I've been discussing the DNA uptake issues by email with a colleague who studies type four pili. She's pushed me into checking some things I thought I knew, and into making my ideas about uptake clearer.
First the retraction issue: I had been thinking that we knew that H. influenzae and other Pasteurellacea could do 'twitching motility', which meant that they must be able to actively retract their pili. But I rechecked and found that nobody has directly shown that they move. Strains of H. influenzae that have pili do form the same 'rafts' at the edges of their colonies that cells with known twitching motility do, but nobody's watched them doing it. I don't know how difficult this would be, but in any case we can't do it until we have cells that we know are piliated.
Second, the DNA-kinking issue: I had been thinking only at an intuitive level about this (hand-waving and doodles), and her questions pushed me into coming up with a physical model.
We can reasonably the DNA and pilus with a long rubber band (= circular DNA) and a pencil (= the pilus); the relative diameters are appropriate. If a loop of DNA is going to be pulled through a pore that's only slightly wider than the pencil, then all of the loop has to become closely appressed to the pencil. For this to happen the DNA must, at some point, make a 180 degree turn in a distance no longer than the diameter of the pencil. If we were to let the DNA detach from the pencil the turn could open up to a larger diameter, but then the pencil plus DNA wouldn't fit through the pore.
The 180 degree turn is a problem because the persistence length of DNA is about 50nm (150bp), whereas the pilus diameter is only about 6nm. Nucleosomes manage to bend DNA smoothly through two full turns over distances of 165bp; this is a diameter of about 9nm (radius of curvature of 4.3nm). Perhaps not coincidentally, part of the uptake signal sequence resembles a nucleosome-positioning sequence.
So the DNA loop needs to bend on the pilus a bit more sharply than it does in a nucleosome - maybe not too big a problem. And if the DNA is lying in the positively charged grooves of the pilus, it may not stick out too far to fit through the pore (our model needs a pencil with grooves). But where the DNA makes its turn, it will have to leave its snug grooves and cross over at least one of the raised parts of the pilus, making an awkward bulge on the surface that's going to have a hard time fitting through the pore.
So uptake of a loop raises two problems. One is forcing the DNA to bend sharply, and the other is fitting the pilus back through the secretin pore once it has a loop of DNA attached to it. I don't think this is impossible, but it probably takes some specialized interactions. I think the role of the uptake signal sequence is to interact with the pilus and secretin to facilitate this.
I'm going to see if I can buy some tubing of different sizes to use for a demo of the problem.
Friday Fabulous Flower - Geranium?
2 days ago in The Phytophactor