Vibrio cholerae harpooning DNA
Pictures Considered #60
by Christoph
Let me start with an adage: a picture is worth a thousand words. Can this also be valid for video snippets? I think so. Here you see a few seconds of a looping video showing how a cholera bacterium extends a pilus, attaches the tip to extracellular DNA, and then retracts it. Worth a thousand words? Yes, sure, but also deserving 1,000 words, and not the few with which I first mentioned this video in STC.
Figure 1. This video shows a pilus shoot out from the bacterial cell to latch onto a piece of DNA. The image on the right shows the bacteria and pilus in green and the DNA in red. The image on the left shows the same cells visualized using phase contrast microscopy. Credit Ankur Dalia, Indiana University. Source
A hint: if the fidgety video in Figure 1 makes you dizzy, take a look at the stills taken from it in Figure 2. You will admit, though, that the dynamics of the process only really come into their own in the video.
One of the first things you will notice in the video taken by Ankur Dalia, corresponding author of the Ellison et al. (2018) paper, is that the wriggling motion of the pilus is completely different from that of a rotating flagellum. This is more obvious in the video than in the stills! It has long been known that type IV pili "move" by extension from their base in the cell wall followed by retraction. But it has rarely, if ever, been shown "in the act" in such detail. What you cannot see but can rightly assume: both pilus extension and retraction are ATP hydrolysis‑driven just like flagellar rotation.
Figure 2. Clockwise from upper left: this series of four still images shows a pilus stretch out from a bacterium, in green, to catch a piece of DNA in the environment, in red. This is the first step in the DNA uptake process. Credit Ankur Dalia, Indiana University. Source
The Vibrio cell stays put in the video and pilus retraction brings the attached DNA closer to it. That's because the cell has significantly more inertial mass than the DNA snippet. This is quite different in twitching motility, where the cell is pulled towards a solid substrate such as an agar surface by retraction of its pilus.
Pilus retraction is triggered when the minor pilins at the tip meet resistance, here Cy3-labeled DNA. You may notice that the pilus tip seems to lose its "red dot" as it retracts. Probably the Cy3 DNA label slipped out of focus here. Ellison et al. (2018) show convincingly in Figure 3 that pilus retraction reliably results in delivery of the attached DNA to the cell's outer membrane (vibrios are Gram-negative).
You may wonder why the Cy3-labeled DNA appears as red dot rather than as the thin line you're familiar with from DNA depictions in diagrams. The 6 kb PCR fragment carrying Cy3 labels randomly every 20−60 bases that Ellison et al. (2018) used in their experiments would have a length of ~2 µm when fully stretched between optical tweezers and should be visible as a faintly fluorescent reddish thread (see scale in Figure 3). However, under the experimental conditions (temperature, ionic strength of the medium), 2 µm long DNA pieces don't stretch but tend to form the small irregular blobs you see (and by "blobbing" graciously increasing the fluorescence signal by locally concentrating it).
You also see in Figure 3 the size scale and the timer that are missing in the video. The time required for an extended pilus to fully retract (~20 s) shows that the video in Figure 1 must have been sped up a bit (not really a time lapse though) compared to the up to 1,700 rps (revolutions per second) of V. cholerae flagella under optimal conditions, which drive the cells to swimming speeds of ~50 µm/s, pili extension and retraction are downright sluggish processes.
Figure 3. Type IV competence pili bring bound DNA to the cell surface. Montage of time-lapse imaging of pilA-cys strain after labeling with AF488-mal and incubation with Cy3-labeled DNA in a wet mount. Scale bar, 1 μm. Images are representative of three independent experiments. Source
Neither the Cy3-labeled DNA nor the pilus are visible by phase-contrast bright-field microscopy (left side in the video). How could they make the pili "visible" by fluorescence microscopy while keeping them fully functional? Maybe you recall that electron microscopists could make F pili visible by "decorating" them with F pili‑specific phages (look up Figure 2 in our Pictures Considered #55, or click here), but this approach would not be suitable for studies of pili dynamics. Briefly, Ellison et al. (2018) had adapted and refined a method developed for fluorescently labeling flagella (described in detail here) that uses click-chemistry coupling of a bifunctional, thiol-reactive maleimide carrying a fluorophore, AF488-mal, to a cysteine knock-in mutant (S81C) of the major pilin, PilA, of V. cholerae. They found that neither the S81C mutation per se or coupling of the fluorophore to Cys81 of PilA lead to any detectable impairment of pili functioning during extension or retraction. Note, however, that the brightness of the label makes it impossible to even approximately measure the width of the pilus.
It should not go unmentioned that the Vibrio strain so eagerly wielding its pilus in the video (Figure 1) was genetically manipulated to not express any other pili or flagella, and to be constitutively competent. In addition, the strain was cultured under conditions that lead to pili formation in narrow time window of ~ 1h prior to observation.
Sometimes, though rarely, molecular tinkerers get unexpected but most welcome perks. Here: the bright green, fluorescent cells. No extra labeling was necessary to make them visible! Ellison et al. (2018b) had found in their previous study of Caulobacter pili that the continuous staining of the cell cytoplasm is a consequence of the repeated extension and retraction of AF488-mal stained pili whose PilA subunits are recycled without losing the covalently bound label. Go figure!
One question remains: what is the prominent yellow spot at the base of the pilus? In response to my question, Courtney Ellison explained: "...about the fluorescence, the yellow spot is the overlay between the green and red channels. So, for the V. cholerae cell that is red labeled DNA that has been taken up into the cell by [retraction of] the pilus." (I should have studied their methods paper more closely, which mentions this.)
Addendum
Francis Collins was so enthusiastic about the video that he wrote in his NIH Director's Blog: "Now that we can visualize exactly how horizontal gene transfer works, it may be possible to develop new and better strategies for interfering with this process." I share his enthusiasm, but humbly point out that this visualization is only one step, albeit a crucial one, in understanding how horizontal gene transfer (HGT) works by one of the known possible pathways, namely transformation (others are conjugation and transduction).
In an earlier post, Dial "V" for Murder, I described HGT via transformation of naturally competent Vibrio cells like a script for a crime movie, and divided it into scenes. I leave it to you to figure out where in that story Ankur Dalia's video fits in (spoiler: it does).
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