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 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.