One component of the CIHR proposal we're working on is identifying mutants that are unable to retract their pseudopili ('retraction mutants') or to preventing DNA from sliding back out after it has been partly pulled in ('anti-backsliding mutants'). These mutants are expected to have similar but not identical phenotypes. The retraction mutant should be able to bind DNA at the cell surface but not take up any of it into the periplasm, so when cells are incubated with 32P-labeled DNA, all of the 'cell-associated' DNA should be removed by DNase I. The anti-backsliding mutant should also bind DNA, but it would be able to bring some DNA into the periplasm. This uptake should be inefficient, especially if the next step (translocation into the cytoplasm) is blocked by another mutation or by using circular DNA. So uptake should be reduced but probably not eliminated.
But the screens for these mutants aren't very good, so I need to think more about this.
Consider the retraction mutants first: These mutants should be able to bind DNA, but that doesn't mean they will have the same level of cell-associated radioactivity as wildtype cells. That depends on whether the uptake machinery is reused to take up more than one DNA fragment. To explain this better, here's an extreme example: Imagine that wildtype cells have only one DNA-binding structure on their surface. After DNA binds to this structure, the DNA is passed to an uptake machine (again only one per cell) which pulls it across the outer membrane into the periplasm. In species A, the structure is now free to bind another DNA fragment and pass it to the uptake machinery, and then another, and then another. In species B, each structure (or each uptake machine) can be used only once. When we compare the amounts of 32P-DNA associated with cells of species A and species B, species A will have four times as much. Now consider a mutant of each species whose uptake machine is unable to pull DNA in at all. These cells can still bind DNA but they can't take it up. The species A mutant will only have 25% as much cell-associated DNA of wildtype species A cells, but the species B mutant will have 100%.
Is H. influenzae's DNA uptake like species A or species B? We have suspected it's more like species B (i.e. uptake machinery isn't reusable), but the evidence for this is mainly just that the number of transformants doesn't keep increasing with increasing time or increasing DNA concentration, as if the uptake machinery had been used up. But the same effect may not be seen with DNA uptake experiments (I have one example where it isn't), which would mean that the bottleneck is at a later step (DNA translocation or recombination). I think we need to carefully investigate this first, so we'll know what phenotype to expect of our postulated retraction mutants. The post-doc has some newer uptake data that may address this.
The other issue is how we measure cell-associated DNA. The standard procedure is to incubate competent cells with a 'saturating' amount of DNA labeled with 32P (or 33P) and wash the cells by centrifuging them, resuspending them in fresh medium (with vigorous vortexing), centrifuging them again, resuspending them again, and probably centrifuging and resuspending them one more time. The goal is to remove all the DNA that's in the medium but leave all the DNA that's stuck to or inside the cells. This procedure should be fine for DNA that's inside the cells, but we don't really know how well this works for DNA that's just bound to the outside of the cells. Does the vigorous vortexing pull loosely bound DNA off the cells? The answer probably has something to do with Reynolds numbers, but that's beyond my expertise. In Neisseria, does it break off the pili that the DNA may be bound to? The RA probably can answer this, not because she knows about Reynolds numbers but because she's worked with Neisseria.
So I'm considering a different way of washing the cells. We routinely use filtration to collect and wash cells when we're transferring them to competence medium, so why not also use it to collect and wash cells in DNA-binding assays? We could first dilute the cells+DNA into a large volume of medium (100-1000-fold dilution, and then collect the cells by filtration (perhaps using only gentle suction to minimize shearing forces at the filter). We can then easily wash the filter with lots more medium to make sure all the unbound DNA is removed. Then we can just pop the filter into a scintillation vial for counting. To detect only DNA that has been taken up, we can add DNase I to the cells, dilution mix or wash. The dilution and washing medium should be cold or at room temperature to stop uptake of DNA that's already bound. Clogging of the filter isn't a problem because we'd be using only ~1 ml of cells, and the medium is cheap so the dilution and washing volumes are limited only by the capacity of the collection flask under the filter. The filters cost a couple of dollars each, but I think this would be well worth the cost.
We also can't be sure that the mutant's defect will be due to defective retraction of the postulated pseudopilus, but that should be a separate post.
Bedazzled by alternate truth
4 hours ago in The Phytophactor