We need a hypothesis.

We have lots of hypotheses, Lord knows, but I've just read the NIH advice for preparing a strong proposal, and the first thing they emphasize is the importance of starting with a clear hypothesis that the proposed work will test. Of course at one level I know this, but at another level I've been ignoring it. The series of experiments I would like to propose fit together nicely, building a coherent picture of the limits to recombination at both molecular and population levels, but it's not obvious to me that they test a specific hypothesis.

I can come up with a hypothesis for the first parts (the ones covered by the current post-doc's NIH fellowship proposal). "Every stage of transformational recombination is limited by sequence biases." This hypothesis might well be false - we only know of biases at two steps: DNA uptake and mismatch repair. But it provides a clear explicit framework for finding out where and what the biases are.

But I would like to extend the research to include the biases due to the differences in different isolates. Well, that's a way of saying it that sort-of fits the hypothesis but doesn't really capture what I want to propose. Instead I should first describe what I would like to propose, and then try to find a way to describe it that can be part of a unified hypothesis.

I would like to propose to find out the molecular reasons why different isolates of H. influenzae differ so much in their ability to transform. This should be fairly straightforward, given the molecular and genetic/genomic tools we have. We can also try to estimate how long these strains have had their specific transformation phenotypes - did a very recent ancestor get a mutation (or recombination!) that inactivated a competence/transformation gene, or is it descended from a long lineage of non-competent ancestors.

But I would also like to find out how different are the recombination histories of strains that can and can't transform - looking not just at the individual sequences they have acquired, replaced, or lost, but at the pattern underlying these events. Do strains that don't transform at all in the lab have a history of greatly reduced recombination, relative to strains that transform very well in the lab?

I don't know how do-able this is. If we get genome sequences of enough strains, can we build up a detailed picture of their recombination histories? The previous post-doc used a program called Mauve to identify recombination tracts...

I also don't know to what extent this analysis will be facilitated by the information we'll get from the first parts described above. We'll first build up a complete picture of the relationship between (i) a pair of donor and recipient genomes and (ii) the recombinant genomes that result from DNA uptake. (If resources permit we can do more than one pair of genomes.) Then we try to use this knowledge to infer the recombinational histories of other genomes. Is there a hypothesis in there? Maybe the hypothesis is that this will be possible. We could hypothesize that "Understanding the limits to transformational recombination in lab cultures will let us identify the recombination histories of natural isolates."

But as science this is what I consider a 'pseudo-hypothesis'. It's not a hypothesis about the nature of reality, but a hypothesis about the nature of our abilities. How about "The limits to transformational recombination in lab cultures explain the recombination histories of natural isolates."

Hmm, if that's our hypothesis then maybe this proposal does belong in the Evolution of Infections Disease program after all, and not in the Prokaryotic Cell Growth, Differentiation and Adaptation program. I think I'll handle this question by putting together a decent summary (once we have a single hypothesis) and asking both Program Directors where they think it belongs.