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

Abstract

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

Recipient UvrD helicase is involved in single- to double-stranded DNA conversion during conjugative plasmid transfer

“…After a conjugative plasmid transfer event has taken place, the ds plasmid (or a partially replicated intermediate containing a ds parS pMT1 site) is formed and mCherry-ParB pMT1 signals convert into foci (Figure 4A ). As ParB does not bind to the ssDNA version of parS ( 37 , 38 ), this system acts as a sensor for the conversion of ssDNA to dsDNA during plasmid establishment in the recipient ( 39 ).…”

“…Plasmid genes at the first region to enter the recipient cell (leading region) are usually repressed in the donor cells and expressed upon the entry of plasmid DNA in the recipient cell ( 13 , 39 , 47 , 48 ). A recent paper showed that translocation of proteins encoded in the leading region from the donor to the recipient cell through T4SS barely happens, even if they are overexpressed ectopically ( 49 ).…”

Addressable and adaptable intercellular communication via DNA messaging

“…One potential class of applications where the use of our addressing system may not always be appropriate, however, is in cases where a transient amount of off-target expression would be detrimental. Because our system blocks transfer by degrading the message after it has entered the recipient cell, it is possible that genes on the message could be expressed in an off-target recipient before the message is cleaved and degraded—indeed, some genes carried on the F plasmid have been observed to express as soon as 10 min after the plasmid’s initial entrance into a receiver cell 55 , 56 . Preliminary experiments with our F HR -based system, however, suggest that when genes are expressed weakly from the message plasmid, Cas9-mediated cleavage can occur quickly enough to prevent any detectable activity of these genes within off-target recipients ( Supplementary Note ; Supplementary Fig.…”

Bacterial Subcellular Architecture, Structural Epistasis, and Antibiotic Resistance

“…The conjugative process of plasmids (which eventually encodies antibiotic resistance), might produce (probably small) alterations in the bacterial architecture of the donor cell, because of the relaxosome protein complex (docked to the Type IV secretion system) and the entire conjugative apparatus, forming an envelope structure bridging the IM and OM (86). In the donor and recipient cells, the exit and the entry of the ssDNA plasmid in triggers the local recruitment of Ssb (singlestrand binding protein) molecules, and the formation of membrane conjugative foci, which are apparently located at specific membrane positions, possibly related to the density and stability of the outer membrane protein OmpA, a beta-barrel porin, collaborating in the process (87). Eventually, mobile genetic elements (possibly small plasmids?)…”