I've been thinking more about one reviewer's concern that our CIHR proposal was 'unfocused'. Although part of the problem was that we were proposing to do too much, I think the underlying problem was indeed a genuine lack of focus.
We identified three major gaps in the understanding of DNA uptake by gram negative bacteria, and proposed a series of experiments that would help fill in these gaps. BUT, we didn't tie these experiments together very well, and we weren't proposing to completely fill any of these gaps. They were all parts of a big problem, but they weren't the whole solution (that will likely take many years of work by more than one lab).
Ideally, our proposal should identify one well defined problem that our set of experiments will address, and then we should show how all of our experiments will come together to solve it. I think the way to do this is to focus on first determining whether uptake consists of distinct steps, and then on characterizing the steps as much as possible. So we'll propose a distinction between the initiation of DNA uptake, an event that happens at one place on the DNA fragment (we think at the USS), and the subsequent uptake, an ongoing process that gradually pulls the entire fragment into the periplasm.
We can further ask whether there is an initial binding step that's distinguishable from the initiation of active uptake. That is, does the uptake machinery at the cell surface first bind to DNA, either at the USS or first elsewhere and then at the USS, and then kink the DNA and pull the initiating loop across the outer membrane? The alternative is that binding is not an independent step, but that DNA is kinked and pulled immediately on contacting the uptake machinery.
All of our experiments contribute to clarifying these distinctions, in one way or another. So we just need to situate them in this context, and emphasize how they reinforce each other. The second goal, of characterizing the steps, is open-ended, but I don't think this creates a 'focus' problem.
The distinction between binding and initiation has implications for the evolution of uptake specificity too. We've previously considered two alternative explanations for uptake specificity. Either the protein responsible for uptake specificity is an add-on, evolved to screen DNA for relatedness before allowing it to be taken up, or the uptake specificity is intrinsic to the initiation process, occurring because specific sequences are easier to process. Identifying a separate binding step would favour the former.
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