We have a plan (again, but it's a new plan)

Specific Aims, in the form of questions:

Aim I. Characterizing the genetic consequences of transformation:

A. How do transformant genomes differ from the recipient genome? We want to know the number and length distributions of recombination tracts. This will be answered by sequencing a number of recombinant genomes (20? more?), preferably using multiplexing. We have preliminary data (analysis of four) showing 3% recombination.

B. How much do recombination frequencies vary across the genome? This will be measured by sequencing a large pool of recombinant genomes. The sensitivity of this analysis will be compromised by various factors - some we can control, some we can't.

C. Are these properties consistent across different strains? We should do 2 or more transformants of 86-028NP and of a couple of other transformable strains.

D. How mutagenic is transformation for recombined sequences? For non-recombined sequences? Is mutagenicity eliminated in a mismatch repair mutant? If not, is it due to events during uptake or translocation?

Aim 2. Characterizing the genetic differences that cause strain-to-strain variation in transformability: (The results of Part A will guide design of these experiments.)

A. What loci cause strain 86-028NP to be ~1000-fold less transformable than strain Rd? (Are any of these loci not in the CRP-S regulon?) We will identify these by sequencing Rd recombinants (individually and/or in one or more pools) pre-selected for reduced transformability.

B. What is the effect of each 86-028NP allele on transformability of Rd, and of the corresponding Rd allele on 86-028NP? Are the effects additive? Do some affect uptake and others affect recombination?

C. Are transformation differences in other strains due to the same or different loci? This can be a repeat of the analysis done in Aim 2A. Does each strain have a single primary defect?

D. How have these alleles evolved? Have they been transferred from other strains? Do defective alleles have multiple mutations, suggesting they are old?

In the Approach section we'll explain how we will accomplish these aims, and why we have chosen these methods. In the Significance and Innovation sections we'll need to convince the reader that these aims will make a big difference to our understanding of bacterial variation and evolution.

Aim 1 will provide fundamental information about bacterial recombination (and associated mutation), which will put almost all studies of bacterial evolution on a more solid footing. Aim 2 will help us understand why natural transformation has such dramatic variation in populations of many different bacteria, and thus how natural selection and other processes act on transformability.