I've added a new (but obvious in retrospect) question to our CIHR proposal: Which protein creates the retraction force that pulls the DNA across the outer membrane?
The PilT protein that does this in other bacteria, but H. influenzae and the other Pasteurellaceae have no pilT gene. We expect that the same retraction force is responsible for both initiation and continuation of uptake, and we can't begin to investigate its function until we know its identity (duh!). We have a full set of nonpolar knockout mutants, covering all the genes that are implicated in competence by either direct mutations or by their membership in the CRP-S regulon, so we will screen these for the expected phenotype: no transformation, no DNase I-resistant DNA uptake, but binding of DNA to competence-induced cells (DNase I-sensitive radioactivity bound to cells.
Testing for transformation and for DNA uptake are routine in our lab, but binding will be a bit trickier because we don't know what to expect. Tests for DNA binding are hard to interpret in normal competent cells, because DNA is taken up soon after being bound. So 'binding' is measured as the relative difference in cell-associated radioactivity ± treatment with DNase I ((noDNaseCPM - DNaseCPM)/noDNaseCPM). This isn't very sensitive, because noDNaseCPM isn't much larger than DNaseCPM. We don't know how much DNA should bind to the surface of cells that can't pull it in, nor how strong this binding should be. But DNaseCPM should be negligible (background in these experiments is usually <1% of DNaseCPM), so even if binding is only 10% of uptake it should be readily detected.
Should this binding be USS-specific? I think yes, but we won't throw out any uptake-minus mutants that bind DNA non-specifically.
This screen will be the first set of experiments in the proposal. The second set of experiments will be the DNA-protein crosslinking, and we will test any candidate retraction proteins for crosslinking to DNA, and test the corresponding knockout mutants for effects on the binding of other proteins. A genuine retraction knockout should increase the crosslinking of the binding/initiation proteins that act upstream of (before) it.
What if this screen for retraction mutants doesn't find anything? We won't conclude that no retraction protein exists, but rather that there is no real binding step distinct from the initial initiation that pulls the first DNA into the periplasm.