Shining a light on Vibrio DNA uptake
by Rachel Diner
Vibrio cholerae is "kind of a big deal" in the bacterial world and a popular topic here on Small Things Considered. Beyond being the causative agent for the disease cholera, it's a model bacterium for studying horizontal gene transfer and interbacterial competition.
A recent study in Nature Microbiology sheds new light (literally) on how this happens. Using advanced fluorescence-based microscopy techniques combined with classic microbial genetics, the authors present exciting new insights into how V. cholerae takes up DNA from the environment. The study confirms previously hypothesized mechanisms of DNA uptake, and also shows through stunning videos just how fast and flexible this uptake mechanism.
Seeing is believing
There are two common models proposing how bacteria bring DNA from the environment into the cell during natural transformation. A dominant theory poses that competence pili bind to DNA outside of the cell and then retract, pulling the DNA with it into the cell. These type IV competence pili are distinct from the type IV secretion system and other pili the bacteria produce. An alternative theory suggests that the pili act as "gatekeepers," essentially signaling the cell to allow passive diffusion of environmental DNA through the membrane.
Figure 1. Proposed ComEA dependent mechanism by which double stranded DNA is forcefully pulled through the PilQ pore via a molecular ratchet mechanism. Source
The evidence that would distinguish between these models: does the pilus actually bind to DNA?
Previous microscopy studies suggested that DNA binding is exactly what happens. Using electron microscopy (EM), researchers had previously observed DNA co-localizing with the pilus. But there are two major problems with this type of evidence. First: it's static. EM gives you a snapshot that doesn't allow real-time observation. It just shows a potentially fortuitous moment in time. Secondly, how can you tell if the DNA is actually attached, or just-co-localized?
Using real-time fluorescence microscopy, Ellison and co-authors captured the entire "catch and reel" process. First, they altered a hyperpiliated pilT mutant strain so that it could become labelled with a fluorescent dye. They did this by replacing an amino acid residue of PilA, the major pilin subunit (Figure 1), with a cysteine that could be fluorescently labelled with a thiol-reactive maleimide dye. Then, they labelled both the DNA and the pili using a constitutively competent V. cholerae strain (Figure 2).
Modifying is (also) believing
Having observed this interaction, the authors next sought to disrupt it using targeted mutations. First, they identified mutations that would reduce the DNA binding activity. By altering positively charged residues in major and minor pilins, they identified specific minor pilin mutations (VC0858R165Q, VC0859K148Q, and VC0858R168Q, at the tip of the pilus in Figure 2) that inhibited or abolished DNA binding and in some cases altered transformation efficiency and pilus dynamics.
Figure 2. Diagram of the proposed Type IV pilus in V. cholerae including minor VC pilins at the pilus tip that, when mutated, can abolish DNA binding capability. Supplementary Figure 1a of Source
Sterically inhibiting pilus retraction (a method necessary because knocking out these essential genes impacts pilus function and gene expression) did not affect DNA binding. However, transformation frequency was massively impaired. More than 99% of DNA uptake events required this pilus extension-retraction mechanism. Interestingly, cells could capture linear or circular DNA fragments equally well, though they seem to prefer double-stranded DNA.
Additional experiments that examined cellular proteins involved in DNA uptake into the periplasm and pilus retraction, showed that pilus retraction is necessary but not sufficient for DNA uptake. A fluorescently-fused ComEA protein, a protein essential for natural transformation that binds to DNA in the periplasm, was frequently and readily observed, but only after retraction of the pilus (Figure 3). Confirming previous studies, ComEA knockout mutants could not internalize DNA even with normal type IV pilus dynamics. This important role of ComEA and other mechanistic features in V. cholerae DNA uptake is discussed in a previous STC article.
Together these results allude to a natural transformation process, where DNA binds to the tip of an extended pilus which then triggers the ComEA-dependent mechanism to pull DNA through the outer membrane and into the cell (Figure 4).
Wiggle with it
Now that the authors confirmed the importance of pilus binding and retraction for DNA transport into the cell, they generated and published several real-time videos (one example at the end of this post) showing spectacular features of this well-studied system. They are pretty amazing, so give your cat videos a rest (do as I say, not as I do) and check them out!
Figure 3. Extension of the green-labelled type IV pilus (middle pane), followed by retraction and subsequent visualization of red-labelled comEA protein, signaling DNA internalization. Source
One notable observation: the type IV pili that V. cholerae use aren't rigid, static structures: they writhe, wiggle, and move. In fact, what first caught my eye about this story was a striking clip showing a flexible and fast moving pilus capturing the DNA and "reeling" it in. While many news articles described this as a "harpoon-like" system, it seems to me much more closely related to fly fishing.
This is in contrast to the murderous type VI pili of V. cholerae , which are indeed harpoon-like. Zoued et al. eloquently called these "nano-cross-bows." These can be used to kill other bacteria, at which point V. cholerae can then then take up their precious DNA. However, the type VI pilus doesn't actually bind to it. That being said, the systems are related, and common cues can trigger both the killing and DNA-uptake mechanisms.
Rocket speed
To catch this potentially valuable DNA, V. cholerae generates these pilus assemblages at a staggering rate, and then retracts them even faster. The pili are about 1 µm long, and about 47% of cells grown in competence-inducing conditions generated one or more within one minute. Since the bacteria are typically about 1 – 2 µm in length, this is roughly equivalent to a human generating a limb the length of their entire body. Spectacular but really, really weird.
Figure 4. Time-lapse images of fluorescently-labelled Vibrio cholerae strains showing green pilus assemblages binding at the tip to red-labelled DNA. Source
While V. cholerae grow its pili quickly, they retract them twice as fast, in roughly 30 seconds. This allows the bacteria to quickly and efficiently pull the captured DNA to their surface. In fact, some cells generate multiple pili at the same site, suggesting that some of the components may be reused locally.
So, what's the rush? This is a major expenditure of cell resources (just thinking about my example of growing a new limb every minute makes me exhausted). It may allow bacteria to take advantage of potentially useful "pulses" of free DNA in the environment. Or perhaps disassembly is easier than assembly, and the resources are needed desperately for additional pili.
What's new about this news?
Natural transformation is a way in which bacteria, including V. cholerae , take up useful DNA from the environment, including antibiotic resistance genes and virulence factors. A convincing mechanism for DNA uptake in V. cholerae was previously hypothesized and partially demonstrated. But the exact role of the type IV pilus in this process, which we knew was required for natural transformation, wasn't substantiated until now.
With stunning visuals and extensive experiments, this study goes a long way towards a comprehensive understanding of this DNA uptake in bacteria. In sum, it's fantastic to see (and believe) new discoveries about this amazing natural transformation system.
Reference
Ellison CK, Dalia TN, Ceballos AV, Wang, JCY, Biais N, Brun YV, Dalia AB. (2018). Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae . Nature Microbiology, 3, 773 − 780. PMID:29891864 (paywall)
Rachel Diner is a Ph.D. candidate in Andrew Allen's lab at UCSD and the J. Craig Venter Institute in La Jolla, CA. She studies marine microbes and how they interact with each other, humans, and the environment. She is also a new mom, which gives her ample time for running her own blog (really?).
