- "What is the H. influenzae uptake specificity? A pool of USSs that have been intensively but randomly mutagenized and then selected for the ability to be taken up by competent cells will be sequenced to fully specify the uptake bias."
- What forces act on DNA during uptake? Laser-tweezer analysis of USS-dependent uptake by wild type and mutant cells will reveal the forces acting on the DNA at both the outer and inner membranes.
- Does the USS polarize the direction of uptake? Using magnetic beads to block uptake of either end of a small DNA fragment will show whether DNA uptake is symmetric around the asymmetric USS.
- Does the USS increase DNA flexibility? Cyclization of short USS-containing fragments will reveal whether the USS causes DNA to be intrinsically bent or flexible, and whether ethylation or nicking can replace parts of the USS.
- Which proteins interact with incoming DNA? Cross-linking proteins to DNA tagged with magnetic beads, followed by HPLC-MS, will be used to isolate and identify proteins that directly contact DNA on the cell surface.
- Which proteins determine USS specificity? Heterologous complementation with homologs from the related Actinobacillus pleuropneumoniae (which recognizes a variant USS) will identify the proteins responsible for this specificity.
Before any of this, I or the post-doc should carefully test whether H. influenzae can indeed take up closed circular DNA intact. I've been citing an old report of this for years, but it's critical to my model of DNA uptake so I really need to test it myself. I'll write a separate post about this.