Learning from the experts, part 2

The other expert I consulted this morning works on type four pili, the filaments we think are responsible for pulling DNA across the outer membrane. Here are some things I learned from her.

We shouldn't put too much faith in the present measurements of, for example, secretin structure (secretin is the protein pore the filaments pass through). It may be wider or more flexible, which would allow a pilus plus DNA to pass through.

She agreed that, if H. influenzae uses short pseudopili to bring in DNA, the pilus has to function as a ratchet that (i) binds DNA at the cell surface, (ii) retracts and pulls the DNA across only the thickness of the periplasm, (iii) releases the DNA (perhaps because the pseudopilus has become too short to hold on to it), (iv) elongates, and (v) binds the same DNA at a more distant position before retracting and pulling again. She thought this quite unlikely.

We need to revisit the question of whether competent cells might make long pili, rather than just short pseudopilus stubs that don't protrude beyond the outer membrane. We can look for pilin subunits in the culture medium of competent cells, but we will need a good anti-pilin antibody. We may be able to get this from a colleague, or she can help us design a peptide for a commercial antibody. We also may need to add BSA to the mix to prevent the intrinsically stickly pili/pilin from being lost by binding to the tubes and tips. And to add EDTA to inhibit any secreted metalloproteases that would degrade the pili (Vibrio has these but I don't know if H. influenzae does).

If cells make long pili, the DNA might wrap around them, eliminating the need for the improbable pseudopilus ratchet mechanism.

She agrees with a reviewer of our earlier proposal that secretin is quite stable, so that we may be able to purify it to test whether it binds DNA. But this would be a part of the proposal that I don't think would be very strong, so we might instead eliminate it in favour of the search for long pili.

She thinks that H. influenzae (and B. subtilis?) might have an as-yet-unrecognized protein that does the same job as PilT, using ATP hydrolysis to power pilus retraction. She mainly works on the toxin-coregulated pili of Vibrio cholerae - these don't themselves retract, and they have no PilT, but they are responsible for pulling filamentous phage into the cell so must be able to exert a similar force.

She thinks that the pilC gene encodes a crucial inner membrane protein that makes the connection between PilB/PilT (on the cytoplasmic side of the inner membrane) and the pilin subunits at the base of the pilus. This is likely to be the weak link in the strength of the pilus, as the pilin subunits are tightly braided together.