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 conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding 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 fluorescent protein gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescent protein 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 fluorescent protein gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescent protein 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.

Automated summary

In this study, a simplified experimental method is introduced for measuring the transfer frequency of conjugative plasmids between bacterial strains and species. A blue fluorescent protein gene is inserted into the conjugative plasmid of interest, and a small non-conjugative plasmid carrying a red fluorescent protein gene is used to label the recipient bacterial strain. The use of a strong constitutive promoter allows for clear distinction between donor, recipient, and transconjugant populations using flow cytometry, enabling precise monitoring of conjugation frequencies over time.