Background section for the CIHR proposal on DNA uptake

I've convinced myself that I need to reorganize the Background section of our upcoming proposal to CIHR (due March 1), but I'm not making much progress on paper so I thought I'd try to outline it here.

The plan is to first give a very brief overview of natural competence, saying what's generally true for all bacteria. This could also give some H. influenzae-specific information but I think it's better kept general.

Then I'll have a drawing of our current model of DNA uptake in gram-negative bacteria (applicable to both H. influenzae and N. meningitidis, and maybe to most Gram-negative bacteria). The figure below is one from a review we wrote - I'll modify it for the grant. I was originally (i.e. yesterday) planning to just briefly point out that this 'model' is really only a static picture of the known and hypothesized players. But I'm beginning to think I should give some description of the mechanisms illustrated the figure, also telling the reader that it's all hypotheses based on limited information (what does and doesn't happen to transforming DNA in wildtype and mutants, what properties proteins are predicted to have based on their sequences, what homologs of the proteins are thought to do in Tfp function).

1. Initiation of uptake has a strong sequence bias towards a motif called the uptake sequence.
2. Initiation occurs internally on DNA fragments.
3. Force for uptake is produced by shortening of a protein multimer called the pseudopilus, which is closely related to long filaments called Type IV pili (more details below).
   
4. Transforming DNA enters the periplasm through an outer membrane secretin pore like those through which type IV pili exit the periplasm.
5. DNA can bind nonspecifically to type IV pili; this may be how the force is transmitted.
6. Once DNA is in the periplasm, a fragment end interacts with translocation machinery.
7. One strand is degraded (probably on the periplasm side of the membrane) and the other enters the cytoplasm.
   
8. The competence protein Rec2 may form a pore in the membrane.

The model is not informative about many points. We don't know:

1. What are the ultimate sources of the forces that pull DNA in (ATP? PMF?, other?).
2. How the pseudopilus is disassembled.
3. How the DNA fits through the pore?
4. What role the sequence specificity plays .
5. What is the full uptake specificity.
   
6. Whether sequence specificity only matters at initiation.
7. Whether uptake and translocation are usually coupled.
8. What most of the genes in the H. influenzae competence regulon do
   
9. What prevents backsliding.
   

The rest of the Background is headed by the three gaps I propose to fill.

Gap 1: Who (what?) are the players?
This section will describe what we know and don't know about the proteins that might contribute to DNA uptake. I need to also say what other proteins might do (process DNA in the cytoplasm)? End with an overview of what we'll do in Aim I.

Gap 2: What is the uptake specificity?
This section will describe why I think the uptake specificity is an important component of the mechanism (i.e why I don't think it's just a Haemophilus-specific artefact). Emphasize that properties (genomic and uptake) are shared by Neisseria (just not the actual sequence itself) and because these are the two best studied systems we have to take them as exemplars. Then I can describe what we know: crude uptake assays, detailed genomic analysis, two types in the pasteurellaceae, and give an overview of what we'll do in Aim II.

Gap 3: What are the forces?
This section will describe the unknowns about what forces act on the DNA, in the contexts of the B. subtilis and Helicobacter analyses. Here I'll describe the absence of PilT, and the apparent need for a ratchet mechanism. Also the backsliding problem. And the need for an uptake force that is independent of translocation.

Proposed dynamic model
The Background will end with my dynamic ratchet-based model of uptake.