Friday, June 11, 2010

Degenerate Uptake: Pilot Study

Something to blog about! (Wow; it's been over a month... sorry to my three loyal blog readers.)

I’ve gotten around to doing a pilot-scale experiment on the specificity of H. influenzae DNA uptake for the “uptake signal sequence” (USS). The USS is a ~29 base pair motif highly abundant in the H. influenzae genome, and sites that match the consensus USS are known to be preferred substrates for DNA uptake by competent cells. The presence of many USS in the chromosome is presumed to be why H. influenzae competent cells prefer H. influenzae DNA over DNA from other organisms.

However, little is known about how the structure of USS contributes to uptake of USS-containing fragments: Limited analyses of mutations of a DNA fragment containing a consensus USS suggests that some but not all informative positions in the USS motif are important to uptake, indicating that other forces (perhaps later steps in transformation) contribute to the structure of the USS motif.

To carefully dissect uptake specificity for the USS motif, we have devised an enrichment experiment:
(1) A complex pool of DNA fragments containing a degenerate USS library is incubated with competent cells.
(2) The fragments preferentially taken up by cells are purified from the periplasm.
(3) DNA sequencing is used to compare the input and periplasm-purified pools of sequences.

Details and Pilot-scale Results:

I’ve previously discussed the design of the input DNA pools. The control 200 bp construct is designed to already contain the sequences needed for Illumina single-end sequencing, along with a 32 bp consensus USS site near the middle of the fragment. The test construct is the same, except the USS is degenerate, having a 24% chance of a non-consensus base at each position. Thus in the degenerate-USS pool, the average site has ~7-8 mismatches from the consensus sequence.

The expectation is that
, while the consensus-USS construct (USS-C) will be taken up by cells well, the degenerate-USS construct (USS-D) will be taken up more poorly, since it contains many suboptimal sequences (i.e. it is less uniformly delicious). Indeed this is the case, with USS-C being taken up about 10 times better than USS-D at sub-saturating DNA concentrations (see below). The notion is that comparing the USS-D input to that taken up by cells will provide a precise measurement of uptake specificity for the USS (i.e. which sequences are tastiest). We think this will tell us a lot about the mechanism of uptake.

It occurred to me a couple weeks ago that before moving on to the data collection (i.e. the DNA sequencing), I should first make sure that the USS-D fragments recovered from the periplasmic purification are taken up better than the original USS-D input (i.e. the competent cells selected more delicious sequences). This would provide the clearest indication that the experiment worked and the material is worth sequencing. It is!

I compared the uptake of USS-C and USS-D before and after periplasmic purification of taken up DNA from rec-2 competent cells across a range of DNA concentrations. Here are the results:
A and B show the % DNA uptake for USS-C and USS-D, respectively, for different amounts of added DNA (to 200 ml competent cultures). C and D show the same data: C is a dose-response curve, and D is a double-reciprocal plot (since I used 2 ng of hot label, along with an additional amount of cold label for these experiments).

Input USS-C and periplasm-purified USS-C were quite similar, while periplasm-purified USS-D was taken up substantially better than input USS-D.

Notably, at low (sub-saturating) concentrations of DNA, periplasm-purified USS-D is taken up less well than USS-C, while at high (saturating) concentrations similar amounts of DNA are taken up. Also of note is that the input USS-D does not saturate until higher concentrations than the other three samples.

This is all good news. I left out a fair number of details, but this pilot-scale experiments is extremely encouraging. Next week, I plan to repeat the experiment, but this time on an appropriate scale for recovering samples for sequencing. I will also investigate how periplasm-purified USS-D samples behave when recovered from uptake experiments with varying amounts of DNA. I expect that at sub-saturating concentration, the cells will be less “picky”, such that periplasm-purified USS-D will be taken up less well than that purified from saturating concentration. This would provide a useful experimental condition, as in the sequence analysis we would be able to investigate the role of competition in shaping USS specificity.

I think this might end up working swimmingly... Onward!

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