DOI: 10.5061/dryad.12jm63z2t
title: A streamlined approach for fluorescence labelling of low copy-number plasmids for determination of conjugation frequency by flow cytometry

description: 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.
description: <strong>Flow
 Cytometry</strong> 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
 OD<sub>600</sub> 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.
 <strong>Data
 Analysis</strong> 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
 <em>ebfp2</em> 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, 10<sup>5</sup>
 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<sup>+</sup> and
 <em>ebfp2</em><sup>+</sup> 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
 (<em>ebfp2</em><sup>+</sup>) and J53Az +
 p_mCherry-stable control (mCherry<sup>+</sup>) populations.
description: 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