and detected through a 610/20 nm bandpass filter. The ebfp2 fluorophore was excited by the violet laser and detected through a 474/25 nm bandpass filter. Fluorescent beads with 0.88 µm and 1.34 µm diameters from the Size Standard Kit (Spherotech, United States) were used to validate the performance of the flow cytometer in detecting particle sizes. For each sample, 105 events were acquired and recorded by the flow cytometer within a time limit of 10 minutes. Flow cytometry data analysis was performed using the FlowJo software (v10.7, FlowJo LLC, United States). Briefly, a universal rectangular gate was used to separate bacterial cells from background noise events in all the SSC-A vs. FSC-A plots (Panels A and B, Figure S2, Supplementary Materials). The autogating tool was used to capture approximately 90% of all events based on the contour of each bivariate plot within each rectangular gate (Panel C, Figure S2, Supplementary Materials). Doublet discrimination was performed on the SSC-H vs. SSC-A bivariate plots by drawing a narrow rectangular gate along the diagonal to retain single cells (Panel D, Figure S2, Supplementary Materials). Compensation matrices were calculated for single-color control strains for mCherry+ and ebfp2+ from the 4 h time point and were applied to all samples. A spider gate was drawn on the compensated mCherry vs. compensated BFP bivariate plots to distinguish between J53Az (double negative), UB5201Rf + pConj_blue-strong control (ebfp2+) and J53Az + p_mCherry-stable control (mCherry+) populations.
Usage notes
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