Bacteria caught live in action while smuggling genes from their surroundings.

Whenever a big discovery is made in the lab, you will hardly hear them saying "Eureka" ; it will mostly be -"wtf just happened". Probably, something similar happened with Courtney Ellison, at Ankur Dalia's lab in Indiana. I imagine a reaction like something like - wtf , did this bacteria just go fishing for DNA in its surrounding.

Figure 1 For the first time they captured the bacteria live in action as it threw its appendages around and uptook the DNA on binding.
Image showing a cartoon summary of the study, showing DNA(red) bound to tip of the pilius(green) of a bacterium Vibrio cholerae, which then retracts pulling the DNA inside the cell. You can see the live video of pili in actio here

What is so great about this study? Why should I care?
To make long story short, the bacteria, has ability to take up extracellular DNA floating around in environment. While it is not clear why they take up DNA from the environment in the first place, that ability has been pretty well known to most people in the field. There are multiple competing hypothesis to why it needs to take up the DNA. Whatever it maybe, it seems that the bacteria takes up DNA, not just for nutrition but it also uses this DNA to repair and update itself. For instance, if the bacteria takes up DNA very close to its own it uses it repair its own genome via homologous recombination. If bacteria has taken up a plasmid that has some antibiotic resistance gene, it becomes resistant to that antibiotic. It gives bacteria a survival advantage over other bacteria, when exposed to antibiotics. And this is what makes this study even so important.

The antibiotic resistant strains of bacteria creates a havoc for human health. And rise in antibiotic resistant strains of TB, pneumonia, meningitis and what not is already of huge concern (See Fair et al., 2014). Given bacteria grows and evolve faster than the speed at which we can design new antibiotics, the most optimal strategy to fight against bacteria would be to stop the spread of antibiotic resistance. So in theory, if you understand mechanism of how bacteria takes up DNA you can slow these bitches down.

(On the other hand ability of bacteria to take up DNA is heart of molecular biology and genetic engineering. Almost all biological studies need playing around with genes. And, like it or not even the genes made to be inserted in model animals or mammalian cells or in those CRISPR viruses are first cloned inside a bacterial plasmid. The industrial insulin you get for diabetics, or even your vitamins such as B12 which you need if you are on vegan diet, are produced by bacteria which are genetically engineered to make best possible product. While there are different methods to make bacteria artificially competent to take up DNA in the lab, a complete picture of different mechanisms of naturally competent strains will come as an add on.)

The research I mention here has beautifully dissected the mechanism of how a naturally competent bacteria - Vibrio cholerae takes up the DNA. In fact for the first time scientists has watched this process as it happens live under the microscope.

What did we know before? Whats so cool or new about this study?

Well to summarise there were different competing models for how naturally competent strains of bacteria such as Vibrio choleraetakes up the DNA through it's membrane. To summarise you need a pore through which DNA can go in. However as it turns out the pore the bacteria uses, called the secretin pore for this purpose is way too thin for DNA to enter. However, what it rather interesting is that it is the same pore through which bacteria make this tiny thread like appendage called type IV pili. Type IV pili has previously been hinted to important for DNA uptake in bacteria. However the active role of this pili had not been clear before.(Muschiol et al., 2015). For instance we did not know that whether the binding to pili is compulsory for DNA uptake. And after binding to the pili whether the DNA passively translocates through the pore, or if pili actively pulls it inside(see figure 2a). Other possibility is that the pili actively comes on and off based on binding of pili to pore or pili (See figure 2b).

Figure 2: Competing models of DNA uptake in bacteria: (a)Trap and retraction model - in this model the DNA is bound and trapped on the pili. However after being trapped the DNA can be passively translocated via the ComEC pore by binding to ComEA (the DNA receptor) without retraction of pili. In this case pili just facilitates the interaction of ComEA with DNA. Alternatively, pili can retract and apply the force to pull the DNA inside. (b) Hole in the wall model - in this model the pili may again just facilitate the binding of DNA to ComEA if anything. However the pili falls off because of some ATPase enzyme acting up , creating a hole in the membrane through with DNA just moves in.
Cartoon inspired from reading: Muschiol et al., 2015

This lack of this knowledge can be attributed to lack of live imaging that captured DNA uptake live in action. Previous attempts such as by Hepp et al., 2016 have used indirect methods to measure the speed and force of DNA uptake to get the hint. However the picture remained incomplete as speed does not tell you which component molecule is generating that speed and force. For instance Hepp et al., suspected that retraction of pili might not play a major role in DNA translocation but binding to DNA receptor ComEA does. Motivated to resolve the actual mechanism the current study established an active role of type IV pili in uptake of DNA. If you watch the video, you will see that , they very cleverly labelled pili and the DNA , and imaged the bacteria live in action. This study clearly shows that the pili actively pulls the DNA inside. The force of pulling is generated by an enzyme called PilT, which is important, however not sufficient. The DNA uptake requires both the force of retraction plus binding of DNA to ComEA (Figure 3). Almost as if a lizard is grabbing its prey by its tongue and pulling it inside, just that in this case the tongue is a nano-machine called pili.

Figure 3: The model from the current study, showing that DNA is bound to tip of the pili which then retracts. And since ComEA DNA receptor is localised behind the membrane the force of retraction is rather important in making sure that DNA is pulled inside this small pore. While the pili is retracting the DNA receptor ComEA gets assembled at the pore and binds to DNA if the retracting DNA pulls the DNA inside along with it.
Cartoon inspired from Ellison et al., 2018

So how did they do it? What was the fancy trick?

Like I mentioned earlier, that the gap in the knowledge majorly existed because no one has seen DNA uptake happen live before. The used a smart trick of site specific labelling. Which means they made a pili with a mutation which can be specifically labeled by dye of their choice. They used another dye to label the DNA and Bazinga! Now they could watch it happen all live. They can measure speeds and see mutating which component of pili affects binding of DNA or measure speed or pili extension or retraction. Using the same dye binding technique they can rather bind heavy molecules to the pili and physically stopping the retraction to see how important retraction is. And this is what help them elucidate what previous studies did not. Keep reading if you want to know the details of how they did this.

Site specific labelling of Pili and live imaging
Well one of the rate limiting step when you want to specifically image a cell appendage live in action is to find a way to label it. Think if you were the one doing this experiment how would you do it? Well I will find a protein specific to pili and tag it or label it with a dye, and look under the microscope. Thankfully molecular structure of type IV the structure of the pili is pretty well known. A pili is made by polymerization and extension small units of protein called PilA. The extension is made possible by an enzyme called pilB which is ATPase like PilT but functionally antagonistic. So you can technically take a strain of a bacteria which makes a lot of these pili structures and label PilA. Luckily, there also exists a technique in which you can dye label a protein, with any (thiol reactive) fluroscent dye of your choice if the protein has a an amino acid cysteine on its surface(Kim et al., 2008).

So these guys took a mutant strain of Vibrio cholerae which makes a lot of pili. Now PilA doesn't have cystine residue on its surface for labelling. So they created a bacteria with mutant PilA which has a cysteine on the surface. After confirming that there are no unwanted side affect of mutation, they added a fluorescent dye on bacteria which would now bind to PilA and make the pili green. They saw that these pili had dynamic activity in which they kept extending and retracting even in absence of DNA. They then used DNA dye to color red and the rest is history. So now you clearly see in the video the the pili extends and retracts and if DNA is bound at the tip of pili it just pulls it along with it during retraction.

Pili is required for binding of DNA
However, that is just the visual aspect of it and doesn't tell you much about what is going on at the scale of molecules. And if you were a scientist you would want to validate what you see by making predictions and testing them. For instance you would want to know if DNA binding was specific to the pili fibre. Since pili fibre is made by PilA, they made a mutant PilA bacteria which failed to form pili. The loss of pili prevented the Vibrio cholerae from binding to DNA in its environment. They also ruled out that this loss of DNA binding is due to any abbe ration in any secretin pore proteins, which is implied in DNA uptake. This establishes the crucial role of pili in context of DNA binding and DNA uptake. And this kind of makes sense because the secretin pore itself is way too small. The diameter of the pore is merely equal to DNA double helix folded once. This make probability of passive entry of DNA via outer membrane highly unlikely. The binding of DNA to pili may either serve the function of properly orienting DNA for entry into the pore. Alternatively the retraction of pili can generate a force to drag in the DNA along with it (figure 2a).

Importance of pili retraction
Well a simple experiment to test how important is retraction of pili one might think of way to simple physically hold back the pili from retracting . The authors did this by load the pili with heavy load. They used the same cysteine mutation they made to pilA to bind the dye with it. Except this time other than the dye they used the load of biotin and neutravidin to put weight on the pili. And it seems that blocking the retraction physically led to a 100 fold decrease in DNA uptake.

You may predict now that if retraction is really important then critical proteins in DNA uptake such as ComEA might work hand in hand with pili retraction. And that is what the authors see as well. ComEA assembled at the pore in periplasm (space between inner and outer membrane of bacteria) only when pili was retracting.

Speed and force of retraction matters

Since I told you earlier that PilT is an enzyme important for retraction of pili, you many ask what happens if we mutate PilT. Well to begin with they show that retraction doesn't completely stop even when you make a strain with loss of function mutation in PilT. Nonetheless DNA uptake is reduced by 10000 folds. Now it is possible that there is some basal level of retraction and PilT just speeds it up. Now if speed and force applied by PilT is important it may explain why PilT mutants did not uptake DNA even though pili was retracting. In fact this is what the authors find , that speed and force of retraction in PilT mutants was significantly lower.

Based on these experiments we can safely assume a model in which these type IV pili keeps extending and retracting randomly fishing for DNA in the surrounding. PilT provides a force for retraction that is capable of pulling the DNA inside the secretin (PilQ) pore. At the same time as retraction it is important that ComEA DNA receptor also gets assembled at the pore behind the membrane. If the retracting DNA pulls the DNA inside it is bound to ComEA and from here onwards ComEA and other machinery at the pore takes the charge to pull the DNA inside. (Figure 3).

Now getting back to the significance of this study, you may ask - so this is what happens, now what could scientists make to stop the spread of antibiotic resistance. Well they can make a drug that stops pili retraction, or they can make a drug that can inhibit binding of DNA to the pili itself. If such a drug is given in combination with antibiotics it can in theory reduce the socioeconomic burden of widespread antibiotic resistance.

PS: There are other cool experiments in the paper as well which you may love to read. I just highlighted the most crucial ones , which I think makes the point. For instance they also elucidated the small pilins required for binding of DNA to tip of pili. They also beautifully validated that the pulling of DNA you see in video is in fact the DNA being pulled by the pili.

References
Ellison CK, Dalia TN, Vidal Ceballos A, Wang JC, Biais N, Brun YV, Dalia AB. Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae. Nat Microbiol. 2018 Jun 11. doi: 10.1038/s41564-018-0174-y. [Epub ahead of print] PubMed PMID: 29891864.

Fair RJ, Tor Y. Antibiotics and Bacterial Resistance in the 21st Century. Perspectives in Medicinal Chemistry. 2014;6:25-64. doi:10.4137/PMC.S14459.

Muschiol S, Balaban M, Normark S, Henriques-Normark B. Uptake of extracellular DNA: competence induced pili in natural transformation of Streptococcus pneumoniae. Bioessays. 2015 Apr;37(4):426-35. doi: 10.1002/bies.201400125. Epub 2015 Jan 15. PubMed PMID: 25640084; PubMed Central PMCID: PMC4405041

Kim Y, Ho SO, Gassman NR, Korlann Y, Landorf EV, Collart FR, Weiss S. Efficient site-specific labeling of proteins via cysteines. Bioconjug Chem. 2008 Mar;19(3):786-91. doi: 10.1021/bc7002499. Epub 2008 Feb 15. PubMed PMID: 18275130; PubMed Central PMCID: PMC3086356.

Deceleration
No copyrighted images has been used in this article. All cartoons used are illustrations created by me based on the literature cited.