One issue we need to deal with better in our revised CIHR proposal is the identity of the H. influenzae protein that retracts its type 4 pili and/or pseudopili (short stb pili).
In the well characterized bacteria (Neisseria meningitidis, Pseudomonas aeruginosa), pilus retraction is done by the protein PilT, using energy it gets by hydrolyzing ATP. I'm just going to summarize the things I think are true, but once I've done that I'll need to read the latest papers to find the evidence for my statements, and to check what I may have gotten wrong.
The problem is that we (and others) haven't been able to identify a PilT homolog in H. influenzae, although everything we know about DNA uptake in other systems, and about the need for other proteins of the type 4 pilus system in H. influenzae, predicts that a PilT homolog should be needed to pull the DNA in (by pulling the pilus or pseudopilus in). The competence regulon (CRP-S regulon includes all of the other proteins with recognizable T4P-family signal sequences ('prepilin protease-dependent leader sequences'), but none of these are good homologs of the PilT proteins identified in other bacteria (nor of the related PilU). Nor are there recognizable PilT or PilU homologs among proteins that don't have this leader sequence.
The closest H. influenzae relative of PilT in other systems is a protein assigned as the PilB homolog. In other bacteria PilB is essential for assembly of the T4P, and we know that H. influenzae PilB is essential for DNA uptake. I'm pretty sure that PilB can't also do the job of PilT, because both proteins are ATPases. That is, in the bacteria where its function has been studied (mainly N. meningitidis and P. aeruginosa) PilB uses energy from hydrolyzing ATP to assemble pilin subunits into a pilus fiber, and PilT uses energy from hydrolyzing ATP to disassemble the pilus fiber into its subunits. From the perspective of the pilus, the PilT reaction is a reversal of the PilB reaction, but from the perspective of the ATP these are very different reactions - the energy requirement tells us that PilB will not be able to carry out pilus disassembly.
But some protein must do the work of pulling in the DNA. One possibility is that H. influenzae has a cryptic PilT homolog - maybe an ATPase that gets to the right place in the inner membrane without having a recognizable T4P targeting sequence. Another is that this function is done by an unrelated protein. I'd expect such a protein to be an ATPase,
(Here's a link to a couple of movies of P. aeruginosa cells whose pili have been made visible with fluorescent antibody. In one you can see the pili (~5 times longer than the cell) shortening, and in the other you can see the tip of an elongated pilus attaching to the slide surface at a point distant from the cell, and then shortening, pulling the cell to the attachment point.)
I can't think of any way to select or screen for a defect in pilus disassembly. This is partly because pili have not been detected on strain Rd - the group that showed pili only did this work in the clinical strain 86028-NP. That strain does detectable twitching motility under the alkaline conditions where it does produce visible pili, but it also doesn't have a PilT homolog. In fact (I think), none of the Pasteurellaceae have them. And I think that Pasteurellacean cells typically don't have type 4 pili at all. We do have a pilus-associated phenotype that we can screen for defects in - DNA uptake - and we expect a PilT mutant to be defective for this. But we have already identified lots of genes whose knockouts prevent DNA uptake and thus would be found in such a screen, so this is a lousy way to look for PilT mutants.
But let's think about this a bit more. Say there are about 25 genes needed for transformation. In principle we can do random knockouts using some well-behaved in vitro method and use transformation to put these into the chromosome (just like Gerry Barcak and Hanna Tomb did in Ham Smith's lab 20 years ago). Then we'd do a massive screen for strains that don't transform, and screen the clean nontransformers for DNA uptake, then check where the knockout was in each strain that didn't do uptake. Then we'd check out any new genes. (This strategy is looking more and more lousy with each additional screening step. If I had unlimited money I might do this, but I don't think I'd fund it over competing projects.)
An alternative plan is to start by screening the genome for proteins with ATPase motifs (Walker boxes) and then pick out the ones that have signals to target them to the inner membrane and don't have any other assigned function. If there are only a few candidates this would be reasonable, but there might be very many.
Another plan is to wait for someone else to solve the problem. Another bacterium lacking PilT is also able to retract type 4 pili - I thought it was Myxococcus, but there's a 2003 paper describing Myxococcus pilT gene - Oh, it's that Myxococcus pilT mutants can still retract their pili, though not as well as wildtype cells.
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