Something got me thinking that, rather than kinking during uptake, the USS might facilitate DNA uptake by wrapping itself around the 'type IV' pilus.
We know that these pilus filaments on the cell surface are required for DNA uptake in many bacteria including H. influenzae. Some of these bacteria make long pili, and others (including our H. influenzae strain) just make what are probably short stubs, too short to be seen by scanning electron microscopy. We also know that DNA can bind to the pili of Pseudomonas aeruginosa.
I have been discounting this idea because I remember reading an article that, in a species whose uptake had sequence specificity, substituting the normal pilin gene with one from a species with no uptake specificity didn't change the cell's uptake specificity. BUT, I can't remember the details, and I can't find the article, so maybe I was wrong. The bacteria were probably Neisseria gonorrhoeae or Neisseria meningitidis; it wasn't H. influenzae or one of its close relatives, and the Neisserias are the other group of bacteria with uptake specificity. I should probably email either the Neisseria people or the woman who did the pilin-binding work in P. aeruginosa to see if they can point me to the paper. In any case, the Neisseria USS is unrelated to the H. influenzae USS, so I should consider testing this for H. influenzae.
I was thinking that the DNA would need to wrap 'paranemically' around the pilus - this term means that the DNA and the pilus aren't topologically interlinked like links in a chain, but just snuggle up to each other like... like... (can't think of a good analogy). But if the pili are just short stubs this distinction doesn't matter.
How could we test this? The conceptually simplest way (i.e. the only idea I have right now) is to mix USS-containing and control DNAs (circular? linear?) with purified H. influenzae and look for evidence of conformation change ( pililigatability of the ends) or of DNA-protein interactions (protection from nucleases? interference by ethylation?).
Unfortunately there's no way to purify the hypothetical short stub pili that our strain probably makes, but I can think of several possible solutions. One is to overexpress the pilin gene (pilA) under conditions where pili can assemble. This might work in our H. influenzae strain, or we could overexpress the gene in a different species whose own pilin gene is knocked out. If the pilin subunits won't assemble into pili, we might be able to purify the pilin subunits from such an overproducing strain and make them reassemble in vitro. Maybe the best solution would be to use a different strain, as some H. influenzae strains are reported to produce long pili.
(Hooray! While searching for the paper about H. influenzae strains that produce type 4 pili, I found the paper that showed that replacing a Neisseria pilin with one from P. aeruginosa didn't change uptake specificity. I'll need to read it carefully - it's quite dense.) I found the H. influenzae pili paper, and the strain they describe is one that we have in the lab. The authors don't report purifying the pili, but maybe we wouldn't need to do this. The next step is to get in touch with them, in case they are working on pilin-DNA interactions themselves. We wouldn't want to compete with them, but maybe we can collaborate.
13 hours ago in Variety of Life