Through The Pilus

by Roberto
 

Among the many mechanisms of bacterial horizontal gene transfer, conjugation, the plasmid-mediated cell-to-cell movement of DNA, holds a special place in the heart of this old-timer E. coli geneticist. Its discovery not only allowed genetic mapping through "interrupted mating" experiments, it also opened the door to studies on the regulation of gene expression (witness the famed PaJaMo experiment).

 

At the molecular level, the mediator of conjugation went from "F" the fertility factor to the F-plasmid, encoding the machine needed for conjugal transfer. A key component of this machine is the F-pilus. Charlie Brinton's iconic electron micrograph of the E. coli F-pilus connecting donor and recipient cells, which Christoph considered in STC, provided the "icing on the cake" of conjugation. The beauty of that image allowed the imagination to picture the donor DNA going through the pilus. But did it really?

 

Subsequent findings, from work that spanned many decades, provided mixed evidence for exactly what was the role of the F-pilus in mediating DNA transfer. The fact that the F-pilus retracts, along with detailed description of an DNA-transfer complex at the surface of the cells led to a change in interpretation. The F-pilus may just serve to contact recipient cells and "lasso" them into close contact with the donor. But then, in 2008, Babic and colleagues published a paper indicating that the donor DNA could indeed go through the F-pilus (see here in STC). Yet, critics were quick to note that the experimental setup used in that paper could not rule out that the conjugation events occurred prior to imaging. Importantly, in those experiments the F-pilus was not directly visualized. So, the concept of DNA going through the F-pilus began to fade away. Until about a year ago...

 

Fig. 1. Time-lapse images of distant transfer events between E. coli ssb-ypet/F-parS/p traAS54Cdonors labeled with AF-Mal488 and E. coli ssb-ypet recipients producing mCh-ParB. Distant transfer is reported by both the formation Ssb-Ypet (Yellow) conjugative foci at each end of the pilus (Green) connecting the mating pair cells and the subsequent the formation of mCh-ParB (Red) foci, which confirms the acquisition of the dsDNA plasmid by the recipient cell. Top: Fluorescence micrographs. Bottom: Schematic Diagram, D – donor, R – recipient, T – Transfer Scale bar 1µm. Adapted from source. Frontispiece: Fluorescence micrograph of cells expressing TraA^S54C and labeled with AF-Mal⁴⁸⁸. Source

In late 2023, Kelly Goldlust and colleagues published a paper that, once and for all, provides direct evidence that DNA can and does go through the F-pilus. To accomplish this, the authors developed a clever setup. For one, they used living cells trapped in a microfluidic chamber making it possible to generate time-lapse movies of individual mating pairs using fluorescence microscopy. For these movies, they developed three conjugation-related fluorescent markers in the cells. They visualized the F-pili by labelling the pilin protein with a stain that fluoresced green. (They did this by first changing a serine to a cysteine in the pilin and then binding the fluorescent dye AF-Mal⁴⁸⁸ to all surface-exposed cysteines.) They visualized single stranded DNA (which is the form of DNA that gets transferred) in donor and recipient cells through its being bound by a yellow-fluorescing single-stranded-DNA-binding protein fusion (Ssb-Ypet). And, to visualize the formation of double stranded DNA in the recipient, they used a different protein fusion which fluoresced red (mCh-Par). The images on the figure tell the story. At 0 minutes, a mating pair is spotted. Donor and recipient cells are physically separated but connected by the F-pilus. They will remain separated throughout the whole movie. At 6 minutes, right at the junction points where the F-pilus connects with both donor and recipient, foci of single stranded DNA become apparent. Single stranded DNA must be going through the pilus. And by 12 minutes, a focus the newly formed double-stranded DNA is seen in the recipient. Yes indeed, during conjugation, the DNA can go through the F-pilus. When it is all said and done, turns out only about 6% of the mating pairs they observed transferred their DNA through the F-pilus. The rest? Those mating pairs were closely adjoining cells. So, it's not always that the "Brinton" through-the-pilus transfer occurs. But for those of us initially fascinated by that concept, it's nice to see direct proof that it can and does happen!
 

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