Live-Cell Visualization of DNA Transfer and Pilus Dynamics During Bacterial Conjugation

Real time visualisation of conjugation reveals the molecular strategy evolved by the conjugative F plasmid to ensure the sequential production of plasmid factors during establishment in the new host cell

“…During conjugation, the intracellular localisation of Ssb changes dramatically. As previously reported (Goldlust et al, 2022;Nolivos et al, 2019), the entry of the ssDNA plasmid in the recipient cell, now called a transconjugant, triggers the recruitment of Ssb molecules and the formation of bright membrane-proximal foci, we termed Ssb conjugative foci (Figure 1B, Figure S1). Here, we also observe the formation of Ssb conjugative foci in the donor cells, thus revealing the presence of ssDNA plasmid on each side of the conjugation pore during transfer (Figure 1B, Figure S1).…”

“…The conversion of the newly acquired ssDNA plasmid into dsDNA by the complementary strand synthesis reaction and the subsequent plasmid duplication events were analysed using the parS/ ParB DNA labelling system (Goldlust et al, 2022; Nolivos et al, 2019). The parS binding site is inserted in the F plasmid, while the ParB binding protein fluorescently labelled with the mCherry (mCh-ParB) is produced from a plasmid in recipient cells only.…”

“…Our current knowledge of conjugation mainly emerges from experimental genetic, biochemical and structural studies that provided a well-documented understanding of the molecular reactions and factors involved in DNA transfer, while genomic and computational studies uncovered the diversity of conjugative plasmids and their importance in the epidemiology of antibiotics resistance dissemination. It is only recently that the application of optical microscopy has started to provide insights into the organisation of conjugation at the cellular scale (Aguilar et al, 2011; Babic et al, 2011; Babić et al, 2008; Carranza et al, 2021; Clarke et al, 2008; Goldlust et al, 2022; Lawley et al, 2002; Low et al, 2022; Nolivos et al, 2019). In this study, live-cell microscopy combined with specifically developed fluorescent reporters offers a unique view of the cellular dynamics of conjugation while providing insights into the timing and localisation of each key step.…”

“…In this study, we use live-cell microscopy imaging to visualise the complete transfer sequence of the native F plasmid between E. coli K12 strains. We inspect the key steps of conjugation using specifically developed genetic reporters, including a fluorescent fusion of the chromosomally encoded single-strand-binding protein Ssb (Ssb-Ypet) to monitor the ssDNA transfer, the mCherry-ParB/ parS system to reveal the ss-to-dsDNA conversion and subsequent plasmid duplication, and translational fluorescent fusions to quantify and time plasmid-encoded production in the new host cell (Goldlust et al, 2022; Nolivos et al, 2019). This approach uncovers the choreography of conjugation reactions in live bacteria and provides new insights into the interplay between plasmid processing and gene expression.…”

Real-time visualisation of the intracellular dynamics of conjugative plasmid transfer

“…In this study, we use live-cell microscopy imaging to visualise the complete transfer sequence of the native F plasmid between E. coli K12 strains. We inspect the key steps of conjugation using specifically developed genetic reporters, including a fluorescent fusion of the chromosomally encoded single-strand-binding protein Ssb (Ssb-Ypet) to monitor the ssDNA transfer, the mCherry-ParB/ parS system to reveal the ss-to-dsDNA conversion and subsequent plasmid duplication, and translational fluorescent fusions to quantify and time plasmid-encoded production in the new host cell 58 , 59 . This approach uncovers the choreography of conjugation reactions in live bacteria and provides new insights into the interplay between plasmid processing and gene expression.…”