Thursday, March 11, 2010

What have I got?

Okay, grant planning part 2... Below is a dense description of the preliminary data I have/will have for writing this next grant...


Transformation frequency depends on chromosome position:
DNA from a multiply marked derivative of Rd (MAP7) was briefly incubated with competent Rd cultures. The resulting transformation frequency at each of four loci was evaluated by selecting for cells that acquired the corresponding MAP7-specific antibiotic resistance allele. MAP7 DNA transformed each Rd locus at a different frequency. (Repeat experiment in progress… stay tuned but looks good.)

Sequence divergence decreases transformation frequency:
DNA from an antibiotic-resistant derivative of NP (1350NN) transformed Rd competent cells less efficiently than did DNA from MAP7, and vice versa. NP differs from Rd by ~2.4% per alignable base position (and an additional 10% of each genome is absent from the other, contained in indel polymorphisms) while the transformation frequencies at two loci were affected ~2 to 4-fold. (Data in hand.)

Co-transformation frequencies are non-random due to congression and linkage:
(Wish I didn’t have to describe this, but it’s too fundamental. Data mostly in hand.)

Transformants acquire hundreds of donor-specific alleles:
Several large DNA fragments recombined into the chromosomes of four individual Rd competent cells, as revealed by genome sequencing. Each of the four transformants was selected for resistance to one of two antibiotics encoded in the 1350NN strain (two NalR and two NovR), and the corresponding donor-specific allele was present in each of the four. In all, 24 donor segments (contiguous stretches of donor-specific alleles) were found across the 4 transformants, with an average of 1.4% of each recipient chromosome replaced with donor DNA (~25 kb and ~600 SNPs each). Mismatch repair is likely responsible for the disruption of contiguous stretches of donor-specific alleles in the transformants; assuming that for closely adjoined segments this was true, a total of 10 (instead of 24) independent transformation events occurred across the four transformants (6 of which were unselected; notably two of these were overlapping in independent transformants). (Data in hand; re-analysis in progress.)

DNA uptake signal sequences (USS) are densely distributed in the two genomes:
Both the Rd and NP chromosomes contain USSs nearly every kilobase and most are syntenic. (Cursory data only. Need a better analysis.)

Sequence preferences in DNA uptake can be captured by periplasmic DNA purification:

DNA fragments containing uptake signal sequences are efficiently taken up into cells, and taken up fragments can be cleanly purified away from both free DNA and chromosomal DNA. The use of rec-2 and rec-1 mutations will facilitate separating sequence biases at different stages of natural transformation. (Data in hand, except rec-1.)

  • Four/five marker transformation rates
  • Rd vs NP transformation rates
  • SNP spacing histogram with embedded SV table
  • Genome sequencing figure (pool data)
  • USS analysis
  • Molecular biology figure (uptake data)


  1. Sequence divergence paragraph: preface by a few words about how much divergence is thought to limit recombination (I think the previous experimental numbers might have suggested that 2-3% wouldn't matter). So there is much need for higher-resolution analyses.

  2. Previous experiments with defined levels of divergence said that recombination is pretty much blocked at ~10%, but 2-3% still matters. It is homologous recombination, after all. The relationship looks linear out to 5-8%, then got much worse in the paper I'm thinking of.

    Also, our analysis will not really be higher resolution, so much as investigate a lot more sequences.