Articulating why us, why H. influenzae

The research associate has been going through the reviewers' comments on our unsuccessful CIHR research proposal. Much of the criticism was along the lines of 'Why should you do this when it's already been done in Neisseria*?", "Why should you do this in H. influenzae instead of Neisseria*?" and "Why should anyone try to do this, when other scientists have been unsuccessful?"

So yesterday she took the devil's advocate position, pushing me to defend our plans. One argument we will make is that we're in a much better position than the Neisseria researchers to use uptake sequences as a tool to study the uptake mechanism. For example, we have a resource that they lack - the availability of related species with variant uptake specificity. We also have done more investigation (in H. influenzae) into the details of the uptake specificity, measuring uptake with a series of uptake sequences altered at single or double positions. And Aim I of our proposal will give us an immensely detailed characterization of how strongly every base at every uptake sequence position contributes to uptake.

Another defense is that we have thought more deeply about how uptake could work than others have. Nobody else has considered that it's not enough to have a type four pilus or pseudopilus pull on the DNA (a ratchet is needed). Furthermore, nobody else has recognized the significance of the ability to take up circular molecules intact.

These issues are better addressed in H. influenzae. The need for a ratchet is not obvious in Neisseria, because it has long external pili. But most competent bacteria appear to pull DNA to the inner/cytoplasmic membrane with short stubby structures (pseudopili). Neisseria doesn't need long pili either, but this is only seen in mutants, whereas in H. influenzae we're studying the natural mechanism, not an aberration. We know that H. influenzae can take up circular DNAs that remain supercoiled in the periplasm, but we don't know that for Neisseria.

We will downplay testing whether competent H. influenzae have external pili. None are visible in the few published electron micrographs of competent cells, but nobody has ever specifically looked for them. We will begin with the reasonable assumption that competent cells lack external pili but will check this assumption using an anti-pilin antibody.

We may also downplay the search for the proteins that bind to DNA. It's intellectually messy work and we don't have any experience with mass-spec.

* The reviewers' emphasis on Neisseria made us wonder if one or both of them might have a Neisseria background. But I just checked the member lists for the previous two versions of this review committee, and none of them have any obvious connections to Neisseria or natural competence or pili. (These are previous committees, the membership list for our actual committee won't be released for months.) But whoever our reviewers were, they were surprisingly knowledgable!