Tuesday, March 9, 2010

Another day, another attempt to get a dollar

Sigh... another grant due soon; this time, it's my last attempt to get an NIH postdoctoral fellowship. My last reviews mainly took issue with my proposal, which they found to be overly ambitious and somewhat unfocused. So below, is my first attempt at a summary/specific aims page, followed by a couple of preliminary data collection things I'd like to do before it's due (on April 8)...

The introduction:
Naturally competent bacteria take up intact DNA from their surroundings and can incorporate it into their chromosomes by homologous recombination. Akin to sexual recombination in eukaryotes, this natural transformation pathway moves alleles and genes between otherwise clonal lineages; and human bacterial pathogens have used this pathway to share antibiotic resistance genes, antigenic determinants, and virulence factors. To better elucidate the mechanism of transformation and to inform population/epidemiological studies, the proposed work will use the opportunistic Gram-negative bacterium Haemophilus influenzae to disentangle the sequence biases intrinsic to the DNA uptake and DNA recombination mechanisms by combining classical microbiology with modern DNA sequencing.

The specific aims:
  1. Define the genetic consequences of natural competence to H. influenzae. Transformation frequencies vary for different sequences and at different chromosomal locations, and this could strongly influence the rate of sequence evolution and adaptation along the genome. I will transform competent cultures of the standard lab strain with the genomic DNA of a clinical isolate and use deep sequencing to measure transformation across the lab strain’s chromosome for all the ~40,000 sites differing in the clinical isolate. This will provide an unparalleled dataset for investigating the sequence factors that promote and limit genetic exchange between bacterial cells.
  2. Measure the contribution of DNA uptake specificity to natural transformation. In several human pathogens, including H. influenzae, the uptake machinery prefers DNA fragments containing short “uptake sequences”, and abundant sequence motifs in many bacterial chromosomes suggest that biased DNA uptake has had a profound influence on genome evolution. I will purify the intact DNA molecules taken up into the periplasm and cytosol of competent cultures and use deep sequencing to measure the sequence biases of the uptake machinery. In combination with (a), this will disentangle the contributions of DNA uptake from those of DNA recombination during natural transformation.
The platitudes: The proposed work will link molecular studies of transformation to the growing genome sequence data being collected from many isolates of many bacterial species. By establishing my approach with completely sequenced chromosomes and using a well-defined experimental system, later studies could include a greater diversity of sequences or mimic more and more natural conditions. As a directly applicable outcome, the work will also produce the beginnings of a new type of genetic resource for mapping traits that differ between natural bacterial isolates (as in eukaryotic quantitative genetics) by generating fully genotyped recombinants. In the future, such studies will give empirical underpinnings to population genomic studies of bacterial genetic exchange, as well as provide new testable hypotheses for investigating the molecular mechanism of transformation.

Preliminary data I would like: (besides what I’ve got)
  • Properly replicated transformation frequencies for several markers. (I did this before, but it hasn’t been properly replicated.)
  • Follow a few molecules through uptake and recombination?
  • Population genetic inferences of “recombination” in H. influenzae? (I did this before, but it sucked.)

No comments:

Post a Comment