There is a unified set of analyses to be done. We have a complete set of knockout mutants that have been only partially characterized (transformation assays, preliminary DNA uptake assays). So we're proposing to do a more thorough analysis of all the mutants whose DNA uptake is defective (because DNA uptake is the overall focus of the proposal), using two new methods.
- One problem of working with the standard lab strain of H. influenzae is that it doesn't make long pili that can be seen by electron microscopy (EM), which means we can't tell whether our mutants block the assembly of pilin subunits into the pseudopili that pull DNA into the cell. (Well, we might be able to devise an assay for pseudopili, using crosslinking, but this will be a fallback.) So we're going to use transformation to put each of our knockout mutations into the NP strain, which does make pili, and then use EM to see if the mutation prevents pilus assembly. We'll be especially interested in mutants with abnormal pili.
- Our DNA uptake assays use centrifugation (pellet cells, resuspend pellet in fresh liquid, repeat twice) to wash unbound DNA away from cells, with or without first adding DNase I to digest DNA that's not been taken into the cell. But a B. subtilis paper I read on Saturday described instead washing cells by filtration, using special 96-well plates that have a filter in each well. This allows more thorough washing and is gentler on the cells. We need to repeat and replicate the uptake assays on our uptake-defective mutants anyway, because we've decided these are critical to detecting whether the mutants can still bind DNA at the cell surface. So we're now proposing to use the filtration assay to get very solid data for all these mutants.
The hard part is deciding what other investigations to include in this section. One issue raised by a reviewer of the previous submission is that knockout mutants are a very crude tool for investigating function, especially for processes that depend on concerted work by many components. In competence, a knockout that eliminates the pre-pilin peptidase has the same uptake and transformation phenotype as one that eliminates the pilin subunits or the assembly ATPase or the outer membrane pore used by the pseudopilus. We've added another phenotype to our screen (production of pili by strain NP), but we would like to have at least one experiment showing that we can use less-drastic mutations to investigate the specific function of a gene.
I think we should describe several analyses we know we want to do (each a short paragraph), and explain that similar techniques can be applied to other genes, depending on the phenotypes revealed by assays 1 and 2 above. I was planning to organize these by the questions they address, but it might be more effective to organize them by the techniques they illustrate.
- Different DNA substrates for the uptake assays: DNA concentration, ±USS, short vs long DNA fragments to detect retraction and retention defects (e.g. for the comE1 mutant), end blocked with a bead to detect polarity...
- Double mutants: To see if the DNA uptake by the comE1 knockout depends on DNA translocation across the inner membrane we'll test a comE1-rec2 double mutant.
- Reisolation of DNA from the periplasm: Test whether comF is blocked at the same step as rec2.
- Truncation mutations:
- Point mutations that change specific amino acids: We can make specific mutations to change proposed DNA-binding residues in the secretin subunits that form the outer membrane pore (comE). We can test whether pilF2 encodes a pilotin homolog by mutating the specific residue that should be its lipidation site.
- Random mutagenesis of a gene or part of a gene, followed by screening for a desired phenotype: We'd mutagenize the gene in an expression plasmid, and then put the plasmid into the corresponding knockout mutant to look for the desired phenotype. We're considering doing this to pilB to screen for an effect on retraction, but this is a long shot.
- Cross-species complementation and optical tweezer experiments will be described in separate sections, but they might be mentioned here.