Our solution the problem of lacking preliminary data for the crosslinking experiments is to eliminate these experiments from the proposal. But now I'm concerned that the proposal lacks scientifically interesting experiments. Here's a summary of the work we're proposing:
Q. 1: Which genes/proteins are required for DNA uptake:
We have made marked knockouts (= antibiotic-resistance cassette in a deletion) of all but one of the 25 genes in the competence regulon, and of the two other genes implicated in competence. Most of these have been tested for transformation frequency, but 10 haven't. Converting these marked mutants into unmarked mutant (by deleting the cassette) has been much more difficult than expected. Seven have been made and tested for transformation defects, the rest haven't been made yet.
The work of Q. 1 is to make the rest of the unmarked mutants and test them all for both transformation and DNA uptake. This will be quite boring work and would be best done by a junior technician.
Q. 2: Which genes contribute to uptake specificity:
Any cell-envelope mutants that retain substantial DNA uptake will be tested for decreased uptake specificity using 200 bp fragments with ideal or randomized uptake sequences.
Strains containing combinations of
H. influenzae and
A. pleuropneumoniae competence genes will be tested for uptake specificity with 200 bp fragments containing the
Hin and
Apl USSs. Any with altered specificity will be tested using deep sequencing of degenerate USS pools before and after uptake.
Q. 3: What are the uptake specificities of H. influenzae and A. pleuropneumoniae?
(This used to be Q. 1 and maybe we should restore this order so Q. 2 can follow this work.)
This will be more sequencing of degenerate USS pools before and after uptake. It's more of the same (though a lot more work and money), so we need to emphasize how preliminary the preliminary results are. The post-doc now has NIH funding for other work, so we could designate this as a grad-student project.
Q. 4: Does DNA conformation or flexibility contribute to uptake?
This work examines the physical properties of the USS and tests the effects of various physical permutations of it. It's a short section and should be built up a bit more.
Q. 5: Which genes power pseudopilus retraction and prevent backsliding?
This is an extension of the analysis in Q. 1. Mutants found in Q. 1 to have completely blocked uptake (~15 mutants?) will be tested for the ability to bind DNA to the cell surface - any mutants able to bind DNA but not take it up are candidates for defects in retraction of the pseudopilus. Mutants with some uptake (~5 mutants?) will be tested for the ability to take up very short fragments (~50 bp?) better than long ones, and maybe for the ability to retain bound DNA under shearing stress. Mutants that can take up short DNA but not long DNA will be candidates for defects in the hypothesized anti-backsliding function.
This section seems weak to me. I think we should find a way to go beyond the basic uptake experiments. Maybe we could reintroduce the crosslinking analysis as a way to test them?
Q. 6: What forces do competent cells exert on DNA?
This is the optical tweezers work. This time we explicitly propose the work needed to get the system functioning well, and then the measurements that will tell us about the uptake forces exerted by wildtype and mutant cells. The plan is to frame this as a way to test the candidates identified by the work in Q. 1 and Q. 5.
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